ABSTRACTS
THE ROLE OF REGULAR LOW-AMPLITUDE MOVEMENTS
IN THE FORMATION OF VIRTUAL RECEPTIVE STRUCTURES
OF PERCEPTION ELEMENTS AND SIGNALS IN THE RETINA
G.M. Agadzhanyan and G.E. Dronenko
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia
The investigation of eyes tremor by means of trace reflection of visual information (TRVI) method reveals a new and
important role of low-amplitude movements in the process of perception and transformation of image elements. The authors
studied the structure-forming role of regular low-amplitude movements in retinal cones transformation into virtual receptors –
cones with another geometric, amplitude-frequency and spectral characteristics of transformation of elements focused on the
retina. Fundamental differences in the perception of static and dynamic structures of reception have been shown in such processes as formation of image receptive fields and primary visual signals; exchange of optical information between the receptors; formation of orientation characteristics of image perception elements.
A new hypothesis is offered to clarify the mechanism of regulation and performance of tremor movements. The influence of regular low-amplitude movements trajectory on formation of dynamic structures was studied. The following aspects of
the virtual structures of perception elements (VSPE) influence on human visual perception were analyzed:
 Identical perception of visual elements regardless of color type of retinal receptors-cones.
 Formation of intensity-modulated, space-frequency spectral and orientation signals of image elements directed to the
CNS.
THE FINE STRUCTURE OF THE MEMBRANE LIPOPROTEIN SUBUNITS AND THEIR BIOLOGICAL
MOBILITY
T.S. Aglintsyan
L.A. Orbeli Institute of Physiology,
National Academy of Science of Armenia, Yerevan, Armenia
The lipoprotein subunits of the biological membranes are the globules of 5-9 nm in diameter. The thickness of protein
coat is 1.5-2 nm. The investigations conducted on the biopsed myocardium of the heart disease patients allowed to elicit the fine structure of these particles [4] and to propose their three-dimensional model [1,5]. According to this model the protein coat
of the phospholipid micelle forms the equatorial filaments and the cone-like "tail". From the electron microscopic pattern of
the intramembranous particles (IMP) revealed in different human myocardial cell membranes it is possible to say about exclusive flexibility and mobility both of the "tails" and filaments of IMP. The vesicles forming from the external nuclear membrane
are edged by three IMP. The latter on the tissue section have the ring-like head with the filaments on two sides and the "tail"
with a length, which is less longer than the head diameter. In the several sites the internal nuclear membrane has the crossstriated form owing to perpendicular orientation of the "tails" in relation to membrane plane. Earlier we supposed the free orientation of the particles in the membrane thickness and that its perpendicular orientation are connected with the membrane
globular organization and the cross-striated form [1,4]. But as it turned out the membrane proteins cannot make flip-flop. In
contrast, they can have rotational or lateral diffusion ability [6]. Therefore we found the compromising choice and confined a
mobility of the filaments and "tails". The latter are able to fold at right angles and straighten up giving the membrane crossstriated form. Probably the "tails" of the two pairs of IMP have the opposite directions in the case, when they are straightened
up. As we suppose, these pairs of IMP form then pores providing the opposite flow of Na + and K+-ions via the membrane as a
result of the filaments folding in "tails" direction. What kind of protein forms the coats of IMP and its derivates? This is probably the amphipathic structural protein that may imitate transmembrane integral protein -integrin [7], when the "tails" of IMP
are straightened up. The electron histochemical investigations [2] established that besides the myofibrils myofibrillar ATPase
is revealed in sarcolemma, in membranes of both T-system and sarcoplasmic reticulum. The mitochondrial ATPase is localized
in the internal membrane of the mitochondria. The protein coats of the IMP and their derivates have also a myoglobinperoxidase activity in rat myocardium [1]. The biological mobility of these particles is caused probably by enzymatic activity of the
above mentioned protein in the presence of cations The folded "tails" of the IMP are apparently connected with the head of the
neighboring or adjacent row particles providing the membrane integrity and creating the similarity of the sieve holding up the
transport of large particles.
It is known [3] that by the addition of lipids to the amphipathic protein solution the lipoprotein micelles are formed
spontaneously. The transition from the globular to the lamellar organization of the membrane occurs apparently as a result of
the opposite process: the protein layer of IMP slips down and the uncovered lipid micelles become invisible under the electron beam or turn into the true bilayer. In these cases the folded "tails" of the IMP imitate the discrete protein layers o f the
membrane. The globular membrane is more permeable for the water dissoluble substances and the lamellar membrane on the
contrary is more permeable for the lipid dissoluble substances and subjected to the influence of the phospholipases.
Thus, the biological mobility is inherent in evolution aspect of such ancient structure as biological membrane.
References
1. Aglintsyan T.S. Proc. of 9th Ann. Conf. of AEMS "Electron microscopy - 2000", p.13.
2. Gousacova N.F., and Aglintsyan T.S. - Ibid., p 39.
3. Singer S.J., and Nicolson G.L. - Science, 1972,v. 175, p 720.
4. Aglintsyan T.S. Vestnik khirurgii Armenii, 1999, №1- 2, p. 140 (in Russian).
5. Aglintsyan T.S.Proc. VI Russian Conf. On Cell Pathology, M., 2000, p. 63 (in Russian).
6. Alberts B. et al. Molekulyarnaya biologiya kletki, M. 1994 (in Russian).
1
7. Bykov V.L. Funktsional’naya morphologiya kletki, St.-Peterburg, 1995, 55p. (in Russian).
TISSUE ENGINEERING OF HEART VALVE ALLOGRAFTS
V.S. Akatov, 2R.M. Muratov, 1E.N. Ryabokon’, 1A.V. Chekanov, 2I.I. Scopin
1
1Institute
of Theoretical and Experimental Biophysics,
Russian Academy of Sciences, Pushchino, Russia;
2Research Center of Cardiovascular Surgery, Ministry of Public Health
of Russian Federation, Moscow, Russia
Tissue engineering methods are actively utilized for creation of new generation of heart valve allografts. One of the
approaches consists in implantation in vitro of cells of the recipient in tissues of allografts. This modification is done with the
aim to decrease the immune response against the allograft, to avert its calcification, and eventually to rise biocompatibility of
allograft and to increase duration of its normal operation after an implantation. One of stages of the allograft modification by
cells of a recipient is obtaining smooth muscle cell (SMC) culture from a human vein and support of conditions providing their
migration in vitro through basal membrane into media of leaflets and vascular wall of heart valve allograft. In the report the results of development of technology intended for implantation of SMC into the allograft tissues are presented.
It is shown, that SMC in vitro are capable to attach to basal membrane of leaflets and vascular wall of an allograft and
to spread on them. It was found the ability of SMC to migrate through a basal membrane into media in 2-3 day of in vitro cultivation. Based on the results obtained the method of cell seeding on wall and leaflets of allografts have been developed. The
roller regime of cultivation was used for this purpose. The method of perfusion cultivation of cells seeded on the allograft is
circumscribed which provided the migration of cells in tissue. It is shown that SMC after migration into tissue synthesize collagen and proliferate.
As a whole, the technology of obtaining human SMC and their implantation in leaflets and vascular wall of human
heart valve allografts is designed.
PLURIPOTENT SIGNALING IN CHANNEL/ENZYME MULTIMERS: ATPase TRANSITIONS IN SUR MODULE
GATE ATP-SENSITIVE K+ CONDUCTANCE
Alexey E. Alekseev, Leonid V. Zingman, Martin Bienengraeber,
Denice Hodgson, Amy B. Kim, Petras P. Dzeja and Andre Terzic
Mayo Clinic, Mayo Foundation, Rochester MN, USA
Traditionally, channels have been recognized as passive conduits for ion permeation regulated by membrane potential,
ligand binding or covalent modification. The recent discovery of bifunctional protein complexes that combine enzymatic and
ion conductance properties raises the possibility of a novel principle in channel regulation. It is well established that interconversion of an enzyme through transitional states during the catalytic reaction governs specialized protein functions. However,
linkage between catalysis at the enzymatic module and ion permeation through the pore remains elusive. In fact, it is unknown
whether the channel catalytic property is a remnant of evolution or a true determinant of gating amenable to regulation and capable of integrating cellular chemical and electrical events.
The cases in point are hetero-octameric ATP-sensitive K+ (KATP) channels critical in the regulation of vital cellular
functions, such as neurohormonal release and cytoprotection. These channels are formed by association of the pore-forming
Kir6.2 subunit and the sulfonylurea receptor SUR, an ATP-binding cassette (ABC) protein harboring a recently uncovered intrinsic ATPase activity. By virtue of nucleotide-dependent gating, KATP channels act as metabolic sensors setting membrane
excitability in response to oscillations in cellular metabolism. It is generally accepted that channel inhibition by intracellular
ATP at Kir6.2 can be antagonized by replacement of MgATP with MgADP at the second nucleotide binding domain (NBD2)
of the SUR subunit. Yet, the similar affinity of NBD2 for MgATP and MgADP suggests that nucleotide competition at the
binding site is not solely responsible for K ATP channel regulation in a cellular environment with exceeding ATP over ADP levels. Therefore, rather than ligand competition per se, an ATPase-driven transition between nucleotide-liganded states in the
NBD2 of SUR may provide a more effective governance of channel gating. However, the real relationship between conformational rearrangements in the ATPase cycle and channel gating has not been established.
Coupling of discrete conformations in the SUR catalytic cycle with channel gating was solved using nucleotide trapping procedures in conjunction with current recording to monitor in real-time the outcome of ATPase transitions on channel
behavior. Capture of intermediates was achieved using -phosphate (Pi) analogs, rthovanadate (Vi) and beryllium-fluoride
(BeFx), which readily undergo changes in coordination geometry and stabilize the ATPase cycle in distinct conformations: E +
MgATP  E*MgATP  E**MgADPPi  E***MgADP + Pi  E + MgADP + Pi, where E is the ATPase molecule
and stars indicate transitional states during ATP hydrolysis. We establish that trapping MgADP at the SUR ATPase with beryllium-fluoride mimicked a prehydrolytic MgATP-bound state (SUR*MgADPBeFx=SUR*MgATP), and translated in pore closure (negative channel gating). Recruitment of a posthydrolytic MgADP-bound state was accomplished by orthovanadate
(SUR**MgADPVi=E**MgADPPi), and promoted opening of ATP-inhibited KATP channels (positive channel gating). Trapping the SUR ATPase by beryllium-fluoride and orthovanadate required divalent cations, hydrolyzable ATP or externally applied ADP, and structurally intact NBD2. Conformations linked with channel gating were revealed only following arrest of sequential discrete steps during hydrolysis. Thus, in the normally occurring ATPase cycle the short lifetime of each SUR transition may not translate into regulation of channel gating. Yet, the spectrum of conformational intermediates, with different potential outcomes on channel gating, would endow SUR with positive or negative regulation of ion permeation.
This pluripotent gating property of SUR could, thus, be exploited by prolonging the lifetime of specific conformations
during the ATPase cycle. In fact, KATP channel regulators, such as potassium channel opening drugs, target this subunit. We
have demonstrated that entry of SUR into posthydrolytic conformation plays a permissive role in mediating the action of potassium channel openers. MgADP-dependent stabilization of posthydrolytic states implicates this endogenous metabolite as a
2central regulator of ATPase cycling. The intracellular metabolic messenger, MgADP, suppressed the ATPase activity of
NBD2. The effect of ADP in slowing the rate of ATP hydrolysis was concentration-dependent with an IC50 at 9724 M.
Therefore, increased cytosolic levels of MgADP under metabolic stress would effectively decelerate ATPase cycling arresting
it in conformational transition associated with channel opening. In this way, nucleotide exchange between the SUR ATPase
and intracellular enzymatic pathways could couple K ATP channel function with the cellular metabolic status. In the cell, the
high rate of intracellular creatine kinase flux facilitates disengagement of posthydrolytic conformations, and promotes SUR
ATPase cycling. Here, creatine kinase doubled the rate of ATP hydrolysis at NBD2 and reversed MgADP-induced KATP channel opening underscoring a critical role for a phosphotransfer reaction in regulating the lifetime of conformational transitions in
the ATPase cycle. Assigning to the channel catalytic module a role in coupling ion permeation with intracellular enzymatic
pathways identifies a novel principle in the regulation of cellular excitability, and defines an operational paradigm for channel/enzyme multimers.
THE ROLE OF NITRIC OXIDE IN INSULIN-DEPENDENT REGULATION OF RAT HEART CONTRACTILITY
L.A. Andrejeva, N.A. Chumaeva, O.V. Nakipova, S.G. Kolaeva, Y.A. Andrejev
Institute of Cell Biophysics, Russian Academy of Sciences,
Pushchino, Moscow Region, 142290 Russia
Nitric oxide (NO) is a free radical endogenously produced by variety of mammalian cells and shown to be ubiquitous
signal transduction molecule. It is synthesised by constitutive (1 and 3) and inducible (2) NO synthases (NOS), enzymes that
use l-arginine and molecular oxygen as substrates. NO interacts with soluble guanilate cyclase, leading to activation and elevation of cGMP levels. NO has been shown to be important physiological regulator of calcium homeostasis and myocardial contractile function through interaction with both L-type calcium channels in sarcolemma and Ca2+-ATPase and ryanodine receptors in sarcoplasmic reticulum. However, although there is general consensus that NO-related activity can exert significant
chronotropic and inotropic effects on the heart, both positive and negative effects have been reported. The molecular basis for
modulation of heart function by NO is only partly understood.
Recent studies have shown that the activation NO/cGMP pathway stimulates glucose transport, glycolysis and glucose
oxidation in skeletal and cardiac muscles and possible this cascade is required for maximal insulin sensitivity. It was shown
that some diseases such as ishemic heart disease, heart failure, diabetes mellitus, hypertension alter NO synthesis. The maximal
activity of NOS was significantly decreased in obese insulin resistant skeletal muscle compared with lean insulin-sensitive
skeletal muscle. However, the interrelation between NO/cGMP and insulin signalling pathways in regulation of heart contractile function has not be established.
In the present study the effect of N(G)-nitro-L-arginine methyl ester (L-NAME), inhibitor of NOS, L-arginine and insulin on the steady-state force-frequency relationship (FFR) in isolated rat ventricular papillary muscle (PM) was examined.
L-NAME (1 μM) had relatively minor negative effect (10%, P > 0,05) on PM. This finding indicates that basal release
of NO does not play considerable role in inotropic state of the rat myocardium.
L-arginine (1 nM - 1 μM), substrate for NOS, exerted a monophasic concentration-dependent negative inotropic action at all frequencies tested (0.1-3 Hz) with maximum effect at concentration 1 μM (P<0,05). It was shown that high levels of
NO induce large increases in cGMP and negative inotropic effect mediated by PKG-dependent reduction in myofilament responsiveness to Ca2+.
Administration of insulin (0.1 nM – 10 nM) led to a dose-dependent decrease of force of contraction at all frequencies
tested. After inhibition of NOS (L-NAME, 1 μM) the negative response to insulin of PM was significantly enhanced at frequencies range from 0.1–1 Hz (P<0.05) and did not change from 1–3 Hz. It was shown that in adult isolated cardiac myocytes
NOS3 activity is regulated in part by change in pacing frequency or [Ca 2+ ]0. The increase of beating rate induced activity of
NOS3 and the associated increase in endogenous production of NOx.
Pretreatment of PM with insulin (10 nM) prevented the negative inotropic effect of L-arginine (1 μM), conversely pretreatment of papillary muscle with L-arginine unmasked effect of insulin on the force of contraction. This results suggest that
insulin stimulates maximally NO/cGMP system, but has also any other metabolism for the negative inotropic action.
The possible mechanisms of interrelation between insulin and NO/cGMP pathways are discussed.
This study was supported by the Russian Foundation for Basic Research, project № 01-04-48199 and the Federal Central Program “Integration”, project № A0055.
THE SEASONAL PECULIARITIES OF FORCE-FREQUENCY RELATIONS IN THE MYOCARDIUM OF
GROUND SQUIRREL CITELLUS UNDULATUS
L.A. Andrejeva, O.V. Nakipova, N.A. Chumaeva, S.G. Kolaeva, N.I. Kukushkin
Institute of Cell Biophysics, Russian Academy of Sciences,
Pushchino, Moscow Region, 142290 Russia
The heart of hibernators is capable of functioning without arrhythmias and calcium overloads within the body temperature range from 37 to 0ºC. In contrast to that, the body temperature decrease to 32ºC induces an atrial fibrillation in nonhibernating mammals and its further decrease below 20ºC exerts extrasystole and ventricular fibrillation. The reasons of such a
unique cardiac resistance have not been established.
The rhythmoinotropic relations (dependence of contraction force on stimulation frequency) is an important characteristic of myocardium contractility. It was found that the type of force-frequency relations (FFR) in myocardium of hibernators
varies in accordance with the animal state (hibernating-active). However, the mechanisms providing for such phenomenon are
obscure, whereas the available results are negligible and controversial.
The goal of our work was to study the steady-state FFR in myocardium of the ground squirrel in different periods of
activity: summer, preparation to hibernation, winter and posthibernation.
Papillary muscles (PM) of right ventricle of the heart in ground squirrel C. Undulatus were studied. Amplitude of contractions was measured at a temperature of 30±1ºC. The steady-state FFR was measured within the frequency range from 0.1 3
to 1 Hz. Frequency scanning was performed from lower to higher values with a step of 0.1 Hz. The amplitude of PM contraction at the frequency of 0.1 Hz was taken as 100%.
The following groups of squirrels were studied: summer (June-July); autumn (October-November); active winter (December-February); and spring (April-May). Winter squirrels were taken between bouts.
The contraction amplitude dependence on stimulation frequency in active squirrels changes drastically during annual
cycle. Typical curves of FFR were measured in different periods of activity. According to our findings, the FFR in active
ground squirrels in summer is negative, whereas in autumn and winter squirrels this curve is multicomponent. There is a frequency range within which the dependence is nonmonotonic: an increase in the contraction force is followed by a decrease. As
a result, there is a positive extremum in the curve of the FFR. The nonmonotonic FFR emerges in October, whereas the positive extremum position ranges from 0.2 to 0.5 Hz. In December-February, the extremum in the FFR curve is in more narrow
frequency range (0.3-0.4 Hz). In some cases, the extremum amplitude is more than 20% higher than the contraction amplitude
at 0.1 Hz. In April-May, the amplitude of the extremum decreases and its position is shifted toward a lower frequency (0.2-0.3
Hz). In July, the multicomponent character of the FFR is not observed usually, and the slope of the curve is negative.
Thus, three FFR types were found in active ground squirrels. In summer and a part of autumn squirrels, the slope of
the FFR curve is entirely negative (negative force staircase). This character of the FFR is similar to that in adult rats. The majority of autumn squirrels contained three components: a positive component, within the frequency range 0.3-0.4 Hz, and two
negative components, at frequencies higher than 0.5 Hz and within the frequency range from 0.1 to 0.2 Hz. The force staircase
in myocardium of active winter squirrels contains two components: a positive component within the frequency range from 0.1
to 0.3 Hz and a negative component at higher frequencies.
The rate of uptake and accumulation of Ca2+ in sarcoplasmic reticulum of Richardson's ground squirrel cardiomyocytes increases in autumn animals and reaches the maximum level during hibernation. Perhaps, seasonal differences of the
rhythmoinotropic relations are associated with changes in the ratio of calcium-transporting systems activity. The fact of appearance of a positive component of the FFR curve in the ground squirrel myocardium in autumn, its presence in winter, and
disappearance in summer is a specific feature of active animals during hibernation period. This feature can be regarded as an
indicator of preparation to hibernation and awakening from hibernation.
This study was supported by the Russian Foundation for Basic Research, project № 01-04-48199 and the Federal central program “Integration”, project № A0055.
MONOCYTIC CELL ADHESION TO INTACT AND PLASMIN-MODIFIED FIBRINOGEN: POSSIBLE
INVOLVEMENT OF MAC-1 (CD11B/CD18) AND ICAM-1 (CD54)
T.I. Arefieva, *T.L. Krasnikova
Institute of Experimental Cardiology, Cardiology Research Center,
*Institute of Children Haematology, Moscow, Russia
Fibrinogen is a blood-borne glycoprotein known to play basic role in hemostasis. At sites of tissue injury fibrinogen
forms an insoluble fibrin clot, thus controlling a blood loss. Subsequently, fibrin matrix is degraded by plasmin into number of
fragments, known as the early, X and Y, and the terminal, D and E, fibrin(ogen) degradation products (FDP). Besides controlling the hemostasis, other properties have recently been discovered. Fibrin(ogen) and FDP, accumulated at sites of acute and
chronic vessel wall injury, regulate cell adhesion and spreading, and also possess vasoconstrictor, chemotactic and mitogenic
activities.
Mononuclear phagocytes play a significant role in vessel wall remodelling in atherosclerosis and restenosis orchestrating inflammatory reactions in the damaged vessel wall. 2-integrin Mac-1 and immunoglobulin-like ICAM-1 adhesion molecules are expressed by these cells and are both known to bind fibrinogen and its degradation products.
Here, we investigated whether fibrinogen cleavage with plasmin modulates cell adherence and what types of adhesion
molecules are involved. Using several cell types with the different patterns of Mac-1 and ICAM-1 expression we have demonstrated that plasmic cleavage of fibrinogen inhibited the adhesion of monocytic THP-1 cells expressing Mac-1 as a predominant fibrinogen receptor. On the other hand, the maximal adherence of cells with the predominant ICAM-1 expression, namely
monocytic U937 and B-lymphocytic Daudi cells, was observed on plasmin-pretreated fibrinogen. THP-1 cell adhesion to fibrinogen was significantly inhibited by more than 60% when the cells were pre-incubated with function-blocking anti-2integrin antibody, whereas anti-ICAM-1 monoclonal antibody had no inhibitory effect. In contrast, attachment of U937 cells to
plasmin-degraded fibrinogen was similarly inhibited by anti-ICAM-1 antibody, while anti-2-integrin antibody was ineffective.
To reveal what fibrinogen derivatives are responsible for the modulation of cellular adherence, U937 and THP-1 cells were
seeded on plates coated with FDP, characterized by electrophoresis in SDS-polyacrylamide gels. THP-1 cells adhered more effectively to non-cleaved fibrinogen and fibrinogen partially cleaved to X and Y fragments. The maximum U937 cell adhesion
was associated with generation of X and Y fragments, while the complete fibrinogen transition to D and E fragments led to a
loss in stimulation of cell adhesion.
Our data suggest, that  2-integrin-mediated cellular adherence requires an intact or minimally modified fibrinogen
structure, whereas ICAM-1 is more effective in mediating the cell attachment to the early products of fibrinogen degradation,
fragments X and Y, rather than to the intact fibrinogen. We speculate, that depending on the extent of fibrin(ogen) breakdown
monocytes can utilize either Mac-1 or ICAM-1 receptors to ensure effective cell trafficking in the injured artery wall where fibrinogen derivatives are deposed.
AGGREGATION PATTERNS OF FRAGMENTS OF DROSOPHILA LIGHT MEROMYOSIN (LMM) EXPRESSED
BY E. COLI
G. Beinbrech*, G. Ader*, C. Ziegler*, S. Hellmann* and K.A. Taylor#
*University Münster, Institute für Tierphysiologie,
4
Hindenburgplatz 55, D48161 Münster, FRG
#Institute of Molecular Biophysics, FSU, Tallahassee, Florida 32306-4380, USA
Drosophila LMM fragments, consisting of the C-terminal third of LMM (MHC-1; 195 AA), or two thirds (MHC-1,2;
390 AA) or three C-terminal thirds (MHC-1,2,3; 585 AA), respectively, are expressed in E. coli in large amounts. They may
form up to 90 % of the total bacterial protein. E. coli expressing LMM fragments with a C-terminus coded by exon 18 (coding
for 1 AA, Isoleucin, and present in flight muscles) or exon 19 (coding for 27 AA, present in leg muscles) were fixed for electron microscopy with glutaraldehyde solutions (Smith D.S., 1966, J. Cell Biol. 29, 449) or by a multistage fixation procedure
(Ashton F.T. and Pepe F.A., 1981, J. Microsc. 123, 93). Electron micrographs were digitized and used for Fourier transforms
(FFT), filtering and image reconstitution (programs of H.-P. Winkler, Tallahassee). In the case of crystals, SPECTRA
(Schmidt M.F., et al., 1993, Ultramicroscopy 48, 251) was used to calculate the dimensions of the unit cells and to reconstitute the image.
All fragments form large aggregates within the bacteria. The structures of these aggregates differ considerably from
the pattern of those obtained by slow dialysis of fragments in vitro (Ader et al., 1997, J. Muscle Res. Cell Motil. 18, 246; Ziegler et al., 1999, J. Muscle Res. Cell Motil. 20, 817): The C-terminal third of LMM (MHC-1) formed paracrystals with a striation pattern causing pronounced layer lines of about 12.5 nm in the FFTs. The observed striation pattern was independent of
the presence of the exon 18 coded or of the exon 19 coded, non-helical tail piece. A 14.3 nm periodicity (cross bridge periodicity along the myosin filaments) is missing. However, layer lines corresponding to a 28 nm to 30 nm periodicity, which is close
to 2 x 14.3 nm, could be observed in the FFTs of aggregates of the large fragments (MHC-1,2,3).
A complex striation pattern could be observed on electron micrographs of aggregates of MHC 1,2-18 and MHC 1,219 fragments with lines in three directions. The diffraction pattern is 2-dimensional. This indicates that the molecules form
crystalline structures. Filtering and image reconstitution by SPECTRA yielded distinct super coiled-coils within the crystals
with diameters of about 10 nm. SPECTRA calculated unit cell dimensions with an angle  = 151° and side lengths of 24.9 nm
and 14.2 nm, respectively. The diameters of the coiled-coils is 3 nm to 4 nm. That is too large for single molecules and might
rather represent subfilament-like elements instead.
Acknowledgements: Supported by the Deutsche Forschungsgemeinschaft (Be 347/12-1). We thank Dr. S.I. Bernstein,
San Diego State University, San Diego, CA, for the cDNA.
EFFECTS OF CHRONIC 2G ACCELERATION
ON M. SOLEUS STRUCTURE IN RAT
I.N. Belozerova, T.L. Nemirovskaya, B.S. Shenkman
SRC Institute for Biomedical Problems, Moscow, Russia
It is well-known that exposure to real or simulated microgravity brings about the structural changes in muscle fibers,
most pronounced in m. soleus (increase of connective tissue content, fiber atrophy, increase of amount of centronucleated fibers etc) [Ilyina-Kakueva, 1976; Belozerova et al., 2000]. In order to prevent the development of such changes a number of
countermeasure means are considered. It is believed that one of such technologies is the application of artificial gravity by
means of centrifuge. Rat studies performed on-board biosatellite KOSMOS-936 [Ilyina-Kakueva et al, 1979] and Rhesus monkey study [Belozerova et al., 2000] using bed rest simulation model gave evidence on the certain positive countermeasure effects of artificial gravity on muscle tissue.
The present study was purposed to investigate the influence of 5 day chronic 2g centrifugation on rat soleus muscle.
14 adult male rats were divided into two groups: control (C) and experimental (E) ones. E rats were subjected to 2g
acceleration on the centrifuge lever for 5 days. Then all rats were decapitated and soleus samples were processed for histology
and histochemistry.
It was shown that 5 day centrifugation induced the sufficient increase in the area percentage engaged by connective
tissue elements (from 4  1% in C rats to 11  3% in E rats, p<0.05). The content of the centronucleated fibers increase from
0.4 to 1.7% (p<0.05).
Thus the number of structural changes in rat soleus was observed after 5 day chronic centrifugation. The study was
supported by grant № 99-01190 of INTAS.
T-JUMP INDUCED DISORDER-ORDER TRANSITION
IN RELAXED RABBIT THICK FILAMENTS OBSERVED
WITH ELECTRON MICROSCOPY AND X-RAY DIFFRACTION
Pauline M. Bennett1, A.K. Tsaturyan2, M.A. Ferenczi3, Natalia Koubassova2, D.A. Shestakov2 and S.Y. Bershitsky4
1The
Randall Centre, King's College London, UK;
of Mechanics, Moscow University, Russia;
3Imperial College, London, UK;
4Institute of Ecology and Genetics of Micro-Organisms,
Urals Branch of RAS, Yekaterinburg, Russia
2Institute
The tension that muscle exerts upon activation increases dramatically when the temperature is raised. This increase in
tension is accompanied by a structural rearrangement that gives rise to changes in the X-ray diffraction pattern. These changes
are consistent with the movement of the tension-producing crossbridges to a state where they are more stereospecifically bound
to actin (Bershitsky et al., 1997, Nature, 388, 186-190; Tsaturyan et al., 1999, Biophys. J., 77, 354-372). One way to investigate this transition further is to capture the two states before and after the change in temperature by cryofixation. To establish
that rapid cooling can preserve the high temperature structure in activated muscle, we have used relaxed rabbit skeletal muscle
as a control. In the rabbit thick filament there is a rapid transition from a poorly ordered to a helical structure as the temperature is raised (Wray, 1987, J. Musc. Res. Cell Motil., 8, 62). When monitored by X-ray diffraction this transition results in in5
creased intensity on the myosin layer lines and, in addition, a loss of intensity of the 1,1 equatorial reflection from the hexagonal filament array (Lowy et al., 1991, Biophys. J., 60, 812-824; Xu et al., 1997, Biophys. J., 73, 2292-2303; Xu et al., 1999,
Biophys. J., 77, 2665-2676).
In our experiments, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump (T-jump) of
15oC-30oC from the dew point (~5oC) in air. We have developed a freezing method using a modified Gatan Cryosnapper in
which a pair of liquid nitrogen-cooled copper jaws are projected under pressure and close on the fibre between 50 and 70 ms
after the temperature jump. The frozen fibre was then freeze-substituted and embedded in Araldite for electron microscopy.
Transverse and longitudinal sections of relaxed cold (~5oC) and T-jumped fibres, as well as rigor fibres were obtained. The
Fourier transforms of well preserved regions were calculated to obtain the ratio of the intensity of the equatorial reflections,
I1,1/I1,0, and the layer-line intensities. For the rigor fibres the I1,1/I1,0 ratio was between 2 and 3, for cold relaxed fibres, 0.5-1.0,
and in warm relaxed fibres, 0.1-0.5. The intensity of the 1st layer line at ~43 nm-1 increased in the warm relaxed fibres compared to the cold ones.
In a parallel set of experiments, T-jumps from 5oC to 35oC-40oC were applied to small bundles of relaxed rabbit muscle fibres. The X-ray diffraction pattern was monitored with a time resolution of 1ms using a new RAPID 2D electronic detector on beam line 16.1 at the Synchrotron Radiation Source, Daresbury Laboratory. Following T-jumps, I1,1 decreased while I1,0
and the intensity of the first myosin layer line (M1) increased with a time constant of ~2ms. These results show that changes in
the intensities of the 1,1 and 1,0 reflections tightly correlate with the changes in the myosin layer lines and hence the disorderorder transition in the myosin arrangement in the thick filaments. In addition, the rates of change are such that by the time of
cryofixation in our EM experiments, the structural changes are essentially complete.
The results of electron microscopy are in agreement with those from X-ray diffraction. In particular the equatorial intensity profiles at ~5oC and 35oC were very similar to those observed in the EM experiments. These results, as well as direct
observations of the original micrographs and their averaged images, are consistent with our having preserved the hot structure
and indicate that cryofixation is sufficiently fast to prevent the transition back to the cold state.
The work was supported by INTAS and HHMI.
FORCE GENERATION IN MUSCLE: PROGRESS AND PROBLEMS
S. Bershitsky
Yekaterinburg Filial of Institute of Ecology and Genetics of Microorganisms,
Pervomayskaya 91, Yekaterinburg, 620219, Russia
Thirty years ago A.F. Huxley & Simmons proposed a mechanism of force generation in muscle 1 based on mechanical
experiments where contracting fiber was subjected to fast changes in length. Force generation was assumed to happen due to
myosin S1 run along the thin filament through a number of discrete steps changing its angle and stretching an elastic element
that links it to the thick filament. Structural evidences for this model has been obtained later in the X-ray diffraction experiments in whole muscle2 and single fibres3 where transitional drop in the intensity of meridional reflection, M3, at 14.3 nm synchronous with fast partial tension recovery was interpreted 4 as a tilting movement of the cross-bridge. Changes in angle orientation of fluorescent labels on the S1 light chains5 also support this interpretation though these changes are too small to correspond to mechanical data.
In vitro motility6-7 and optical trap8-9 techniques permitted experimental measurements of parameters of power stroke
of myosin head at single molecule level. Size of the step as well as force produced by a single myosin molecule were recorded.
However due to uncertainty in the mechanical conditions, the results are still far from complete clarity and often contradict to
the data obtained in mechanical experiments with the fibres. The most reliable characteristic of the actin-myosin interaction is
velocity of movement of actin filaments along myosin coated surface but observation of the filaments moving without load
does not allow to extract energetic parameters of the interaction.
During last 10 years, a several crystallographic structures of the myosin S1 have been obtained 10-13 which permitted
speculations about mechanism of force generation, particularly a “lever arm” hypothesis14. According to this hypothesis, power
stroke is produced by a swinging motion of a “neck” region of S1 actuated by a “converter domain” while the motor domain is
firmly attached to actin.
Data obtained in the temperature jump - X-ray experiments15-16 show however that the motor domain of S1 changes its
stereo-specificity in respect to actin helix during force-generating transient. This result shows that force generation in muscle
seems more complicated and consists probably of two different mechanisms working in parallel19.
3D tomographic reconstructions of quickly frozen contracting insect flight muscle 17 are also distinct from rigor actoS1 model based of crystallographic reconstruction18 and suggest that the motor domain on actin tilts from weak (nonstereospecific) to strong (stereo-specific) binding.
Author thanks for support INTAS, RFBR and HHMI.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
6
Huxley A.F. & Simmons R.M. 1971. Nature 233: 533-38.
Huxley, H.E., Simmons, R.M., Faruqi, A.R., Kress, M., Bordas, J. & Koch, M.H. 1983. J. Mol. Biol. 169: 469-506.
Irving, M., Lombardi, V., Piazzesi, G. & Ferenczi, M.A. 1992. Nature 357:: 156-58.
Irving, M., Piazzesi, G., Lucii, L., Sun, Y.B., Harford, J.J., Dobbie, I.M., Ferenczi, M.A., Reconditi, M. & Lombardi, V. 2000.
Nature Struct. Biol. 7: 482-85.
Irving, M., St. Claire Allen, T., Sabido-David, C., Craik, J.S., Brandmeier, B., Kendrick-Jones, J., Corrie, J.E., Trentham, D.R.
& Goldman, Y.E. 1995. Nature 375: 688-91.
Sheetz, M.P. & Spudich, J.A. 1983. Nature 303: 31-5.
Harada, Y., Noguchi, A., Kishino, A. & Yanagida, T. 1987. Nature 326: 805-8.
Finer, J.T., Simmons, R.M. & Spudich, J.A. 1994. Nature 368: 113-19.
Molloy, J.E., Burns, J.E., Kendrick-Jones, J., Tregear R.T. & White, D.C. 1995. Nature 378: 209-12.
10. Rayment
I.,
Rypniewski,
W.R.,
Schmidt-Base,
K.,
Smith,
R.,
Tomchick,
D. R., Benning, M.M., Winkelmann, D.A., Wesenberg, G. & Holden, H.M. 1993. Science 261: 50-8.
11. Dominguez, R., Freyzon, Y., Trybus, K.M. & Cohen C. 1998. Cell. 94: 559-71.
12. Houdusse, A., Kalabokis, V.N., Himmel, D., Szent-Gyorgyi, A.G. & Cohen, C. 1999. Cell 97: 459-70.
13. Houdusse, A., Szent-Gyorgyi, A.G. & Cohen, C. 2000. Proc. Natl. Acad. Sci. USA. 97: 11238-43.
14. Holmes K.C. 1997. Curr. Biol. 7: R112-R118.
15. Bershitsky, S.Y., Tsaturyan, A.K., Bershitskaya, O.N., Mashanov, G.I., Brown, P., Burns, R. & Ferenczi, M.A. 1997. Nature
388: 186-90.
16. Tsaturyan, A.K., Bershitsky, S.Y., Burns, R. & Ferenczi, M.A. 1999. Biophys. J. 77: 354-72.
17. Taylor, K.A., Schmitz, H., Reedy, M.C., Goldman, Y.E., Franzini-Armstrong, C., Sasaki, H., Tregear, R.T., Poole, K., Lucaveche, C., Edwards, R.J., Chen, L.F., Winkler, H. & Reedy, M.K. 1999. Cell 99: 421-31.
18. Rayment, I., Holden, H.M., Whittaker, M., Yohn, C.B., Lorenz, M., Holmes, K.C. & Milligan, R.A. 1993. Science 261: 58-65.
19. Huxley, A.F. 2000. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355: 433-40. Review.
THE PRIMARY PHYSICAL MECHANISM
OF MECHANOCHEMICAL ENERGY CONVERSION
IN BIOLOGICAL MOTION PROCESS
S.V. Bespalova, A.M. Mishchenko, A.S. Chernenko
Donetsk National University, 46, Schors str., Donetsk 83050, Ukraine;
E-mail: biophys@bio.donetsk.ua
In the proposed model of muscle contraction [1] transportation of the chemical energy of ATP splitting into mechanical energy of tension and movement of biopolymers is realized due to the excitation of hydrogen bond in the actin-myosin
complex.
The A-H...B bonds are characterized by the excitation energies of which their values approximate to that of the energy
E from the hydrolysis of ATP ( 0.4-0.5 eV ). Thus, by conveying a portion of energy to the proton one can keep control
over the hydrogen bond state and consequently over the interaction forces between biopolymers.
If the potential proton energy has the form of a double well
V A (r ) V B (R  r ), min V A  min V B
(1)
in the ground state 0(r) its energy 0 is weakly dependent upon interatomic distance AB=R since the proton is localized in a
deeper well VA(r) , the initial distance AB is comparatively great and the wave function 0(r) makes a weak entry into the region of the well VB(R-r).
The hydrolysis of ATP is assumed to develop proton excitation up to the level
1(R)= 0(R)+E. Its wave function
1(r) penetrates into the region of the second well VB(R-r) and the stronger AB bond appears. This bond proves to be a longrange one. The energy depends essentially on the interatomic distance AB=R. It generates a tractive force f  

, which
R
makes the atoms AB approach each other.
The work of external forces is carried out due to the lowering of the level 1, A=1(R1)- 2(R2), where R2 is initial
interatomic distance and R1 corresponds to min 1.
In [2] the tension force of the hydrogen bond at its different excitation levels have been estimated. As it was shown
that the proton excitation provides force of a longer-range character, generated by a hydrogen bond.
Statistics of the hydrogen bond formation and break in a bundle of actin and myosin filaments realizing the tension
force in the sarcomere of a muscle is studied [3]. Expression for the mean velocity of muscle contraction and for the tension
force as the function of the rate of ATP decay and temperature has been obtained. The dependencies between the muscle efficiency, the heat release, the expenditure of chemical energy and the velocity of contraction under the different load are studied
[4].
In the model of muscle contraction the first stage of the process of transformation of the ATP chemical energy into
mechanical work is the excitation of hydrogen bond in a system actin-myosin. That is the excited hydrogen bond performs the
mechanical work, and the excitation of hydrogen is owing to the hydrolysis of ATP. Thus it is necessary to consider the mechanism of the excitation of hydrogen bond process jointly with the ATP decay.
It was proposed that the thermal fluctuation produces excitation of hydrogen A-H…B bond. As a result the potential
barrier for the break of macroenergetic bond ADP-P is reduced [5]. The probability of such process depending on the mutual
arrangement of H-bond and ATP molecule is considered [6].
The mutual arrangement of ATP molecule and hydrogen bond A-H…B is proposed. System ADP-P is electroneutral
and represents the quadripole. The interaction energy of quadrupole with the proton charge is calculated.
Due to strong inequality of the masses of proton and of phosphate group m<<M for the description of system state the
adiabatic approximation is used. The wave function of the system is:
 (R ,  )   (R ,  )  (  )
(2)
where (R,) - is the proton function. It depends on proton coordinate R and on the phosphate group coordinate  parametrically.() - is the function of a heavy subsystem. The proton energy u i() depends on the coordinate  of heavy subsystem
and is its adiabatic potential.
The proton energy ui() in ground i=0 and in excited i=1 states are:

u 0 (  )    0 ( R,  ) H 0 ( R,  )dR

u1 (  )    1 ( R,  ) H 1 ( R,  )dR
(3)
7

where
H - is a Hamiltonian of isolated hydrogen bond A-H…B in the field of quadrupole.
The potential energy of heavy subsystem has been approximated by the function:
u (  )  V (  )  C exp(   2 (    0 ) 2 )  E
where V() - potential interaction of groups ADP and P . Its energy can be described by Morse potential:
V (  )  A exp( 2 (    0 ))  2 exp(  (    0 ))  1
(4)
(5)
When
=0
and
C=E,
u()=0;
when
(-0)>>1
and
(-0)<<1 (>>), u()=C- it is the height of potential barrier for the break of isolated bond ADP-P.
Thus adiabatic potentials of heavy subsystem ADP+P when the proton is in ground state 0() or in excited state 1(
) are
U 0 ()  u0 ()  u ()
U 1 (  )  u1 (  )  u (  )
(6)
According to the estimates the potential U1() lies lower than U0(), when  has mean values. There two points of
interaction of potentials. Owing to deviation of the system from adiabaticity in these points there are probabilities of transition
of system from state with the nonexcited hydrogen bond into the state when the hydrogen bond is excited.
The question arises: ''What is the source of energy 1-00.5eV for the excitation of hydrogen bond before the energy
release of ATP decay?'' This is the thermal fluctuation. When the fluctuation concentrates sufficient energy on the atoms hydrogen bond A-B…H, the system tries to excite the hydrogen bond and than to overcome the barrier. After that there takes
place the ATP decay and the energy returns in thermostat in the form of kinetic energy of the phosphate group E. If the value
E1-0 there is no violation of the second law of thermodynamics: the work is performed by means of the chemical ATP
energy, but there takes place slight time deviation from the law in the form of the fluctuation.
It is shown that when the proton of hydrogen bond is excited the potential barrier for the break of ADP-P bond is reduced. The excited hydrogen bond produces the decay of ATP molecule.
The proposed mechanism may be the fundamental principle of the realization of biological process. Thus, we obtain a
notion that the physical principle of biological motility is the joint process of exciting hydrogen bond as well as ATP molecule
hydrolysis. Such a process is liable to take place not only in case of muscle contraction, but in case of pulling transfer RNA
through the ribosome when protein biosynthesis takes place.
References
1. S.V. Bespalova and K.B. Tolpygo, J. Theor. Biol., 153 (1991) 145-155.
2. V.A. Telezhkin, K.B. Tolpygo, S.K. Tolpygo, Ukr. Fiz. Zh., 24, (1979) 1979-1988 (in Ukraine).
3. S.V. Bespalova and K.B. Tolpygo, Biophysics, 38, (1993) 7-15.
4. S.V. Bespalova and K.B. Tolpygo, Biofizika, 41, (1996) 22-32 (in Russian).
5. S.V. Bespalova and K.B. Tolpygo, Biophysics, 43, (1998) 457-462.
6. S.V. Bespalova, A.M. Mishchenko, V.M. Shatalov, Biophysics (2001). –In press.
THE EFFECTS OF CATIONIC INHIBITORS OF Ca2+-CHANNELS IN PLASMA MEMBRANE ON
CONTRACTILE OSCILLATIONS OF PHYSARUM PLASMODIUM
S.I. Beylina, N.B. Matveeva, A.A. Kochegarov, V.A. Teplov
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia
E-mail: beylina@venus.iteb.serpukhov.su
It was found that the contractile oscillations in the Physarum plasmodia sustained for about 1 hour in Ca2+-free
EGTA-containing media and then disappeared, presumably, due to depletion of the intracellular stores [1]. These findings suggest that contractile oscillations are independent of external calcium supply, but, paradoxically, they do not exclude that the
transmembrane Ca2+ influxes are involved in the self-sustained oscillations. In the matter of fact, the control of free calcium
level in vicinity of plasma membrane invaginated deeply in the ectoplasm is difficult to attain. The first reason is an ability of
the plasmodium to extrude calcium-contained particles [2], the second one is characteristic of Physarum capability to acidify
an external media [3]. The latter may critically reduce a capacity of EGTA to chelate calcium ions, at least at such EGTA and
pH-buffer concentrations, which are not harmful for the plasmodium. Hence, we employed an alternative approach and investigated an oscillatory contractile behaviour of the microplasmodia under action of some cationic inhibitors of the plasma membrane Ca2+-channels. It should be mentioned that cations Cd 2+, Ni2+, Mr2+, La3+ and Gd3+ used, all are known as blockers of
Ca2+-influx triggered by depletion of intracellular Ca2+ stores [4]. Gd3+ is widely used also as an inhibitor of the stretchactivated Ca2+ channels [5-7].
Physarum polycephalum, strains RCM(F) 3283 and LU 887*MA 362, was grown as a suspension of microplasmodia,
multinucleate cells 100-200 μm in diameter [8]. Microplasmodia were harvested at the logarithmic phase of growth and resuspended in a non-nutritional medium contained 100 µM CaCl2 and 10 mM HEPES, pH 4.5. Cells were used for assays during
first 8 hours of starvation. Cyclic contractions of cells and pulsating movement of its leading edge were monitored optically
[3].
Cd2+ was shown to cause complete suppression the contractile activity of plasmodium at 1-5 mM concentration. Contractile oscillations ceased within 5 to 30 min depending on the state of individual cells. After incubation with 5 mM Cd 2+ for
30-40 min and subsequent washing, the contractile activity of microplasmodia was restored. At more prolonged incubations,
the effect of Cd2+ was irreversible. As to the other divalent cations, Mr2+ had no influence on contractile behaviour, whereas
Ni2+ suppressed oscillations but had a distinct harmful effect detected by a loose of the plasmodial yellow pigment.
Both La3+ and Gd3+ suppressed the contractile oscillations, strongly increased adhesion and inhibited the crawling of
microplasmodia. All these effects were much more pronounced with Gd 3+ than with La3+, and were shown to be dependent on
the starvation-time of microplasmodia. In freshly isolated cells, 0.1-5 mM Gd3+ induced nearly complete oscillation disap8
pearance occurred almost immediately after Gd3 application. Under action of La3+, a temporal increase in frequency of the
damped oscillations was often observed. At 1-5 mM Gd3+, the dumped oscillation continued for about 1 hour, afterward their
amplitude gradually restored. Recovered oscillations are always of pure sinusoidal shape, that was rarely observed in migrating
microplasmodia. Unlike La3+, under action of Gd3 the cessation of crawling still persisted after the renewal of the oscillations.
After 5-7 hour starvation, very short cessation of oscillations was shown to occur in microplasmodia treated with La 3+
or Gd3+. All changes in the oscillatory motile behaviour were similar to that observed in freshly isolated cells after the renewal
of the oscillations.
Although additional studies are required to elucidate the nature of starvation-related difference in the inhibitory action
of these cations, these findings seems to be important for better understanding an interplay between the Ca2+ entry and the release of Ca2+ from internal stores during oscillatory contractile activity of Physarum plasmodium.
This work was supported by The Russian Foundation for Basic Research.
References
1. Wohlfarth-Bottermann K.E., Gotz von-Olenhusen K. 1977, Cell Biol. Int. Reports, 1, 239-247.
2. Beylina S.I., Belyavsky M.A. 1986, Stud. Biophys., 116, 195-203.
3. Matveeva N.B., Beylina S.I., Teplov V.A., Layrand D.B., Lednev V.V. 1981, in Biology of Physarum (ed. Rakoczy, L.), Jagellonian Univ.
Press, Krakov, 235-245.
4. Barritt G.J. 1999, Biohem. J., 337, 153-169.
5. Yang X.-C., Sachs F. 1989, Science 243, 1068-1071.
6. Yoshimura K. 1998, J. Membrane Biol. 166, 149-155.
7. Lee J., Ishihara A., Oxford G., Johnson B., Jacobson K. 1999, Nature 400, 382-386.
8. Daniel J.W., Baldwin H.H. 1964, in Methods of Cell Physiology (ed. Prescott J.), 1, 9-41
DISCRETE SARCOMERE LENGTH DYNAMICS
IN SINGLE CARDIAC MYOFIBRILS
Felix Blyakhman*, Olga Yakovenko** and Gerald Pollack**
*Ural
State University, 51 Lenin Ave., Ekaterinburg, 620083, Russia;
of Washington, Box 357962, Seattle, WA, 98195, USA
**University
In investigating the mechanism of motility and contraction, attention has turned toward the elementary molecular
translation step. Although measurements in the past have yielded steps of broadly variable size, recent experiments with improved signals have shown steps on the order of 5 – 6 nm (Viegel et al., 1999), and experiments on single myosin molecules
translating along single actin filaments have shown consistently a step size of ~5.4 nm (Kitamura et al., 1999, Yanagida et al.,
2000). The value 5.4 nm is equal to the monomer repeat along a single actin strand.
We have carried out similar measurements on single sarcomeres. Previous bumblebee flight myofibril experiments
have shown that activated sarcomere shortening occurred in steps that were integer multiples of 2.7 nm (Blyakhman et al.,
1999). This value is half the 5.4 nm reported above. It is equal to the actin monomer repeat along the thin filament (5.4 nm repeat along each strand, the two strands displaced by 2.7 nm). Because of noise, however, detectability was limited to steps
larger than 4 - 5 nm, so we could not establish whether the implied 2.7-nm quantum actually existed. In more recent experiments we employed a new high-resolution algorithm (Sokolov et al., submitted) and measured steps in activated cardiac sarcomeres during both shortening and imposed stretch.
Single rabbit left ventricle trabecular myofibrils were isolated and mounted in an apparatus built around a Zeiss Axiovert-35 microscope as described previously (Linke et al., 1993). One end of the myofibril was attached to the tip of a fixed
glass needle; the other to a glass tip on the piezoelectric motor, which could impose desired length changes on the specimen.
We applied length ramps to passive (pCa ++ = 8.0) and active (pCa++ = 4.0) specimens. The striated image was projected onto a
photodiode array, which was scanned every 50 ms to produce a trace of intensity vs. position along the myofibril. Tracking the
span between adjacent A-band centroids gave sarcomere length vs. time. Such traces were consistently stepwise.
Step sizes obtained from many traces (253 sarcomeres from 26 myofibrils) were plotted as a continuous histogram.
The distribution of step size during shortening shows a series of clear peaks. The primary peak is at 2.7 nm. Additional peaks
are seen at integer multiples of the primary value. Lengthening steps were analyzed in a similar way. A series of histogram
peaks was observed, each lying at an integer multiple of ~2.7 nm. The largest peak was situated at 5.4 nm. Thus, we confirm
the 2.7 nm quantum.
Experiments were also carried out to determine the effect of ramp speed. 27 myofibrils underwent rapid stretch and
release (10-11 nm/sec). Both of these distributions exhibit a similar tendency: a series of peaks was observed, each lying at an
integer multiple of 2.7 nm. For stretch, higher speed slightly decreased the fraction of smaller steps relative to the larger ones.
Otherwise, the results were similar.
A series of control experiments to check for artifact has already been reported (Yang et al., 1998; Blyakhman et al.,
1999; 2001). Two additional controls in cardiac specimens were carried out here. In the first, we applied a “stepped ramp” to
the myofibril to check resolution. Imposed step size was 10-15 nm per sarcomere. These steps appeared faithfully in the sarcomere-length trace. In addition, smaller spontaneously generated steps were interspersed between the larger steps. For lengthening and shortening alike, the size pattern of the spontaneously occurring steps was the same as for the steps seen during imposition of the ramp, namely multiples of 2.7 nm. Because the steps appear so consistently at integer multiples of 2.7 nm, suspicion arises that the origin of the steps could lie in some unsuspected feature of the apparatus, notwithstanding extensive controls. We therefore carried out experiments identical to those described above, except that the specimens were unactivated. The
stepping pattern was visually similar. However, the step size was no longer an integer multiple of 2.7 nm, but a multiple of
~2.3 nm. Higher multiples of these quanta were easily distinguishable (e.g. 8.1 nm vs. 6.9 nm).
The results show a high level of consistency for both shortening and lengthening—step values of 2.7 nm, and integer
multiples thereof. These observations confirm and extend earlier observations of step size values at high integer multiples of9
2.7 nm, and establish that the previously implied quantum does indeed exist. Because the same quantal value is found in invertebrate and vertebrate specimens alike, the value may be taken as general. Thus, activated sarcomeres shorten in 2.7-nm steps
or integer multiples thereof. The 2.7-nm value is equal to the axial repeat of actin monomers along the thin filament. Hence, a
direct link between dynamics and structure is observed. Details of how such dynamics are generated are open to speculation
(Pollack, 2001), but the simple quantitative result reported here places a signature-like constraint on any proposed mechanism.
COFILIN INDUCED STRUCTURAL CHANGES AT THE INTERMONOMER INTERFACES IN F-ACTIN
Andrey A. Bobkov1, Sergey Vorobiev2, Steven C. Almo2,
Andras Muhlrad1 and Emil Reisler1
1Dept.
Of Chemistry & Biochemistry, Univ. of California, Los Angeles, CA 90095; 2Dept. of Biochemistry, Albert Einstein College of Medicine, New York, N.Y 10461
Cofilin has been shown to change the twist (McGough et al., 1997, J. Cell Biol. 138:433,) and to weaken lateral contacts (McGough and Chiu, 1999, J. Mol. Biol. 291:513) in actin filaments. We studied these effects in solution by investigating
the interaction of yeast cofilin with wild type, Q41C and Q41C/C374S mutant yeast F-actins, and with Q41 dansyl ethylenediamine (DED) labeled and 4-azido-2-nitrophenyl-putrescine (ANP)-labeled and cross-linked rabbit skeletal F-actin. Cofilin decreased the excimer fluorescence produced by the interaction of two pyrenes attached to C41 and C374 on the Q41C mutant. It
decreased also greatly the fluorescence resonance energy transfer (FRET) from tryptophans to DED on Q41 in -actin filaments, and to AEDANS on C41 in the Q41/C374 mutant actin. FRET between tryptophans and ANP attached to Q41 of actin filaments was also strongly decreased by cofilin. However, FRET was much less affected when F-actin was intrastrand
cross-linked by ANP between Q41 and C374. Cofilin accelerated the rate of -ADP release from F-actin and inhibited nucleotide exchange on G-actin. These results indicate that cofilin displaces the DNase loop 1 thereby, weakening the contacts between subdomain 1 and 2 on adjacent protomers as well as lateral interactions in F-actin.
SEROLOGICAL HOMOLOGY BETWEEN ACTIN AND PROTEINS OF BACTERIOPHAGE M13:
IMMUNOLOGICAL AND BIOINFORMATIONAL ASPECTS
V.A. Bogatyrev, M.V. Sumaroka, I.S. Zaitseva, O.I. Sokolov,
V.A. Piskunov, A.S. Shigayev, L.A Dykman
Institute of Biochemistry and Physiology of Plants and Microorganisms RAS,
13 Prospect Entuziastov, Saratov 410015, Russia, E-mail: bog@ibppm.saratov.su
Actins belong to the class of conservative proteins; the degree of homology among them is not lower 97% in remote
species. In this connection, the production of antibodies (Ab) to actins by conventional methods as a rule gives rise to low-titre
antisera. We have a combinatorial phage library of antibodies (Griffin.1 Library, MRC, UK), where was kindly provided by
Professor W.J. Harris, Aberdeen, UK. To obtain phage particles with a high affinity for actin, we screened the library phages
by using chicken-gizzard smooth-muscle actin. Alternatively, the Ab were raised by immunization of rabbits or mice with actin
plus complete Freund’s adjuvant and colloidal gold. Immunodot testing of the preparations (indirect labelling with Protein
A+colloidal gold or direct labelling with polyclonal anti-phage Ab+colloidal gold) showed cross-interaction of the anti-actin
Ab with both actin and phage particles. This phenomenon has been known for GAG-peptides (actin-like) of certain retroviruses.
This work was aimed at: (i) bioinformational search for homologous sites of the actin macromolecule and phage M13
proteins; (ii) isolation and identification of the relevant proteins.
By using the Swiss-PDB Viewer program, we found two sites in the molecule of minor capsid protein g3p that
showed a sequential similarity with primary actin-structure.
The first site of similarity appears as follows:
g3p
257 DFDYEKMA 264
DF
E MA
actin
224 DFENE MA 230.
The second site appears as follows:
g3p
283 DAKGKLDSVATDY 294
D K KL VA D
actin
213 DI KEKLСY VALDF 224.
As can be seen, in protein g3p, the sites of similarity with actin are separated by a site composed of 19 amino acids,
whereas in actin these sites cross. The complete of similarity of actin and g3p is as follows:
DIKEKLCYVALDFENEMA.
However, at the second site, the central tyrosine atom (Y220) is at a distance of not more than 5Å from the neighbouring-chain-proline residue (P309). This gives ground to suggest the presence in this fragment of two small epitopes similar to
g3p.
A similarity site was also found in the molecule of peptide g8p that appears as follows:
G3p
3 GDDPAKAAFNSL 14
GDD : : : A F S :
Actin
23 GDDAPRAVFPS I 34.
Considering the fact that amino acids 1-19 are located on the outside N-end of the peptide, this site may well be responsible for antibody binding.
Thus, we found sequential similarities between the protein sites of phage M13 and actin that are potentially immunogenic. Currently, we are conducting an immuno-electron-microscopy study of phage M13 particles to localize sites of antiactin-Ab binding.
This work was supported in part by CRDF (grant № REC-006) and RFBR (project № 01-04-48736 a).
10
THE EFFECT OF S1 ON MOVEMENTS OF DISTINCT AREAS OF ACTIN
1
2
3
1
Yu.S. Borovikov , I.V. Dedova , W. Van der Meer , N.N. Vikhoreva ,
1
1
2
P.G. Vikhorev , S.V. Avrova and C.G. dos Remedios
1
Institute of Cytology RAS, St.-Petersburg, 194064, Russia;
Institute of Biomedical Research, The University of Sydney, Sydney 2006, Australia;
3
Department of Physics and Astronomy, Western Kentucky University,
KY 42101-3576, USA
2
Muscle contraction is generated by the interaction of myosin cross-bridges with actin filaments and ATP. During
force generation the myosin cross-bridges go through several stages, the most significant of which are so-called “strong” and
“weak” forms of myosin binding to actin (Lymn and Taylor, 1971; Stein et al., 1979, Geeves, 1992). Myosin cross-bridges undergo structural changes during a force-generation process (Cooke R., 1997). Recently, the negative experimental results of the
rotating cross-bridge model have led to suggestions of a more complex model of the muscle contraction. This model involves
large-scale conformational changes of myosin head in the light chain-binding domain that rotates relative to the actin-binding
portion of the catalytic domain (Huxley, 1974; Thomas et al., 1995; Cook 1986; Xiao et al., 1998; Sabido-David et al., 1998;
Hopkins et al., 1998; Palm et al., 1999). The large free-energy change upon binding of the myosin head to actin is also able to
generate conformational change in actin (Geeves, 1991).
Here we studied the movements of different areas of polypeptide chains within F-actin monomers at formation of
strong binding with myosin subfragment-1 (S1). We used myosin-free ghost fibers, where thin filaments were reconstructed by
adding actin labelled with a fluorescent probe attached to different areas of actin molecule. These sites were: Cys-374 labelled
with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labelled with NBD-Cl; Lys-113 labelled with Alexa-488; Lys-61 labelled with
FITC; Gln-41 labelled with DED and Cys-10 labelled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimide. The reconstruction of filaments from exogenous G-actin within the fibers was performed as described earlier (Borovikov et al. 2000). Ghost
fibers were incubated for 20-30 min at 20 C in a solution containing G-actin (2-4 mg/ml) unlabelled or labelled with a fluorescence dye. Then 100 mM KCl, 1 mM MgCl2, 20 mM Tris/HCl buffer, pH 7.5 were added to the bathing solution and the incubation was continued for 20-30 min to polymerize the incorporated G-actin. In some experiments, we used FITC-phalloidin,
TRITC-phalloidin, and e-ADP that were located in filament groove and interdomain cleft, respectively.
Fluorescent probes located in a thin filament repeat a strictly ordered spatial arrangement of this protein and of the
corresponding aminoacid residues they are bound to. The anisotropic arrangement of the probes gives rise to the polarized fluorescence of the muscle fiber. To quantitatively assess changes in the probe orientation we used a model-dependent (Tregear
and Mendelson, 1975; Yanagida and Oosawa, 1978; Kakol et al., 1987; Irving, 1996) and model-independent (van der Meer et
al., 2001) analysis. The data showed that the orientation and mobility of fluorescent probes were changed significantly when
actin and myosin interacted, depending on fluorophore location. Parts of F-actin at high radii, which are responsible for myosin
binding, and parts of F-actin at low radii, move in opposite directions. It is proposed that these alterations in F-actin conformation play an important role in muscle contraction.
This work was supported by Russian Foundation for Basic Research, grant № 01-04-49310 (Y.B.).
THE ROLE OF GTP-BINDING PROTEINS IN MORPHOGENETIC MOVEMENTS OF GASTRULATION
V.P. Bozhkova
Institution for Information Transmission Problems of Russian Academy of Sciences
It is known that near 1% of all babies are born with the major structural abnormalities which arise still in early embryonic development. But the mechanisms of morphogenetic embryopathies remain unrevealed. The goal of this study is to clear
up what regulatory systems play the signal role in normal morphogenesis. Previously we have shown that intracellular injections of myo-inositol-1,4,5,-trisphosphate (IP3) in undividing frog Rana temporaria eggs could initiate the propagation of contraction waves similar the waves normally observed at the different stages of development [1]. More recent experiments have
led us to the notion that phosphatidylinositol (PI) cycle is the main participant of reactive-diffusion system that directs the contractions while calcium waves and cytoskeleton reorganization are secondary events [2]. To reveal PI cycle involvement in
regulation of morphogenesis at the blastula and gastrula stages GTP-gamma-S (in final concentration 80-100 M per embryo)
and GDP-beta-S (in final concentration 5-15 M per embryo) were used, respectively, as activator and the inhibitor of PI signalling pathway at the level of G-proteins. IP3 as intermediate of PI cycle was also tested (in final concentration 5-10 M per
embryo) for their influence on the morphogenetic movements of gastrulation. The work was carried out using fish (loach, Misgurnus fossilis) embryos. All substances were injected into the yolk cell at the blastula-gastrula stages when they can be transfered to the presumptive mesoderm cells via cytoplasmic bridges or gap junctions [3].
The stages of development at which GTP-gamma-S induced the immediate effect after injection were tested. The
cleavage divisions and the second part of gastrulation (50-70% of epiboly) proved to be such stages. The primary effect of
GTP-gamma-S consisted in the overcontraction of the yolk syncytial layer and the blastoderm margin. The late blastula and the
early epiboly stages were not changed after treatment of GTP-gamma-S. In addition to immediate effect GTP-gamma-S, injected at all the stages from early blastula till midgastrula, had remote contractive effect at the second part of gastrulation. The
rate of epiboly was not affected by GTP-gamma-S injections although the epiboly of some embryos was reversed. In opposite
to GTP-gamma-S the injections of the stable analog of GDP – GDP-beta-S which inhibit G-protein function retarded the
epiboly in loach embryos. In opposite to GTP-gamma-S the injections of IP3 did not induce the blastoderm margin contraction
and did not affect the epiboly. So, our results suggest that G-proteins can be involved in stimulation of local cytoskeleton reorganization and contraction at the cleavage divisions stage (perhaps, to contribute to cytokinesis) and at the late gastrula stage to
promote the final phase of epiboly. Their effects on contraction can be connected not only with IP 3. As appears from our data
also in fish embryogenesis the spatial-temporal regulation of G-proteins activity takes place.
Supported by grant № 00-04-48822 of Russian Foundation for Basic Research.
11
References
1. Bozhkova V.P., Voronov D.A. and Romanovsky Yu.M. The initiation of contraction waves on the surface of frog fertilized eggs by inositoltrisphosphate. Biophysica, 1993, 38, 267-277 (in Russian and English translation).
2. Bozhkova V.P., Nikolaev D.P., Potapov M.M., Romanovsky Yu.M. and Strazhevich A.S. Mechano-chemical autowaves on the egg
surface. III.Conditions of travelling pulses existence in phosphatidylinositol cell cycle (in this book).
3. Bozhkova V.P. and Voronov D.A. Spatial-temporal characteristics of intercellular junctions in early zebrafish and loach embryos before and during gastrulation. Dev. Gene. Evol., 1997, 207, 115-126.
12
MECHANO-CHEMICAL AUTOWAVES ON THE EGG SURFACE.
III. CONDITIONS OF TRAVELLING PULSES EXISTENCE
IN PHOSPHATIDYLINOSITOL CELL CYCLE
V.P. Bozhkova*, D.P. Nikolaev*, M.M. Potapov**,
Yu.M. Romanovsky**, A.S. Strazhevich**
*Institution for Information Transmission Problems of Russian Academy of Sciences;
**M.V. Lomonosov Moscow State University
The excitation and propagation of the mechanochemical autowaves (AW) in the frog Rana temporaria eggs after the
injection of 1 mM myo-inositol-1,4,5 trisphosphate (IP3) was analysed. The detection system employed automatic videorecording of microobjects. More than 30 microfilms of various contraction waves (decaying waves, autowaves, repeated waves, etc.)
were shot. The analysis of the microfilms made it possible to calculate the velocity of the wave as a function of the position at
the spherical egg surface. Under the autowave regime, the mean velocity was 7 m/s. In the Rana temporaria eggs, Са2+ (contrary to IP3 ) did not initiate waves and even inhibited them under high concentration. These data are inconsistent with the existing models of the IP3-induced calcium waves known as CICR models. These models are based on the assumption on autocatalytic propagation of the Са2+-wave following the diffuse propagation of IP 3. Thus, the object considered exhibits no positive feedback Ca2+  Ca2+ or Ca2+  IP3.
We developed a model [1] based on the dominating role of the IP 3-cycle in generating AW. According to this model,
Са2+-wave only accompanies the IP3-wave related to the activation of the IP3-dependent calcium channels of endoplasmic
stores. IP3 is one of the end products of phosphatidylinositol 4,5-diphosphate (PIP2) hydrolysis. The action of IP 3 as the secondary mediator ends up upon its dephosphorilation into inositol-di- and monophosphate, and finally, into free myoinositol
(myo-I). After that, myo-I is again involved in the phosphoinositide cycle as an intermediate for regeneration of PIP2. We assume that IP3 stimulates PIP2-hydrolysis and, consequently, the formation of IP 3. In this case, AW can arise in the system considered. Denote the concentration of IP3, myo-I, and PIP2 as I, M, and P, respectively. Consider a "point" model DI  DM  0
for a system in which ( I  I 0 ) is the Havyside function. This function and the parameter C II determine the autocatalytic dependence of IP3. Consider the spherical coordinates with the origin at the pole of the egg cell;  is the azimuth and R is the
radius of the egg cell:
 2
 

 ctg
I  C PI  C II I  I 0 P  C IM I
  2
 


 2
D
M
 
 M 
 ctg
M  C IM I  C MP M
2
t
 
R   2

P
 C MP M  C PI  C II I  I 0 P
t
D
I
 I
t
R2
The system of equations is closed since it complies with the law of conservation of mass (the sum of concentrations
and the integral of the sum of concentrations over a sphere remain constant for the localized and distributed systems, respectively). Assume that the diffusion coefficients of the components are DI  DM  300m2/s. A few parameters of the system are
not known but we can choose them based on the period of the relaxation of the system  e  300 s.
The necessary conditions of forming of the traveling pulses [2] are as follows: a) in the phase plane of the point model
the isocline dI/dt = 0 is s-shaped and intersects with the isocline dM/dt = 0 in a single stable point; b) the pulse is generated if I
exceeds a threshold value; c)  M   I , where  M  500 s and  I  50 s are the characteristic times of the corresponding variables.
After the selection of the parameters of the model, we performed the numerical experiments with the distributed system. Their results are in good agreement with the experimental data (the velocity of AW at the sphere and the dependence of
the velocity on the angle  ). The initial system was represented by a conservative difference scheme obeying the same law of
conservation.
In the further studies, we plan to supplement the model considered by the equations describing the deformation of the
egg surface initiated by the Са2+-AW.
References
1. Nikolaev D.P., Teplov V.A., Bozhkova V.P., and Romanovsky Yu.M., Mechanochemical autowaves in living cells, Proc. of the seminar
"Time, Chaos, and Mathematical Problems", No.1.- M.: Books House ‘Universitet’,1999, Eds. Loskutov A.Yu., Pechencev A.S. pp.
181-202. (in Russian)
2. Vasilev V.A, Romanovskii Yu.M., Chernavskii D.S., and Yakhno V.G., Autowave processes in Kinetic Systems VEB Deutscher Verlag
der Wissenschaften, Berlin 1987; D.Reidel Publishing Company, Dordrecht, Boston, Luncasster, Tokyo, 1987.
DUAL EFFECT OF SECOPALLOIDIN
ON Ca2+ REGULATION IN CARDIAC MUSCLE
Anna E. Bukatina and Gary C. Sieck
Departments of Anesthesiology and Physiology & Biophysics,
Mayo Medical School, Rochester, MN 55905
Striated muscles are known to have two roots of activation: Ca 2+ and cross-bridges (XB). Here we present data on
secophalloidin (SPH), a novel and unique activator of myofibrillar contraction. In skinned cardiac muscle, without Ca2+ and in
the presence of physiological [Mg-ATP], SPH (6-8 mM) activates XB cycling leading to isometric force comparable or greater
than that induced by Ca2+. SPH induced activation is reversible upon washing with a relaxing solution. However, subsequent
Ca2+ activation is considerably reduced (by 30-50%). Subsequent SPH induced activation is also reduced but to a lesser extent.
Thus, SPH has reversible and nonreversible effects on muscle contraction.
13
SPH increases maximal Ca2+ activated force (~25%) indicating to noncompetitive mechanism of action. This potentiation of force is even greater if maximal force was reduced by prior exposure to SPH or by TnC extraction. Mechanisms of Ca 2+
and SPH activation are closely coupled. Each of these activators increases sensitivity to the other. Apparent Kd for SPH is ~0.1
mM at saturated [Ca2+] and ~2 mM without Ca2+. Even a weak SPH activation (15 -20% of maximal force caused by 1 mM
SPH) brings about a dramatic increase in Ca 2+ sensitivity (leftward shift of force-pCa by 0.6-0.7 pCa) and 1.5-2.0-fold reduction in the Hill coefficient. These SPH-induced changes in the force-pCa relationship are twice as much as those induced by
the hypothetical Ca2+-like (competitive) activator. Moreover, in spite of weak saturation with SPH, the force-pCa curve does
not suggest two separate activation processes.
Nonreversible loss in force occurs without changes in the Hill equation parameters, possibly reflecting irreversible inhibition of some sites on myofilaments. This nonreversible effect cannot be solely explained by changes in TnC. Kinetics of
the force response to stepwise increases in [Ca2+] during the 1st application of SPH (which causes a majority of the irreversible
force loss) consists of 2 phases: an initial increase in force followed by a return of force to a new steady state. Such kinetics,
which can be observed several times in consecutive increases in [Ca 2+], suggests that interaction with SPH includes two reactions, which could be consecutive steps of a reaction at the same site: i) initial fast weak binding, causing reversible effects,
and ii) more slow irreversible changes. Ca2+ accelerates this latter step. Furthermore, irreversible force loss was not observed
after SPH application to a rigor muscle at any pCa. Therefore, the SPH modification takes place at a site available only during
XB cycling.
STRUCTURAL AND FUNCTIONAL PECULIARITIES
OF MUSCLE ALLOGRAFTS FORMED AFTER EFFECT
OF LASER RADIATION ON ADRENALS
N.V. Bulyakova, V.S. Azarova
A.N. Severtzov Institute of Ecology and Evolution RAS, Moscow, Russia
The plastic capacity of muscle allografts is extremely limited. The development of differentiated myofibers from allografted muscle tissue and the formation of contractive muscle organ were observed in rats from the same litter, which represented close potential immunological matches. To prevent the allograft rejection in normal non-prepared animals, the taking of
immunodepressants for a long time was needed.
One approach to the solution of tissue incompatibility problem is to change the immune status of muscle allografts.
According to our and literature data, laser radiation may have immunosuppressive effect.
The purpose of present study was to determine the viability of allogenic skeletal muscle tissue under effect of low energy He-Ne laser on adrenals and skeletal muscle allograft before surgery.
The cross allotransplantation of right gastrocnemius muscles was carried out between intact rats and rats in which the
area of projection of adrenals and right hind shin were previously subjected to He-Ne laser radiation (apparatus OKG-12, wave
length 632.8 nm, laser ray was disfocused with lens; power 2.5-3.0 mWt/cm2 ; 10 procedures for 2 weeks, the duration of one
exposure was equal to 5 min; each rat was subjected to laser radiation in total dose 15-18 J/cm2 ).
The experiment consisted of two series. The rats with laser radiated in vivo adrenals and right shin to which gastrocnemius muscle was allografted from intact rats represented the first series. The intact rats to which right laser radiated gastrocnemius muscle was allografted from rats with radiated in vivo adrenals and right shin represented the second series. The
animals were operated under nembutal narcosis. By conditions of experiment, the rats in each series were both donor and recipient and hence a heterogeneity of rats in each series was the same. The study of morphofunctional peculiarities of muscle
allografts was performed two months after surgery.
In both series, the mass of allografted gastrocnemius muscles decreased considerably compared with that of normal
nonsurgical rats. However, in the 1-st series, disintegration and resorption of muscle allografts occured more actively and allogenic muscle tissue was preserved in 38% grafts. They consisted of bunches of wide and narrow myofibers between which the
connective and adipose tissues were formed. Under electrostimulation of n. tibialis, the contractile reaction was revealed in
25% muscle allografts.
The conditions in the 2-nd series were more favourable to survival and development of allografts. The allogenic muscle tissue was revealed in 60% grafts. In some of them, the medial and lateral heads of m. gastrocnemius were preserved. The
myofibers were closely arranged. The allografts showed a moderate lymphocytic infiltration. The sclerotization of allogenic
muscle tissue was less pronounced than in allografts of rats with laser-radiated adrenals. About 43% muscle allografts contracted when stimulating via the nerve or directly.
Thus, the previous effect of He-Ne laser radiation on the area of projection of adrenals and right hind shin, in ratdonor changed immune status of allografted radiated gastrocnemius muscle, increased of viability of allogenic muscle tissue
and promoted to its development when radiated gastrocnemius muscle was allografted to intact non-prepared rat.
Supported by the grant № 01-04-49076 of Russian Foundation for Basic Research.
SCANNING PROBE MICROSCOPY AND SCANNING OPTICAL SPECTROSCOPY FOR BIOLOGY AND
MEDICINE APPLICATIONS
Victor A. Bykov, Alexei V. Belyaev, Sergei A. Saunin, Igor V. Dushkin,
Sergei G. Feklisov
NT-MDT Co., Moscow – Zelenograd, Russia; www.ntmdt.com
Molecular biology, cell biology, biochemistry, virus investigations, medicine materials – that applications are one of
the general application fields of scanning probe microscopy and spectroscopy.
14
To decide the problems of molecular recognitions that have the requirements of the investigations of practically flat
surfaces and very high vertical resolutions and cell investigations that need to measure height up to the 8 – 10 microns – vertical cells size, we have to create different types of scanning probe instruments.
First of them is Solver – BioMol – the device for molecule investigations. This device has to measure either in air –
solid and liquid – solid interface with the scanner parameters 50 x 50 x 3 microns. Electronics of this device have very high
resolution parameters and noise level 0,02 nm on hight. Optical microscopes that have to arrange SPM are Biolam – produced
in LOMO or Olympus – XI.
Second one is Solver – BioCell – the device for rough surfaces investigations. The controller of this device can increase from normal range up to 15 microns and plate scanning range up to 90 microns. In addition tool for this device is liquid
cell thermo stable system in the range room temperature – 50°C. Vertical noise level in this device is 5 times higher than in
that for molecule investigation.
Scanning probe microscopes give the possibility to measure topography, tribology, electrical and force field characteristics of bodies but not chemical conditions of body. To decide these problems we create new type of devices that have to include either high resolution Raman and luminescence spectrometer and scanning probe microscopes. In addition to the solid
probe of SPM this device has small optical probe with the size of 0,2 micron in the plate and 0,5 microns in the high. This
probe can investigate the volume of 100x100x30 microns and in addition to the surface data gives the information for the volume conditions.
The results of the investigations of different objects by SPM and scanning optical spectroscopy device will be reported.
THE ROLE OF BIOCHEMICAL-FUNCTIONAL CORRELATION BETWEEN CARDIAC HEMODYNAMIC AND
ENERGY SUPPLY SYSTEM PARAMETERS IN THE DYNAMICS OF ACUTE HEART FAILURE (AHF)
DEVELOPMENT, CAUSED BY ACUTE CORONARY ARTERY OCCLUSION (CAO)
E.A Chikobava, G.V. Sukoian, D.R. Tatulashvili, N.V. Karsanov
The Republican Research Centre of Medical Biophysics and Introduction of New Biomedical Technologies (Head - Corr. member of Russian Academy of Medical Science, Prof. Karsanov N.V.), Tbilisi, Georgia
ATP content in the normal hearts is maintained constant despite variations in cardiac performance, through balance
changes in the rate of ATP synthesis and utilization, which is also regulated by the level of ATP. Disturbances in ATP content
of the myocardium as the result of energy supply system damage under ischemic stress of the heart remain the central problem
of the AHF development. The goals of the present study were, at first, to determine whether and when ATP content is reduced
under ischemic disease and in second - to identify the structural mechanism(s) explaining the loss of ATP.
To address these goals, the canine model of left descending coronary arthery occlusion (CAO) was used. The dogs
were paced for 1, 2, 5, 15 min CAO to induce the syndrome of early phase of AHF and for 60, 120 min to develop AHF. Each
time at 1, 2, 5, 15, 60 and 120 min CAO we obtained specimens and venous blood samples for measurement of myocardium
content of ATP, ADP, AMP, Pi, CrP, Cr, NAD, NADH, NADP, NADPH, cytochrom C and lactate/pyruvate ratio. Simultaneously intracardiac hemodynamic and contractile ability of isolated contractile protein system (MCPS) was measured also. Data
are expressed as mean  standard deviation. For each parameters Student’s t–test for paired comparison and regression analysis
to determine correlation relationships is used.
The ability of cardiac muscle to increase contractile performance requires concomitant changes in ATP content. The
biochemical correlation of contractile ability (increased workload) with the parameters of energy supply system functioning is
very important. A major result of our study is that the decrease of left ventricular (LV) content of NAD by 15 and 48% in ischemic and non-ischemic areas of myocardium respectively, phosphorylation potential decrease by 15% and decrease of
ATP/ADP ratio by 20% take place even after 15 min of CAO. The decrease of the NAD content in CAO15min correlates with
sharp decrease of force generation and ATP hydrolysis by MCPS. This reversible damage of myocardium is accompanied by
the decrease of mitochondrial coefficient of energy efficacy by 28% and the decrease of content of cytochrom C by 20%. It has
been found that the decrease of dP/dtmax of LV correlates with the decrease of maximal velocity of skinned myocardial fiber
(SMF), but not with the content of CrP or ATP and changes in the Ca-transport system. In case of 120 min CAO in cardiomyocyte the damage of all three subcellular system responsible for contraction-relaxation cycle occurs. In the myofibrils the energy
transduction disturbs and the generated force decreases, that is accompanied with full dissociation between the functioning of
force generation and ATP hydrolysis centers in actomyosin ensemble (r=0.17, NS; in norm r=0.93, p<0.001). A working hypothesis for the role of these changes in the structure of cardiomyocyte is that progressive metabolic vicious circle of cardiomyocyte develops as a results of the loss of cytochrome C and NAD content. The loss of cytochrome C of the respiration chain
in mitochondria plays a key role not only in the energy supply system but an important initial role in the apoptosis. It is clear
that the decrease of NAD and cytochrome C content as well as the ratio of NAD/NADH decreases earlier than ATP falls markedly. Because NAD/NADH is the critical ratio during all the glycolytic and Krebs cycle activities in the cell, maintenance or
restoration NAD/NADH ratio nearly to normal level is indeed more important and essential for cell survival and prognosis of
AHF.
Thus as a result of damage of the energy supply system (loss of cytochrome C in respiratory chain and NAD for
NAD-dependent dehydrogenases of Krebs cycle and glycolysis, as well as many other NAD-dependent processes) the decrease
of ATP, CrP, Pi contents and energetic charge of the cardiomyocyte and cytosole take place. In the basis of such disturbances
lays sharp reduce of cytochrome C content in mitochondria in the ischemic and non-ischemic areas after 60 min CAO and
these changes in myocardium correlate with the increase of NAD (r=-0.73, p<0.005), cytochrome C (r=-0.76, p<0.01), NADP
(r=0.68, p<0.02) in plasma. In peripheral blood through all investigated period of CAO maintenance the correlation between
content of NAD, NAD/NADH and systolic (r=0.76 and 0.78, p<0.005) and diastolic (r=0.91, p<0.0001 and r=0.78, p<0.005,
respectively) functions of the heart occurs.
Obtained data gave ground to conclude that the functioning of the energy supply system of the myocardium could be
15
judged by the determination of the value of redox-potential and content of cytochrome C and NAD in the blood.
16
OXIDANT STATUS IN LOW-FREQUENCY
STIMULATED RABBIT MUSCLE
G.P. Dizhe1, I.Ye. Krasovskaya1, Ya.L. Tolstov1,
R.V. Dyatlov1, L.V. Lislova1, Ye.V. Konisheva1, R. Cusso 2
1
St.-Petersburg State University, St.-Petersburg, Russia;
2 University of Barcelona, Barcelona, Spain
Stimulated contractile activity induced by chronic low-frequency electrostimulation (CLFS) elicits in fast-twitch muscle a series of orchestrated fast-to-slow transitions. After a short electrostimulation period muscle fells in fatigue and is able to
restore its activity in 48 hours. The molecular mechanisms of such fiber transition and mechanisms of fatigue, being very important for understanding of muscle adaptation to work, are still unknown.
There is a view that production of reactive oxygen species (ROS) induced damage in muscle enhanced by CLFS and
could be one of the factors involved in muscle fatigue, so the goal of this study was to investigate the changes in superoxide
dismutase (SOD) activity and lipid peroxidation damage products in rabbit tibialis anterior (TA) muscle after CLFS.
Adult male New Zeland rabbits have been stimulated via electrodes implanted laterally to the peroneal nerve of the
left hindlimb (10 Hz). The TA muscle was examined at the following time points after the onset of stimulation: 1, 2, 4, 7 and
14 days. The contralateral muscle which had not been exposed to CLFS served as controls.
We found that CLFS exposure led to increasing of SOD activity in TA muscle. The most ROS-induced changes were
reversed during 1 day of exposition: SOD activity increased to 60% of control value. Lipid peroxidation was not elevated in
the stimulated muscles. Measurements of lipid peroxidation damage products (conjugated dienes, Shiff's bases, thiobarbituric
acid reactive substances) showed minimal changes in all experimental days suggesting that CLFS occur without the ROS damage.
The results allow to consider that biochemical adaptation to CLFS includes well-developed antioxidant defenses that
minimize or prevent muscle cell damage induced by reactive oxygen species.
THE ROLE OF THE CYTOSKELETAL STRUCTURES
IN THE APOPTOSIS DEVELOPMENT
L. Domnina, O. Ivanova, Ju.M. Vasiliev
A.N. Belozerski Institute of Physico-chemical Biology, Moscow State University, Vorob’evy Gory, Moscow, Russia
The role of the cytoskeleton modifications in the chain of events leading to apoptosis induced by TNF (tumor necrosis
factor) is still unclear. It is known that the characteristic changes in the cell shapes [Mills et al., 1999; Pavlac & Helfman,
2001], probably cytoskeleton-dependent (general rounding, bleb formation, etc) are quite typical for the late stages of execution phase of apoptosis. The mechanisms of those changes are yet unknown.
We attempted to study such changes, using classical system of TNF-apoptosis in Hela cells sensitive to this agent. As
a control we used cultures, transfected by construction containing Bcl 2 gen.
It was shown:
1. In TNF- emetine treated cultures there were pronounced signs of apoptosis: cell rounding, general blebbing and nuclei
fragmentation (detected by the Hoechst staining).
2. Immunomorphological analysis showed, that at the early stages of apoptosis three types of cytoskeletal structures were
damaged. The bundles of the actin filaments and corresponding vinculin-positive focal contacts disappeared, keratin filaments fragmented. The third basic cytoskeletal system – microtubules did not presented significant changes before cell
rounding.
3. The peripheral blebbing, i.e. creation and disappearance of mutual blebs (0.2–0.5 micron) along the active cell edge, (detected by time-lapse videomicroscopy) appears after 2 hours after TNF addition and belongs to the early changes of apoptosis. It was followed by quick cell rounding and general blebbing of the whole cell surface.
4. HA 1077 and H7-inhibitors, which suppress the contractility of actin-myosin by changes in phosphorylation of the light
myosin 2 chain, inhibited the peripheral blebbing. But the speed of the apoptotic manifestations appearance (fragmentation
of nuclei chromatin and cell rounding) did not changed essentially.
5. In HeLa-Bcl2 cells the TNF-induced apoptosis was inhibited, but in the presence of cytochalasine D (which caused actin
cytoskeleton destruction) the HeLa-Bcl2 cells became TNF-sensitive (cells enter the apoptosis).
6. In HeLa cells TNF caused mitochondria concentration in the cell center around the nucleus.
So, in the present work the investigation of the TNF-induced cytoskeletal modifications that caused changes in cell
behavior during the apoptosis progress in Hela and Hela Bcl 2 cells was performed.
The work is supported by RFBR grants № 99-04-49256 and № 99-049126.
17
INVOLVEMENT OF THE MYOSIN LIGHT CHAIN KINASE GENETIC LOCUS PRODUCTS, CYTOSKELETAL
AND EXTRACELLULAR MATRIX PROTEINS IN CARDIAC REPARATION AFTER ADRIAMYCIN-INDUCED
INJURY
T.V. Dudnakova, V.L. Lakomkin, V.I. Kapelko,
B.V. Shekchonin, V.P. Shirinsky
Russian Cardiology Research Center, Moscow, Russia
The aim of the study was to investigate the molecular mechanisms of cardiac reparation upon acute injury. In particular, we studied the involvement of the myosin light chain kinase (MLCK) genetic locus products in this process. In genome of
the higher vertebrates MLCK locus codes for two isoforms of MLCK (MLCK-108 and MLCK-210) and a Kinase-Related Protein (KRP) – the myosin filament stabilizing protein. KRP is expressed in chicken embryo cardiomyocytes but not in adult cardiac cells (Birukov et al., 1998). This observation suggests the involvement of KRP in cardiomyogenesis and, perhaps, in cardiac reparation and hypertrophy.
WK strain rats and cultured chicken embryo cardiomyocytes (CEC) were used in this study. Cardiac injury was produced by adriamycin (AM) the potent antineoplastic agent with the pronounced cardiotoxicity due to the hyperproduction of
free radicals. WK rats received a single AM injection of 2.2 mg/kg and 0.4 mg/kg whereas saline was injected in control animals. Rats were sacrificed after two hours and three weeks after injection. Hearts were clamp frozen in liquid nitrogen and
processed for immunohistochemistry and western blot analysis. CEC were isolated from 7-9 day embryos (Eschenhagen et al.,
1997) and grown in M199 medium supplemented with 0.5% fetal bovine serum (FBS). CEC were treated by AM (0.4 mcg/ml)
for one hour. Four days later CEC samples were processed for Western blot analysis. The levels of protein expression in cardiac tissue and CEC were normalized to GAPDH content.
In rat model 2.2 mg/kg AM exerted cardiotoxic action. The content of MLCK-108, tubulin and fibronectin decreased
by 28%, 20% and 24%, correspondingly, presumably due to the tissue damage by free radicals. After 3 weeks post AM injection tissue content of KRP, tubulin and fibronectin increased by 20%, 50% and 20%, correspondingly, while MLCK-108 content further decreased by 54%. 3 weeks after AM injection at 0.4 mg/kg KRP, tubulin and fibronectin expression increased by
15%, 25% and 23%, correspondingly, and MLCK-108 expression decreased by 25%. Among proteins studied MLCK turned
out to be the most susceptible to AM action, perhaps, because it primarily originates from the vasculature which is first damaged by the drug. MLCK-210 was not detected in rat heart either by immunoblotting or indirect immunofluorescence. Immunohistochemical examination of rat hearts 2 hours following AM administration at either dose demonstrated the normal
deposition of collagen III and IV but decreased content of fibronectin. The latter extracellular matrix protein accumulation was
augmented 3 week following the injection of the low AM dose whereas collagen profile remained unchanged. Cardiac recovery after 2.2 mg/ml injection of AM was characterized by a diffuse cardiosclerosis and increased content of fibronectin and
collagen III.
In CEC culture the source of MLCK isoforms is co-purifying cardiac fibroblasts and smooth muscle cells whereas
KRP comes from CEC per se. After treatment with AM the expression of KRP in CEC culture increased by 20%.
Thus, the results of immunochemical studies of protein expression in adriamycin-treated heart stay in agreement with
the pathophysiological description of this model. Initial stage of tissue damage and decrease in total protein synthesis was followed by the reparation stage which showed the signs of hypertrophic response (increased tubulin expression) and cardiosclerosis (increase in collagen and fibronectin). For the first time we demonstrated that KRP expression is upregulated in cardiac
cells in two independent models after AM treatment. These findings suggest the involvement of KRP in the restoration of the
contractile system in cardiomyocytes. Molecular mechanism of KRP action might include the stabilization of presarcomeric
structures.
Supported by RFBR grants 00-04-48480a (VIK), 99-04-48158 (VPS) and MAC (TVD) and HHMI grant 55000335
(VPS).
EFFECTS OF SERUM FACTORS AND MECHANICAL STIMULATION ON KINASE-RELATED PROTEIN
EXPRESSION
IN CULTURED CHICKEN EMBRYO CARDIOMYOCYTES
T.V. Dudnakova, O.V. Stepanova, V.P. Shirinsky
Russian Cardiology Research Center, Moscow, Russia
Kinase-Related Protein (KRP) is one of the multiple products of the myosin light chain kinase (MLCK) locus in the
higher vertebrate genome. KRP is able to stabilize filaments of smooth muscle and non-muscle myosin and prevent their depolymerization at physiologic ATP concentrations. In chicken heart muscle the significant expression of KRP takes place during the embryonic period whereas the other products of MLCK genetic locus (MLCK-108 and MLCK-210) are not detected in
this tissue. Factors affecting KRP expression in the heart are unknown. KRP content of adult chicken heart is much lower and
the main source of this protein is vascular smooth muscle. Available data allows hypothesize that KRP, as the myosin-binding
protein, is involved in cardiomyocyte differentiation and, possibly, in the hypertrophic response of these cells since both processes demonstrate common de novo sarcomerogenesis. To test this hypothesis and study the mechanisms of KRP expression
in the heart we used cultured chicken embryo cardiomyocytes (CEC) as an experimental model.
CEC were enzymatically isolated from 7-9 day embryos (Eschenhagen et al., 1997) and were maintained in M199
medium supplemented with 0.5% fetal bovine serum (FBS) in the atmosphere of 5% CO2, 95% air at 37 oC. For mechanical
stimulation CEC were grown in elastic bottom multiwells covered with collagen. Cells were subjected to cyclic stretch in a
Flexercell Strain Unit at 1Hz and maximum membrane elongation 25% for 4 days. In parallel experiments CEC were grown in
the presence of 2 ng/ml TGF-beta or 10% FBS. The levels of KRP expression in CEC were assessed by western blot analysis.
Protein loadings were normalized by GAPDH or sarcomeric alpha-actinin content in the samples.
KRP content in CEC increased 2.5-fold in the presence of 10% FBS and decreased by 30% in the presence of TGF18
beta compared to KRP levels in CEC grown in M199/0.5% FBS. Mechanical stimulation of CEC induced 2-fold increase of
KRP expression in these cells.
Thus, KRP expression may be induced by the growth factors present in FBS. These findings are consistent with the
presence of a CArG element (or Serum Response Element, SRE) in KRP promoter region and with the involvement of Serum
Response Factor (SRF) in KRP transcription in A10 cells. Apparently, TGF-beta activates other signaling cascades that do not
stimulate KRP synthesis in CEC. Both growth factors and mechanical load promote differentiation and induce cardiomyocyte
hypertrophy. These conditions are characterized by the active sarcomerogenesis. The increased KRP expression in CEC upon
FBS addition and mechanical stimulation suggests the involvement of this protein in the maturation of cardiomyocyte contractile machinery. Molecular mechanism of KRP action may be the stabilization of non-muscle myosin filaments that form
presarcomeric structures.
Supported by RFBR grant 99-04-48158 (VPS) and MAC (TVD) and HHMI grant 55000335 (VPS).
ACTIN FILAMENT-ENTRAINED CASEIN KINASE
ASSOCIATED WITH THE CORTICAL CYTOSKELETON
DURING XENOPUS LAEVIS OOCYTE MATURATION
S.M. Elizarov* and S.G. Vassetzky**
*Bach Institute of Biochemistry, Russian Academy of Sciences,
Leninsky prosp., 33, Moscow, 117071 Russia;
**Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, Vavilova st., 26, Moscow, 117808 Russia.
Research over the past few years has demonstrated the central role of protein phosphorylation in regulating oocyte
maturation. However, little is known about how the specific protein kinases (PKses) contribute to the positional and temporal
regulation of the actin filament system functioning during maturation. In order to study a possible relationship between protein
phosphorylation and cortical actin cytoskeleton (CSK) reorganization we examined in vitro and in vivo phosphorylation of amphibian actin-associated proteins by homologous actin-binding casein protein kinase-II (CPK-II). It was shown that in the
preparation of actin-binding proteins of full-grown Rana temporaria oocytes, the endogenous CPK-II phosphorylates a set of
polypeptides with molecular masses from 50 to 230 kDa. Vinculin, fodrin, and -actinin were identified among in vitro substrates of this kinase using immunological techniques. Modification of vinculin and -actinin by serine residues to a depth of
0.8 mole phosphate per mole of protein substantially decreases their affinity to F-actin at physiological salt and pH conditions.
These three proteins are also detected as phosphorylated components when full-grown oocytes of Xenopus laevis microinjected
with [-32P]ATP are incubated in the presence of progesterone (PG). The kinase activities in vivo are stimulated by PG and display cell cycle-dependent characteristics. Kinase analyses and in-gel autophosphorylation assays reveal the presence of PGstimulated calcium-independent serine/threonine protein kinases (49 and 45 kDa), as well as CPK-II and CPK-I in the fraction
of CSK proteins. Microinjection of homogenous CPK-II into maturing oocytes leads to further enhancement of 49-kDa kinase
phosphorylation in this fraction. Immunoblot analyses show a distinct decrease in -actinin and fodrin concentrations in CSK
proteins during maturation. Injections of CPK-II enhance the decrease in concentrations of these proteins in the CSK of recipient oocytes. Microinjections of CPK-II result in a delay of PG-stimulated oocyte maturation and provoke dislocation of the
pigment granules in subcortical layers. These results suggest that CPK-II plays a role in the timing of protein kinase activities
associated with CSK and, in ensemble with other kinases, is involved in reorganization of CSK during oocyte maturation.
RESOLVING BIOCHEMICAL CHANGES DURING RAPID FORCE TRANSIENTS
Michael A. Ferenczi, Zhen-He He*
Biomedical Sciences Division, Imperial College, London SW7 2AZ;
* NIMR, Mill Hill, London NW7 1AA, UK
The observation of complex force responses to rapid length perturbations in isometrically contracting muscle fibres
has lead to the well-known model of Huxley and Simmons (1971). Myosin cross-bridges modify their attachment to the thin
filaments in a series of distinct steps, gradually stretching the elastic component in the cross-bridge to give rise to force.
The development of a highly sensitive and rapid method for the measurement of P i release resulting from ATP hydrolysis led us to consider using this technique to determine whether biochemical changes could be detected in muscle fibres
during the force response to mechanical perturbations. This technique makes use of a fluorescently labelled, genetically engineered phosphate binding protein derived from and expressed in E. coli. The fluorescent label is a coumarin probe which gives
rise to a five-fold enhancement of fluorescence upon Pi binding. The Pi-probe is referred to as MDCC-PBP.
The characteristic phases of the transient force response observed in intact fibres for frog skeletal muscle are usually
not seen in permeabilized fibres because force generation by such fibres results in distortion of the sarcomeres and in loss of
the structural integrity. Permeabilised fibres are however necessary to allow incorporation of millimolar concentrations of
MDCC-PBP into the spaces between the myofilaments. To overcome this problem, permeabilized fibres were treated with the
zero-length cross-linker EDC to covalently link a fraction of myosin cross-bridges to actin. This procedure preserves the structural integrity of the sarcomeres, reduces the extent of internal shortening and allows the observation of distinct phases in the
force response to length perturbations. Experiments were carried with rabbit muscle fibres because their responses were slower
than those of frog muscle fibres at the same temperature, thus making measurements easier.
Force transients in response to quick (<0.5 ms) small amplitude (0.5 to 0.8%) length decreases were observed at 5, 12
and 20°C in permeabilized muscle fibres of rabbit psoas muscle. By summing the responses of 8 or more experiments, P i release could be detected with approximately millisecond time resolution. It was found that a quick length release resulted in acceleration in the rate of Pi release at all temperatures. In contrast, quick stretches did not result in measurable changes in the
rate of Pi release.
These results will be presented, and discussed in terms of changes in the distribution of biochemically distinct intermediate states of myosin cross-bridges during force recovery.
19
EPITHELIUM-FIBROBLAST INTERACTIONS IN MIXED HETEROTYPIC CELL CULTURE
E. Fetisova, O. Ivanova, Ju.M. Vasiliev
A.N. Belozersky Institute of Physico-chemical Biology,
Moscow State University, Moscow, Russia
We studied interaction of epithelium and fibroblasts in mixed cultures. Continuous cell lines of epithelial and fibroblast origin were used: MDCK/clone 20 - dog kidney epithelium, AG-1523 and M19 - human diploid fibroblasts. Cells were
cultured in DMEM supplemented with 10% fetal calf serum. Cells of different types were easily distinguished at phase contrast
microscopy. Interference reflection microscopy was used to investigate cell substratum adhesion. Cell-cell interactions were
studied by performing video microscopy. Immunofluorescence staining was used to observe intercellular and cell substratum
adhesions. For simultaneous localization of actin and beta-catenin (E-cadherin, Pan-cadherin, vinculin) cells were rinsed in
PBS, fixed in 3.7 % formaldehyde, permeabilized with 1% Triton X-100 and stained with rhodamine-phalloidin to label actin
filaments and mAbs against beta-catenin (E-cadherin, Pan-cadherin, vinculin), followed by fluorescein isothiocyanate-labeled
goat anti-mouse secondary antibodies. The specimens were studied, using fluorescent microscope Axiophot.
The mode of heterotypic cell interaction depended on the way of their collision. At collision of frontal lamellas of epithelium and fibroblast - epithelium lamellar activity at the place of collision was inhibited. Epithelium formed a stable edge instead of the lamella. Activity of the fibroblast lamella didn't change. The lamella of the fibroblast crept under the stable edge of
the epithelium. Subsequent cell behavior depended on the local situation. If there were no other cells near the fibroblast and the
adjacent substratum was free, then the fibroblast formed a new lamella and its lamella under the epithelium retracted. The fibroblast moved away from the place of collision. If there was a lot of cells near the fibroblast, they complicated its moving.
The fibroblast migrated under the epithelium. Underlying the epithelium the fibroblast formed focal adhesions with the substratum. At collision of epithelium lamella with the lateral side of fibroblast (lack of lamella) - the lamella of the epithelium
crept under the fibroblast lateral side. The fibroblast turned aside. In dense cultures epithelium forced back the fibroblast.
If heterotypic cells were cultivated for 4-5 days without medium change, special intercellular adhesions between epithelium and fibroblasts were formed. Heterophylic adhesions were of various morphology and collocalized with the ending of
actin filaments. Formation of special intercellular adhesions between heterotypic cells was demonstrated for the first time.
The work was supported by RFBR grant № 99-04-49256.
DIVERGENT EXPRESSION OF DELAYED RECTIFIER K+ CHANNEL SUBUNITS DURING MOUSE HEART
DEVELOPMENT
Diego Franco1,2, Sophie Demolombe1,3, Robert Dumaine4,
Jorge N. Dominguez2, Denis Escande4, Antoon F.M. Moorman1
1Experimental
and Molecular Cardiology Group, AMC,
University of Amsterdam, The Netherlands,
2Department of Experimental Biology, University of Jaén, Spain;
3INSERM U533 Hôpital Hotel-Dieu, Nantes, France;
4Department of Molecular Biology and Pharmacology, Utica, NY, USA.
The repolarisation phase of the cardiac action potential is dependent on transmembrane K + currents. The slow (IKs)
and fast (IKr) components of the delayed-rectifier cardiac K+ current are generated by pore-forming  subunits KCNQ1 and
KCNH2, respectively, in association with regulatory -subunit KCNE1, KCNE2 and perhaps KCNE3. We have previously have
investigated the expression pattern of KCNQ1 and KCNE1 in different mouse epithelia (Demolombe et al., 2001). In the present study we have investigated the distribution of transcripts encoding these five potassium channel-forming subunits during
mouse heart development as well as the protein distribution of KCNQ1 and KCNH2.
KCNQ1 and KCNH2 mRNAs (and protein) are first expressed at embryonic day (E) 9.5, showing comparable levels
of expression within the atrial and ventricular myocardium during the embryonic and fetal stages. In contrast, the -subunits
display a more dynamic pattern of expression during development. KCNE1 expression is first observed at E9.5 throughout the
entire myocardium and progressively is confined to the ventricular myocardium. With further development (E16.5), KCNE1
expression is mainly confined to the compact ventricular myocardium. KCNE2 is first expressed at E9.5 and it is restricted already to the atrial myocardium. KCNE3 is first expressed at E8.5 throughout the myocardium and with further development, it
becomes restricted to the atrial myocardium (Franco et al., submitted). The fact that  subunits are homogeneously distributed
within the myocardium, whereas the  subunits display a regionalized expression profile during cardiac development, suggest
that differences in the slow and fast component of the delayed-rectifier cardiac K+ currents between the atrial and the ventricular cardiomyocytes are mainly determined by differential -subunit distribution.
References
Demolombe S, Franco D, de Boer P, Kupershmidt S, Roden D, Pereon Y, Pradal G, Jarry A, Moorman AFM, Escande D. Differential
expression of KvLQT1 and its regulator IsK in mouse epithelia. Am. J. Physiol. Cell Physiol 2001;280(2):C359-C372.
Franco D, Demolombe S, Kupershmidt S, Dumaine R, Dominguez JN, Roden D, Antzelevitch S, Escande D, Moorman AFM. Divergent expression of delayed rectifier K+ channel subunits during mouse heart development. Cardiovasc Res (submitted).
20
THE TRANSCRIPTIONAL DOMAINS
OF THE DEVELOPING HEART
1
Diego Franco, 2Robert Kelly, 3Rafaella de Lisi, 3Marina Campione,
4
Nigel Brown, 3Stefano Schiaffino, 2Margaret Buckingham,
5
Antoon F.M. Moorman
1Department
of Experimental Biology, University of Jaén, Spain;
of Molecular Biology, Pasteur Institute, Paris, France;
3Department of Biomedical Sciences, University of Padua, Italy;
4St. George´s Hospital Medical School,, London, UK,
5Experimental and Molecular Cardiology Group, AMC,
University of Amsterdam, The Netherlands
2Department
During heart development, two fast-conducting regions of working myocardium balloon out from the slowconducting “primary myocardium” of the tubular heart. Three slow-conducting segments of primary myocardium persist, the
outflow tract, atrioventricular canal and the inflow tract, which are contiguous throughout the inner curvature of the heart
(Christoffels et al., 2000). We have previously shown that transgenic mice carrying an nlacZ reporter gene under control of
regulatory sequences from the MLC1F/3F gene provide specific markers to investigate the transcriptional heterogeneity of the
developing myocardium. 3F-nlacZ-2E mice show transgene expression restricted to the embryonic right atrium, atiroventricular canal and left ventricle whereas, in 3F-nlacZ-9 mice, the transgene is expressed in both atrial and ventricular segments
(right and left) and in the atrioventricular canal, but not in the outflow and inflow tracts (Franco et al., 1997). The analysis of
the developmental profile of transgene expression in these mice in conjunction with analyses of endogenous gene expression,
has provided insights into the contribution of the outflow and inflow tract to the definitive ventricular and atrial chambers. The
embryonic outflow tract is incorporated into the ventricular infundibulum, in line with the differential expression of MLC2a
and MLC2v in this cardiac region (Franco et al., 1997; 1999). We have also demonstrated that the genes encoding atrial natriuretic factor (ANF), myosin light chain (MLC) 3F, MLC2V and Pitx-2, and MLC3F transgenic mouse lines display regionalized
patterns of expression in the atrial component of the developing mouse heart (Franco et al., 2001). Four broad transcriptional
domains can be distinguised in the atrial myocardium: 1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles, 2) the atrial appendages, 3) the caval vein myocardium (systemic inlet) and 4) the mediastinal
myocardium (pulmonary inlet) including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in
the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple
molecular compartments within the atrial chambers.
Furthermore, analysis of another transgenic mice, carrying an nlacZ reported gene under control of a minimal cardiac
troponin I promoter have revealed that the atrioventricular canal constitutes an independent transcriptional unit. Moreover,
analyses of the transgene expression of the cTnI-nlacZ mice suggest that a myocardial population of the atrioventricular canal
myocardium contributes to specialized myocardium of the atrioventricular node (Di Lisi et al., 2000).
In summary, analyses of endogenous gene expression as well as transgene expression have revealed an enormous heterogeneity in the transcriptional potential of the developing and adult myocardium. Seven transcriptional domains can be distinguished, outflow tract, systemic ventricle, pulmonary ventricle, atrioventricular canal, atrial appendanges, caval myocardium and mediastinal myocardium, each of them having distinct left and right components (Campione et al., 2001).
References
Christoffels VM, Habets PEMH, Franco D, Campione M, de Jong F, Lamers WH, Bao Z-Z, Palmer S, Biben C, Harvey RP, Moorman
AFM. Chamber formation and morphogenesis in the developing mammalian heart. Dev. Biol. 2000;223:266-278.
Campione M, Ros MA, Icardo, JM, Piedra E, Christoffels VM, Schweichert A, Blum M, Franco D, Moorman AFM. Pitx-2 expression
defines a left cardiac lineage and provides evidence for the existence of ventricular isomerism in iv mice. Dev. Biol 2001;231:252264.
Di Lisi, R, Sandri C, Franco D, Ausoni S, Moorman AFM, Schiaffino S. An atrioventricular canal domain defined by cardiac troponin I
transgene expression in the embryonic myocardium. Anat. Embryol. 2000;202:95-101.
Franco D, Kelly R, Lamers, W.H., Moorman AFM & Buckingham M (1997) Compartment-specific myosin light chain 3F transgene
expression in the embryonic mouse heart. Dev. Biol. 188:17-33.
Franco D, Markman MWM., Wagenaar GTM, Ya J, Lamers WH, Moorman AFM Myosin light chain 2a and 2v identifies the embryonic outflow tract myocardium in the developing rodent heart. Anat. Rec. 1999;254:135-146.
Franco D, Campione M, Kelly R, Zammit PS, Buckingham M, Lamers WH, Moorman AFM. Multiple transcriptional domains, with
distinct lerft and right components, in the atrial chambers of the developing heart. Circulation Research 2000;87:984-991.
21
ADF STABILIZES AN EXITING TWIST OF F-ACTIN
AND CAN CHANGE THE TILT OF OF F-ACTIN SUBUNITS
Vitold E. Galkin1,3, A. Orlova1, N. Lukoyanova1,4,
W. Wriggers2 and E.H. Egelman1
1Department
of Biochemistry and Molecular Genetics, University of Virginia
Health Sciences Center, Charlottesville, VA 22908, USA,
2Department of Molecular Biology, The Scripps Research Institute,
La Jolla, California 92037, USA,
3Institute of Cytology, RAS, St.Petersburg, Russia;
4Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Russia
Actin dynamics play a major role in cell motility, cytokinesis, and endocytosis. The rapid polymerization/depolymerization of actin filaments in the cell, especially under the leading membrane edge, requires efficient protein machinery that can provide a fast response to extracellular stimuli. Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential, conserved, and widespread actin depolymerizing factors that interact with F-actin in a strong cooperative, pHdependent manner. Human cofilin was observed to change the twist of actin by ~5° per subunit when it was bound stoichiometrically to F-actin.
We have used a new approach for image analysis of helical filaments to examine both pure actin filaments and complexes of F-actin with plant and human ADFs. This new approach allows us to analyze tens of thousands of short segments
within filaments, without the need to assume a fixed helical symmetry for a long filament. This approach is therefore sensitive
to variations in helical symmetry between different segments, as well as to differences in the occupancy of the ADF molecules
bound to F-actin. Using this method, we show that segments of pure actin can be found in an ADF-cofilin-like state of twist in
the absence of other proteins. Furthermore, the ADF-actin complex can exist with a twist close to that of the normal actin state.
We find that under conditions where actin filaments are readily depolymerized, two molecules of ADF bind per actin subunit,
and not one as has been believed previously. We also find that under these conditions of filament stabilization some actin subunits undergo a large tilt from their positions in normal F-actin that causes the breakage of the longitudinal contacts within the
actin filament. These results provide new insight into the internal dynamics of F-actin, suggesting that they may be even larger
in magnitude than previously imagined, and suggest that certain actin-binding proteins in the cell may have evolved to regulate
these internal dynamics as part of cellular control of the cytoskeleton.
22
REGULATION BY TROPOMYOSIN-TROPONIN
AND THE DYNAMIC STRUCTURAL ELEMENTS ON ACTIN
Jack H. Gerson, Wenise W. Wong, Tianwei Yu and Emil Reisler
Dept. of Chemistry and Biochemistry and Molecular Biology Institute,
UCLA, Los Angeles, CA 90095
Subdomain 2 and the C-terminus region have been identified as the dynamic structural elements that are involved in
the flexibility and conformational transitions in F-actin. The possible role of these elements and their dynamic changes during
the regulation of actin by Tropomyosin (Tm)-Troponin (Tn) was examined using subtilisin cleaved skeletal actin and yeast actin mutants with fluorescent labels attached at the above sites. Subtilisin cleavage in subdomain 2 (M47/G48) decreases the
binding of S1 but not that of Tm to F-actin. The in vitro motility of subtilisin cleaved filaments is strongly inhibited, but the
normalized pCa curves for the regulation of their motion by Tm-Tn and Ca2+ are indistinguishable from those for the uncleaved actin. Interprotomer cross-linking of subdomain 2 and the C-terminus (Q41 to C374 on adjacent protomers) also inhibits the motility of actin filaments but not the Tm-Tn regulation of acto·S1 ATPase activity. Measurements of fluorescence energy transfer between tryptophan residues and probes attached to C41 and C51 on subdomain 2 in yeast actin mutants are not
affected by Ca2+ and myosin-dependent transitions between the blocked, closed and open states of regulated actin. These results suggest that regulation of actin by Tm-Tn does not involve or depend on motions of subdomain 2.
REACTION OF XENOPUS LAEVIS EARLY GASTRULA ECTODERM UPON A STRETCH WITH A
SUCCESSIVE UNLOADING
N.S. Glagoleva, L.V. Beloussov, N.N. Louchinskaia
Department of Embryology, Faculty of Biology,
Moscow State University, Moscow, Russia
The tissues of the developing embryos are known to be mechanically stressed. The stresses play an important regulatory role and generate, as a response, the active morphogenetic deformations of the given tissue region. The aim of this work
was to study the active reaction of embryonic tissue upon the mechanical stretch.
Explants of a ventral and dorsal ectoderm of Xenopus laevis early gastrula embryos have been stretched by two thin
glass needles to a different extent (10 – 80% from their initial length) and kept under stretch for the different time periods
(from 5 min to 2 h). Then one of the needles was removed thus relieving a tissue piece. By measuring the residual deformations after unloading we could trace a gradual stress relaxation within the still stretched tissue pieces. This was due both to a
planar and radial cell intercalation within a piece.
We could also detect the regional and temporal differences of embryonic tissues in the stress relaxation dynamics. In
the ventral ectodermal explants the residual deformation is accumulated more rapidly than in the dorsal ones. At the same time,
the dorsal tissue pieces demonstrate better correlation of the contractility with the previous stretching. One can conclude that
the dorsal tissues are more elastic than the ventral ones. These regional differences can explain the ventro-dorsal direction of
the ectoderma cell movements which is one of the mostly fundamental properties of the gastrulation.
A prolonged stretching of the ventral explants can generate in these tissue pieces a long-range order providing a coordinated behaviour of all the explants’ cells.
Our data confirm that the morphogenesis can be regarded as the successive interactions between the passive (generated by an outside force) and the active (generated within a given tissue piece) mechanical forces. This is the first attempt to
evaluative these interactions.
ACTION POTENTIAL (AP) RATE AND/OR DURATION AP (DAP100) EFFECT THE NUMBER OF EARLY AP
CAUSED BY NI2+ IN THE CELLS OF THE SINO-ATRIAL VALVE OF RAT HEART
Vladimir A. Golovko
Institute of Physiology, Komi SC, Ural Division Russian Acad. Sci.,
Syktyvkar, 167982, Russia
The aim of the work is to test the hypothesis of what is the major reason of early action potentials (EAP) appearance
under the effect of nickel ions (Ni2+) activity: either a reverse increase of calcium channel permeability (P Ca) of L-type or partial block of Na-Ca-exchanger activity – regulation of intracellular Ca2+ [Ca2+]i concentration?
Method. The experiments were conducted by the method of microelectrode recording on spontaneously contracting
stripes (2 mm x 2 mm) of the sino-atrial region of laboratory rat heart (1.5 month old, n_), control solution contains 2.7 mmol/l
Cа2+; pH 7.2; 281 C. NiCl2 (Sigma) 0.05-1.0 mmol/l was used in the experiment.
Results. In the control solution stripes obtained from the sino-atrial region, valve included, contracted with the frequency of 484 min-1 (group I) or 847 min-1 (group II). The duration of AP cells in this case the duration of AP cells without
slow depolarization phase of the sino-atrial valve of rat heart was equal to 27030 ms (230-330 ms, n!) and 19020 ms (160240 ms, n_), respectively. Addition into the perfused solution of Ni2+ in the concentration of 0.3 mmol/l caused in the preparations generating AP with low frequency single early AP at 151 min. During the following five minutes they were transformed
into multiple early AP from 4 to 47 AP with the general duration from 1 to 12 s in a series. The effects are reversible. It should
be noted that Ni2+ (0.3 mmol/l) in the stripes contracting with a higher rate (group II) didn’t cause multiple early AP but only
occasional single early AP. When intracellular concentration of Ni2+ (0.6 mmol/l) was increased single early APs were observed more often, sometimes a series of multiple early APs from 3-7 APs was registered.
Thus, under the condition of a high rate AP Ni2+ generation block Na-Ca-exchanger – a sensitive regulator of intracellular Ca2+ concentration to a lesser degree and/or less quantity of Ca 2+ enters cells within one period of AP generation.
This work was funded by the Russian Foundation for Basic Research, project N 01-04-49668.
23
CALDESMON LOCALIZATION IN NON-MUSCLE CELLS: ACTOMYOSIN CROSSLINKING AND THE
EFFECT OF PHOSPHORYLATION BY MAP-KINASE
E.A. Goncharova, V.P. Shirinsky, S.B. Marston*,
B.A. Levine# and A.V. Vorotnikov
Institute of Experimental Cardiology, Cardiology Research Center,
Moscow 121552, Russia,
*Cardiac Medicine, NHLI, London SW36LY, UK;
#School of Biosciences, University of Birmingham, Birmingham B152TT, UK
Caldesmon is an actin filament-associated protein that inhibits activation of smooth muscle myosin ATPase by actin.
The actin-binding and inhibitory activities of caldesmon reside in its C-terminal domain, which is phosphorylated by MAPkinase in vitro and in vivo. The phosphorylation by MAP-kinase inhibits the actin-binding and inhibitory activity of caldesmon
in vitro, although little is known whether this is realised in vivo and how phosphorylation affects the protein interaction. The
N-terminus of caldesmon interacts with smooth muscle myosin in vitro and the dual binding activity of caldesmon may result
in cross-linking of actin and myosin filaments. However, evidence for the N-terminal caldesmon activity in vivo has been still
lacking. Using recombinant fragments corresponding to actin- and myosin-binding domains of caldesmon we assessed the
mode of l-caldesmon binding to actin and myosin filaments in non-muscle cells. The effect of phosphorylation by MAP-kinase
on caldesmon interaction with native cytoskeleton and pure actin has been also explored.
Chicken gizzard embryo fibroblasts (CEF) were transiently transfected with cDNA construct encoding for the Nterminal 152 residues of chicken caldesmon (N152) and an additional N-terminal FLAG sequence. The immunofluorescent
distribution of FLAG-N152 and non-muscle myosin revealed their prominent colocalization indicating that N-terminal domain
of caldesmon is capable of myosin binding in vivo.
The mode of non-muscle l-caldesmon interaction with native cytoskeleton was next investigated in triton X-100permeabilised CEF and human HeLa cells using exogenous recombinant N-terminal chicken (N152) and C-terminal human
(H1) caldesmon fragments to dissociate endogenous caldesmon from myosin and actin filaments, respectively. The application
of increasing amounts of N152 alone caused no displacement of l-caldesmon indicating that l-caldesmon did not bind only to
myosin in cells. Incubation of CEF cytoskeletons with increasing concentrations of H1 caused as little as 20% of endogenous lcaldesmon displacement suggesting it represents only the actin-bound fraction. However, simultaneous addition of H1 and
N152 caused up to 60% dissociation of l-caldesmon indicating that at least 40% of the endogenous caldesmon is simultaneously bound to actin and myosin filaments and cross-links them in CEF cytoskeleton. In contrast, H1 alone was sufficient to dissociate most of endogenous l-caldesmon from HeLa cytoskeleton and N152 had little further effect on l-caldesmon displacement. Thus, most of caldesmon appears to be only actin-bound in epithelioid HeLa cells.
When H1 was phosphorylated in vitro by active recombinant p44erk1 MAP-kinase and subjected to NMR analysis, two
serines in positions 759 and 789 of human h-caldesmon were found completely phosphorylated. The phosphorylated H1 was
much less effective in displacement of endogenous l-caldesmon from HeLa cytoskeleton than unphosphorylated H1. Thus
phosphorylation by MAP-kinase inhibits H1 binding to naturally arranged cytoskeleton and likely regulates the caldesmonactin interaction in living cells.
Of the two phosphorylation sites, only the Ser-759 is conserved in primary structure of chicken caldesmon as Ser-702.
Using NMR we studied overlapping regions of chicken caldesmon, residues 658-756 (658C, domain-4b) and residues 693-722
(LW30) that contain two actin-binding sites involving Trp-692 and Trp-722 flanking a backbone turn segment (713-716). This
turn segment constrains the relative disposition of the two actin contact sites and enables their simultaneous docking on subdomain 1 of actin. The turn is preceded by S 702PAPKP sequence that adopts an extended backbone conformation acting as a
rigid ‘spacer’. Phosphorylation of Ser-702 resulted in markedly diminished actin affinity and the loss of dual-sited interaction.
Only the C-terminal contact, centred on Trp-722, was retained whilst the N-terminal actin contacts around Trp-692 were diminished (658C) or lost (LW30). These results are rationalised by the observation that phosphorylation destabilises the turn
segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition, thus abolishing the two-site docking. Importantly, the effect of Ser-702 phosphorylation is not merely a function of the bulkiness of the covalent modification, since the stability of the turn region is sensitive to the ionisation state of the phosphate group. This provides possible explanation for a rather little effect of point mutation that mimics phosphorylation on the inhibitory activity of
658C, corraborated by the observation that single sited phosphorylation of 658C completely abolished its inhibition of actomyosin ATPase.
The work was supported by grants from the Wellcome Trust, HHMI (75195-546901), RFBR (99-04-49209) and British Heart Foundation.
TECHNIQUES FOR BIOMECHANICAL INVESTIGATIONS
OF THE PHYSARUM PLASMODIUM
F.E. Ilyasov, M.A. Morozov, V.A. Teplov
Institute of Theoretical and Experimental Biophysics RAS,
Pushchino, Moscow region, 142290 Russia;
E-mail: teplov@venus.iteb.serpukhov.su
Many of the molecular components of the cytoskeleton and associated motor proteins are now identified, but there
seems no clear linkage of the molecular biology of the cytoskeleton with amoeboid movement of cell. This is primarily a problem of biomechanics, and requires realistic models of cells as mechanical entities and a willingness to contemplate problems
involving both elasticity and viscosity in three spatial dimensions. Much room for solving this problem can be allowed by the
investigation of mechanical properties of the plasmodial strands, which are long tubes with outer gel-like ectoplasm surrounding an inner fluid endoplasm. Their lengths in large plasmodia can reach a few tens of centimeters and the diameters sometimes attain two millimeters. Within ectoplasm of the plasmodium (including its strands) there exists actomyosin microfila24
ment network, which is activated by Ca2+ and is responsible for rhythmical contractions of the strands in the longitudinal and
radial directions and, as a consequence, for vigorous shuttle flows of the endoplasm necessary for plasmodial locomotion.
In order to measure the longitudinal contractile activity of the plasmodial strands and their viscoelastic characteristics,
we have designed a sensitive electronic device. The device consists of two main parts. The first part is a ‘force’ unit, which allows for either measuring the longitudinal tension produced by the strand under isometric condition or imposing the calibrated
load to the strand under isotonic condition. The second part is a ‘length’ unit, which makes it possible either to register longitudinal isotonic contractions of the strand or to change quickly the strand length by definite values (stretching or releasing the
strand) under isometric condition. The excised strand (usually 0.3-0.7 mm in diameter and 4-20 mm long) is held at both ends
in a horizontal position in the moist chamber, being attached to two hooks of these units with cyanoacrylate or warm agar. The
values of viscoelastic parameters are automatically determined on the basis of computer analysis of the transient changes in the
force produced by the strand in response to 1% changes of its length.
The main part of the ‘force’ unit is a transducer, which, due to changes of the moire pattern, converts a very small displacement of the hook, to which the strand is attached, to the electrical signal. The maximum allowable force of 100 mg induces 10 m displacement of the hook. Sensitivity of this transducer is about 10 mV/mg. Another important component of this
unit is an electromagnetic facility allowing for stepwise loading of the strand under isotonic condition. Maximum load of
100mg is quickly achieved with 1000 steps. The ‘length’ unit containing an electromechanical facility allows for either monitoring changes in the strand length under isotonic condition or delivering designated deformations to the strand by moving its
second end for certain distance. Maximum displacement of the hook produced by this unit with an accuracy of 0.1% is 10 mm.
The device also incorporates sources, amplifiers and switches which provide both correct operating of the units and choosing
between the isometric and isotonic modes of measurement. As an interface between the device and computer we used the multi-function data acquisition card, which provides the following advanced features: 8 channel ADC, on-chip sample and hold, 1
channel DAC and 16 digital IO channels.
Inasmuch as analysis of the nonstationary contractile activity distributed throughout the plasmodium requires a large
number of simultaneous measurements in many points, we present a method of the registration and the quantitative analysis of
spatial modes of the cell contractions. The software package has been developed for computer processing of movie pictures obtained with the digital video camera during time-lapse cinematography of the plasmodium. It can sequentially view all the
movie pictures and calculate the optical density averaged over each spots along any selected curve on the plasmodial images.
As a result we have two-dimensional table of optical density, which is considered to be proportional to averaged thickness of
the cell in the spots chosen for analysis. In this table the columns have numbers corresponding to that of the points along the
selected curve, whereas a number of the row corresponds to that of the movie picture, that is, it is straightly proportional to the
elapsed time. Thus, each of the table rows constitutes the distribution of cell thickness along selected curve at fixed instants,
whereas each of the columns presents the time course of thickness changes in the chosen point of the plasmodium.
Using data from this table, the software can create a new movie, in which each picture is a graphical representation of
the distribution of cell thickness along some curve at fixed instants. Such movies strikingly illustrate the dynamics of cell profile changes, which are really produced by the local ectoplasm contractions. Similar movies are created in the course of computer simulations of our mathematical model for the coordinated wave-like contractions of the ectoplasm and shuttle streaming
of the endoplasm in plasmodial strands. Therefore, comparison of these movies has allowed us to judge on the truth of the hypotheses, which underlies the model that deformation-dependent mechanisms of the regulation can play an essential role in
spatial coordination of local contractions.
This technique can be also used in the investigations of coordination of individual cells during their morphogenetic
movements, which are much of the visible aspect of morphogenesis.
The work was supported by the Russian Foundation for Basic Research.
CHARACTERIZATION OF ACTIN BUNDLES FORMED BY FASCIN:
POLARITY, SLIDING AND DISASSEMBLY
ON MYOSIN II AND MYOSIN V
Ryoki Ishikawa, Takeshi Sakamoto, Sugie Higashi-Fujime,
Toshio Andoh and Kazuhiro Kohama
Department of Pharmacology, Gunma University, School of Medicine,
Maebashi, Gunma 371-8511, Japan
Growth cone of developing nerve cell projects filopodia from their leading edge, and their dynamics have been implicated in elongation and pathfinding of axon. Inside filopodia are the bundles of F-actin with barbed (or first growing) ends in
its tip region, and pointed (or slow growing) ends in the basal region. Precise mechanism of the dynamics of filopodia, however, is still unclear. Knockout of fascin, an actin bundling protein in filopodia, caused the loss of filopodia in cultured neuron,
suggesting that fascin is a key player to form actin bundles. Here, we showed that orientation of F-actin bundled by fascin in
vitro were the same as those in filopodium. Furthermore, actin/fascin bundles slid on myosin II with average velocities of 4-6
um/s, under which conditions individual actin filaments also slid with 4-6 um/s. Actin/fascin bundles glided smoothly without
changing the direction, although actin filaments showed frequent changes in direction in the same condition. Sliding velocities
of actin/fascin bundles gradually decreased as concentration of myosin II on coverslip decreased, the profile quite similar to
that of actin filaments. Minimum concentration of myosin II required for attachment of actin/fascin bundles to coverslip was as
low as that of actin filaments. Sliding velocities of actin/fascin bundles also decreased when ATP concentration were decreased. Again, decreased profile was the same as that of actin filaments. However, actin/fascin bundles frequently disassemble to actin filaments when myosin concentrations were increased to 0.6 uM, or ATP concentrations were decreased to less
than 40 uM, possibly because force generated by myosin heads became larger than crosslinking force by fascin. Most of actin/fascin bundles disassemble at pointed end, the dynamics quite similar to that observed in vivo. Actin/fascin bundles also
slid on myosin V as fast as actin filaments (0.47 u/s versus 0.56 u/s).
25
Because myosin II and myosin V localize in filopodia, our results suggest that these proteins may drive the filopodial
actin bundles backward, and cause the disassembly at basal region of filopodia.
DEVELOPMENT OF MYOSIN-COLLOIDAL GOLD MARKER
FOR ACTIN IDENTIFICATION
V.V. Ivanov, V.P. Orlov
Institute of Biochemistry and Physiology of Plants and Microorganisms RAS,
Saratov, pr. Entuziastov 13, 410015 Russia
E-mail: ivanov@ibppm.saratov.su
The past several years have witnessed explosive growth in our knowledge of the myosin superfamily of actin-based
motor proteins, which currently consists of at least 15 distinct classes. In general, the heavy chains of unconventional myosins
are characterised by an ~80 kDa head, a ‘neck’ consisting of one or more repeats of the IQ motif, and a class-specific tail.
These unconventional myosins are defined by myosin-like head (motor) domains attached to class-specific tail domains that
differ greatly from those of myosin-II. The head is responsible for binding to F-actin, hydrolysis of ATP and production of
force, whereas the IQ motifs provide binding sites for calmodulin or calmodulin-like light chains. The tail domains of myosins
are believed to be largely responsible for class-specific functions such as bipolar filament formation for the class II myosins or
GTPase-activating activity for the class IX myosins.
The aim of the work was to construct a marker based on colloidal gold conjugate with myosin molecule fragment
bearing the motor domain. Extraction and further purification of myosin from rabbit skeletal muscle was performed according
to Poglasov’s method [Poglasov B.F., Ivanov G.G., Metlina A.L. (1976). Molekulyarnaya biologiya (russian) V.10, N.175].
The obtained preparation purity was confirmed by SDS PAAG electrophoresis for contaminant proteins and Dot-blot analysis
with phalloidin-colloidal gold marker for F-actin contamination. Additional tests were performed for F-actin activation of myosin ATPase in the presence and the absence of Mg 2+ cations (1mM MgCl2) and was ~19-fold and ~7-fold respectively.
The resulted preparation was used further as a source for heavy meromyosin (HMM) obtaining according to Lowey’s
method [S. Lowey, H.S. Slayter, A.G. Weeds and H. Baker (1969). J. Mol. Biol. V.42, P.1-29]. Electron microscopic examination revealed the characteristic arrowhead configurations on the purified F-actin microfilaments after addition of the HMM obtained.
Colloidal gold (mean particle size, 20 nm) was synthesized as described [Frens G. (1973). Nature Phys. Sci. V.241,
P.2022], by reducing tetrachloroauric acid with sodium citrate. Conjugation was done by simple mixing of gold sols with
HMM (without any coupling agents). The protein concentration was determined by the gold number [Dykman L.A., Bogatyrev
V.A. (1997). Biochemistry (Moscow) V.62, P.350356]. The stability of the conjugate was judged by the absence of aggregation (solution-colour change from red to blue or grey) upon addition of 10% aqueous NaCl to a final concentration of 1%.
The obtained conjugate is assumed to explore for in vitro motility assays and as a marker for plant actin identification.
THE GRADUAL DETERMINATION OF THE VAN'T HOFF COEFFICIENTS IN THE TEMPERATURE
DIAPASON CHARACTERIZED THE AWAKENING OF THE GROUND SQUIRREL CITELLUS UNDULATUS
PALLAS FROM HIBERNATION
T.N. Kalabukhova
Institute of Cell Biophysics, Russian Acad. Sci.,
Pushchino (Moscow region), 142290, Russia
During the awakening of the ground squirrel from hibernation the increase of his body temperature up to 30 C (1),
the 10-fold increase of the frequency of breathing and heart beating (2) are registered along with the 10-fold increase in the rate
of the erythrocyte acid hemolysis evaluated from the chemical erythrograms in the differential form (3,4). The coincidence in
the tenfold increases mentioned above assumes the equality of the mean values of the van't Hoff coefficients for the rates of the
processes in vivo (2) and in vitro (3,4) in the temperature range 8-38 C upon hibernation.
So, the further gradual determination of the temperature coefficient Q 10i of the acid hemolysis of erythrocytes in the
ground squirrel Citellus undulatus Pallas, for the i-th temperature interval, namely 8-18-28-38 C, is a possible approximation
to study the changes of the Q10j of physiological functions, including for example, to investigate the modification of the Q 10j for
the biological motility in general and for its elementary processes also. The prognosis is based on the essential role of the protein component in the structure of the erythrocyte membranes (5) as well as on the data about the sharp transitions in the structure of protein molecules within the physiological temperatures diapason (6). Temperature changes in proteins both the structural ones and in the enzymes may be crucial for the biological motility.
Q10i of the acid hemolysis of erythrocytes for the i-th temperature interval is determined through the t50, i.e. the time,
when 50% of erythrocytes are hemolyzed. t50 is inversely proportional to the rate of hemolysis v50 (7). The former is calculated
from the erythrograms in the integral form.
References
1. Kalabukhov N.I. Hibernation of ground squirrels (Citellus guttatus). Trudy Lab. Eksp. Biol. Mosk. Zooparka.1926, v.1, 1-5.
2. Kalabukhov N.I. The Animal Dormancy. M.-L. :Biomedgiz. 1936, 204 p.
3. Kalabukhova T.N. The Van't Hoff Rule as Applied to Studying Different Phases of Hibernation of Ground Squirrel Citellus undulatus Pallas: Physicochemical Way of Cold Adaptation. Biophysics. 1998, v.43, 1027-1031.
4. Kalabukhova T.N. The Applicability of Van't Hoff's Law to the Investigation of Cold Adaptation. Proceedings of the XV Working
Meeting "Genetic Resource Conservation" (Pushchino, October, 13-15,1998), 1998, 122-125.
5. Structure and Dynamics of Membranes. Vol.1.From Cells to Vesicles, v.1a. Lipowsky, R. and Sackmann, E., Eds. N.Y.: Elsevier.1995, 520 p.
6. Bresler S.Ye. Molecular Biology. L.: Nauka. 1973, 577 p.
26
7. Chernitskii E.A., Senkovich O.A., Slobozhanina E.I. Parameters of Sodium Dodecyl Sulfate-Induced Hemolysis as Indicators of
Structural Changes in the Erythrocyte Membrane. Biophysics. 1999, v. 44, 1, 62-65.
STRUCTURAL-FUNCTIONAL RELATIONSHIP BETWEEN CHANGES IN MAIN PROTEIN OF THIN
FILAMENT, ACTIN, AND CONTRACTILE ABILITY OF HYBRID RECONSTRUCTED FIBERS OF
MYOCARDIUM IN ACUTE MYOCARDIAL INFARCTION (AMI)
N.V. Karsanov, G.V. Sukoian, D.R. Tatulashvili, N.E. Guledani, L.T. Kuchava
The Republican Research Centre of Medical Biophysics and Introduction
of New Biomedical Technologies (Head - Corr. member of Russian Academy
of Medical Science, Prof. Karsanov NV), Tbilisi, Georgia
Earlier it was shown that a sharp decrease in the ability of the myocardial contractile protein system (MCPS) to generate force has a great significance in the development of acute heart failure (AHF). In experiments with hybrid fibers obtained
from ghost myocardial fibers of ischemic and non-ischemic areas of myocardium with coronary artery occlusion and normal
myosin and visa versa was shown, that development of AHF and ability of myofibrils to generate force depends on actin behavior change (decrease its ability to polymerize and take force-generation conformation). Therefore it was found necessary to
investigate the nature of this phenomenon: specifically the role of structural-conformational changes in actin protomer during
contraction of myofibrils from myocardium with AMI in men.
Materials and methods. The work was carried out on 5 practically healthy persons (aged 35-65 years) who died from
acute several physical traumas and 5 persons (aged 35-75 years) who died from AHF caused by AMI. Methods of preparation
of skinned, ghost and hybrid myocardial fibers as well as actin and myosin preparations were described by Karsanov NV et al
(Exp. Clin. Pharmakol., 2000, N4, p.18-25). The methods of tensometric and ATPase measurements, labeling of actin and myosin by fluorescence probe has been done according to Sukoian et al (Bull.exp.biol.med., 1999, 4, 395-399). Obtained data
were expressed as mean  standard deviation. A commercial statistics software package (Microsoft Excel 7.0.) was used for
data analysis. Significant effects were examined using Student’s t-test to compare the groups.
Results. It has been shown that AHF development is associated with the sharp decrease of the ability of MCPS to
generate force and to hydrolyze ATP not only in infarcted, but non-infarcted areas of left ventricle, as well as of right ventricle
and both atrias which are not affected by ischemia – MCPS of the heart is damaged totally. It has been shown, that in AHF in
contradiction to early HF caused by 15 min CAO, the decrease of velocity and the value of generated force of MCPS from infarcted and nonifarcted areas of AMI is accompanied by uncoupling of functioning of force generation and ATP hydrolysis
centers in actomyosin ensemble (r=0.17 and 0.35, NS in case of MCHS from infarcted and noninfacted areas respectively; in
norm r=0.8, p<0.001; G = 8.1 + 0.2 tension) – disturbances in the energy transduction occurs in actomyosin ensemble, energy
transduction in MCPS disturbs as qualitatively as quantitatively.
It has been shown using hybrid myocardial fibers, that the decrease of the ability of MCPS to generate force is related
to actin. The value of force and Ca-Mg-ATPase activity decreases only when reconstructed fiber contains thin filament (with
or without troponin-tropomyosin complex) from patients with AMI and normal myosin and both parameters does not changed
significantly in case of thin filament from practically healthy persons and thick filament from myocardium of patients with
AMI. These results gave new evidence for leading role of structural-conformational changes of actin in the disturbances of
contractile activity of MCPS in patients with AMI. In AHF caused by AMI damages of the force generation centers, which are
localized in outer domain of actin protomer occur in parallel with conformational changes in inner domain of actin (which is
responsible for formation of actin filament [Karsanov NV et al, Basic Res Cardiol. 1986; 81:199-212; Karsanov NV, Jinchvelashvili BG. Gen.Physiol. Biophys.1990; 9:129-146]) and are characterized by change of configuration of external (outside)
domain of actin, disorientation of microenvironment of Cys374 and Cys10 of subdomain 1 and Lys61 and Tyr69 of subdomain
2 in actin - actin loses the ability to take force generation conformation. The distances between Lys61 and Cys374, Cys374 and
Cys10, Tyr69 and Cys374 are essentially increased as well as the correlation lifetime of fluorescence labels to Cys374 and
Tyr69. Formation of actin filament in AHF in contradistinction to norm doesn’t lead to essential change of reciprocal arrangement of appointed aminoacidic residues. Such conformational damages occur not only in the region of Cys374 and Lys61
(which takes place in early HF), but in the region of N-terminal (Cys10) and the cleft near the (Tyr69) as well.
Conclusion. It is concluded, that actin in AHF loses its ability to take conformation needed to generate force and
transduce energy economically in MCPS. These changes affect N-terminal part of actin molecule and excel in depth permitted
fluctuations without overcoming of energy barrier. However it can’t be excluded the existence of the possibility for their reversibility and they must be classified as posttranslational. The significant changes in conformation and function of myosin
head of thick filament have not established in AMI in infarcted and noninfarcted areas. Obtained data should shed the light on
the submolecular mechanism of AHF development and should serve the development of actin hypothesis of force generation
by MCPS.
DIFFERENCES IN EXITATION-CONTRACTION COUPLING MECHANISMS IN FROG AND LAMPREY
STRIATED MUSCLE
I.E. Katina, Y.V. Zhitnikova, G.A. Nasledov
Sechenov Institute of Evolutionary Physiology and Biochemistry,
St.-Petersburg, Russia
The major mechanism responsible for the excitation-contraction coupling (ECC) in skeletal muscle is the Ca 2+-release
from the sarcoplasmic reticulum (SR) through the ryanodine-sensitive (RyR) channels. However, inositol-1,4,5-trisphosphate
(IP3) receptors are present in skeletal muscle and it is possible that IP3-dependent mechanism participates in Ca2+-release from
SR. The purpose of this work is the comparative analysis of relative contribution of two pathways of signal transduction in activation of contraction in intact fast striated muscles of the frog (R. temporaria, m. Extensor digitorum longus IV) and of the
lowest of vertebrates, the lamprey (Lampetra fluviatilis, m. longitudinalis linguae and m. Hyomandibularis glossus).
27
The alterations by selective blockers of RyR-channels – tetracaine (TC, 10 –250 mkM) and dantrolene (DT, 100-300
mkM) in parameters of contractile responses, elicited by short electric stimuli (twitch, Tw), by elevation of external potassium
concentration (120 mM, K-contr.), by external solution containing acetylcholine (ACh, 0.01-0.1 mM, ACh-contr.), or by caffeine (1-10 mM, caff.-contr.), were examined. It was shown that both agents (TC and DT) display doze-dependent inhibiting
action on all types of muscle contraction of both species. Use of these drugs in concentrations more than 150 mM-200 mkM
caused the complete suppression of muscle contractile activity, including caffeine contracture, supporting that they selectively
affect the RyR-channels and that EC-coupling system based on functioning of RyR Ca-release channels is the major mechanism in skeletal muscles of both investigated species. Definite differences in dynamics of development and stationary level of
effect were revealed using sub-maximal concentrations of drugs.
Dantrolene. The treatment of frog muscle with 150 mkM of DT caused the reduction of Tw amplitude to 0.1 of the
control value (Re, related inhibiting effect), while for all types of contractures the extent of inhibition was less significant:
Re=0.35-0.4. In the lamprey muscle application of 150 mkM DT reduced Tw amplitude to 0.25, but in contrast to the frog
muscle, all contractures were more sensitive to DT: 0.05 (K-contr.), 0.13 (Ach-contr), 0.2 (caff.-contr.).
Teteracaine. In lamprey muscle the fastest and quantitatively most prominent effect of TC (30-100 mkM) was on Kcontracture. The sensitivity to TC-action of examined types of contractile responses decreased in a sequence: K-contr.>AChcontr>Tw>caff.-contr. The values of related inhibitory effect for 50 mkM TC were: 0.20.03 (Tw), 0.050.04 (K-contr.),
0.130.05 (Аch-contr.), 0.250.08 (caff.-contr.). Unlike lamprey muscle, frog muscle twitch contraction and ACh-contr. were
most sensitive to the action of TC. The sensitivity row was: ACh- contr.>Tw>K- contr.>caff.- contr., and the Re values (50
mkM) were: 0.080.01 (Tw), 0.340.05 (K-contr.), 0.080.03 (Аch-contr.), 0.500.08 (caff.-contr.).
It is known that TC as a local anesthetic is able to act on different parts of EC-coupling system. In parallel with an inhibition of RyR-channels, it can block voltage-dependent Na- and Ca- channels, and ACh-operated channels of plasma membrane. To separate two types of TC effects, some of experiments were performed in the presence of tetrodotoxin (TTX, 1
mkM) and nifedipine (50 mkM). It was shown that addition of selective blockers of Na- or Ca-channels caused the suppression
of twitch, but did not induce the compatible reduction of ACh- or K- contr., and did not affect the TC effectiveness. Thus, it is
possible to propose that TC should have more prominent effect on twitch and ACh- contr. in comparison with K- and caff.contractures. The observed relation of sensitivity of frog muscle twitch, ACh- and K-contr. is fully consistent with this scheme.
Meanwhile on lamprey muscle ACh- contr. and twitch are suppressing by RyR-channel blockers TC or DT in a smaller degree
than K- contr.
It is known that lampreys muscles possess some primitive properties as compared with muscles of other vertebrates,
and among them – high density of ACh receptors. Supposingly, in lamprey muscles the interaction of ACh with the receptor
and/or fast membrane depolarization activates supplementary system of Ca release from the SR, uncoupled with the RyRsensitive channels, in particular via IP3-operated channels.
This work was supported by RFBR grant № 99-04-49953.
ACTIN DYNAMICS DURING BACTERIAL INVASION OF EUKARYOTIC CELLS
Sofia Khaitlina, Tatiana Efremova and Yan Komissarchik
Institute of Cytology RAS, 194064 St.-Petersburg,
E-mail: skh@mail.cytspb.rssi.ru
One of the exciting models to study actin filaments dynamics is invasion of eukaryotic cells with bacteria. Adhering
the cell surface enteropathogenic bacteria enter the cell either by interaction of a specific bacterial protein with a eukaryotic
surface receptor followed by the bacteria internalization via the mechanism of signal transduction or by the bacteria-induced
localized membrane ruffling in which the bacterium is trapped and internalized via the process resembling macropinocytosis.
Both processes involve actin polymerization and cytoskeleton rearrangements. Once intracellular, bacteria Listeria monocytogenes and Shigella flexneri escape from vacuole and form at their apical ends a comet-like tails of actin filaments used for intra- and intercellular movement which rate is equal to the rate of actin polymerization. It is shown that actin-based propulsion
is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin, activated Arp2/3 complex, actin depolymerizing factor (ADF/cofilin) and capping protein are required for motility.
Previously we have shown that bacteria of spontaneously isolated non-pathogenic strain E.coli A2 produce a new
metalloprotease (protease ECP 32) that specifically cleaves actin. Similar protease activity was found in mutants of Shigella
flexneri. It turned out that bacteria producing protease ECP32 can invade eukaryotic cells and form a bacterium-associated
bundle of actin filaments whereas the corresponding strains of E.coli and Sh. flexnery that do not produce ECP 32 were not
taken up by the cells. We assume, therefore, that protease ECP 32 may be a factor involved in invasion of eukaryotic cells by
these bacteria. If so, a poor polymerization of the cleaved actin might be of a primary importance. The ECP 32 cleaved actin
does not polymerize if it contains Ca2+ as a tightly bound cation. The ECP 32 cleaved actin containing tightly bound Mg 2+ can
polymerize, but with a lower efficiency than intact actin. Efficiency of the cleaved actin polymerization can be further affected
by gelsolin and myosin S1 promoting its nucleation. It is possible, therefore, that bacterial factors that are the strong nucleators
of actin polymerization may target localization of actin filaments in a manner required for invasion.
The work was supported by RFBR grants 99-04-49482 and 00-04-49480.
28
THE CHANGES IN REGULATORY LIGHT CHAIN CONTENT
OF CARDIAC MYOSIN IN RHEUMATISM OF MYOCARDIUM
Ya.N. Khalina, I.M. Iljinsky**, Z.I. Vishnevskaya*, Z.A. Podlubnaya
Institute of Theoretical and Experimental Biophysics RAS,
Pushchino, Moscow region, 142290 Russia,
E-mail: khalina@venus.iteb.serpukhov.su;
*
Institute of Cell Biophysics RAS, Pushchino, 142290 Russia;
** Scientific Research Institute of Transplantology and Artificial Organs,
Ministry of Health of Russian Federation, Moscow, Russia
Light chains (LCs) of myosin play an important role in muscle contraction. Both essential LCs (ELC, LC1) and regulatory LCs (RLC, LC2) of cardiac myosin differ by their molecular weight from those of smooth and skeletal myosins. LC1s in
human myocardium contribute to the regulation of force generation and its Ca2+-sensitivity (1). LC2-deficient myosin is typical
for embryonic myocardium myosin and in this case a myofibrillar disorder is observed. In the postnatal period of ontogenesis
the ratio of LC1:LC2 becomes 1:1 remaining constant in norm. The loss of LCs can cause structural and functional disorder
(1). If compensatory factors are ineffective, the appearance of LC2-deficient myosin can lead to heart failure. This phenomenon has already described for a number of cardiac diseases. For example, in dilated cardiomyopathy (DCM) the LC1:LC2 ratio
in myosin preparations varies from 1:0.1 to 1:0.69 (2). It is known that the LC2 is more sensitive to a process of proteolytic digestion than LC1. In pathology the rate of this process increases due to the activation of proteolytic enzyme mecretin (2).
The goal of this study is to estimate the changes in cardiac myosin LC2 content in rheumatism of human myocardium.
For this purpose comparative analysis of myocardial samples and purified myosin was carried out.
The autopsies of the heart with rheumatic damage were studied. All myosins and myocardial samples were obtained
from right and left atria and ventricles. Analysis of LC content was performed by SDS-PAGE with subsequent densitometry of
the gels to determine the molar ratio of LC1:LC2. The molar ratio of LC1:LC2 in the purified preparations of myosin from
porcine heart was used as control. Comparative electrophoretic analysis of purified myosins and myocardial samples allowed
to determine the content of LC2 bound to myosin and free LC2 in the cytosol.
In norm, the molar ratio of LC1:LC2 for all heart sections averages 1:1 for purified myosin and 1:1.2 for myocardial
samples (i.e. 83% of LC2 are in the bound form and 17% are in free form).
The content of LC2 in "pathological" myosin was reduced considerably. The molar ratio of LC1:LC2 lowers up to
1:0.9 and 1:0.85 for left ventricle and atria, respectively. In contrast, for myocardial samples the content of LC2 increased in
pathology. LC1:LC2 ratio was 1:2.2 and 1:1.5 for left ventricle and atria, respectively. In addition, the distribution of free and
bound forms of LC2 was calculated for myocardial samples. For free and bound forms of LC2 in left ventricle it averaged 59%
and 41%, correspondingly; for free and bound forms of LC2 in atria - 43% and 57%, respectively.
Our data indicate on the marked abnormalities in LCs ratio in pathology due to the increase of free form of LC2 and
the decrease of the content of bound LC2 to myosin. Analysis of purified protein shows that "pathological" myosins are LC2deficient. Atrial myosin contains 85% of LC2 in comparison with the norm, and ventricular myosin - 90 % of LC2. Thus, the
amount of free fraction of LC2 increases in pathology. This increase varies for different heart sections. Samples from left ventricle demonstrate more pronounced changes as compared with norm than atrial samples (59 % and 43 % of LC2 in free form,
respectively).
On the basis of our results it is impossible to explain a reason of the appearance of considerable amount of free LC2
in”pathological” myocardial sample studied by us. However, we cannot rule out an additional expression of LC2. According to
the available data (database of MDC), in human ventricle an overexpression of LC2 up to 220% has been revealed in DCM.
The investigations in this line are in the progress.
The work is supported by RFBR grant № 01-04-48195.
References
1. Morano I., 1999. J.Mol.Med.,77:544-555
2. Margossian S.S. et al., 1992. Circulation, 85:5, 1720-1733
REGULATION OF THE CROSS BRIDGE CYCLING
IN THE SMOOTH MUSCLE: A ROLE OF MGADP
Alexander Khromov, A.V. Somlyo and A.P. Somlyo
University of Virginia, Charlottesville, USA
The experimental data concerning a possible role of MgADP in formation of a latch state, i.e. the ability of the tonic
smooth muscle to maintain substantual force at low (near basal) level of MLC20 (myosin light chains 20 kDa) phosphorylation
will be presented. An increased population of dephosphorylated cross bridges remaining bound to actin after completion of the
individual force generating act (power stroke) are thought to be the major determinant of the latch state. The data supporting
the hypothesis are: 1. Smooth muscle binds MgADP much stronger than skeletal ones (Kd 1-5 microM vs 80-200 microM), 2.
The latch state can be simulated in the permeabilized tonic, but not in phasic smooth muscle strips at the conditions close to
physiological, 3. At the same amplitude of a developed force (which is proportional to number of the cross bridges interacting
with actin) the velocity of unloaded contraction is lower in smooth muscle strips having reversibly phosphorylated MLC20, rather than irreversibly (thiophosphorylated). 4. An affinity of cross bridges to MgADP can be affected by phosphorylation of
MLC20. A molecular basis for the difference between tonic and phasic smooth muscle is related probably to the differences in
the heavy chains (7-amino acid insert) and/or isoform composition of the essential light chains (17 kDa) of smooth muscle myosin.
NUCLEOSIDE DIPHOSPHATE KINASE/NM23:
GENERAL PROPERTIES, TUMOR METASTASIS SUPPRESSION AND CELL MOTILITY
29
Narimichi Kimura1, Mitsugu Fukuda1, Akio Ishii1, Nobuko Shimada1,
Naoshi Ishikawa1, Yohko Takagi1 and Yasushi Ishijima1,2
1) Departments of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo173-0015;
2) the Domestic Research Fellow, Japan Science and Technology Corporation (JST),
4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Nucleoside diphosphate kinase (NDPK)/nm23 is a ubiquitously distributed, housekeeping enzyme involved in the
high energy transfer to produce nucleoside triphosphates pools at the expense of ATP. Metastasis suppression by nm23/NDPK
has been reported for cultured tumor cell lines of various origins, such as human breast carcinoma, human squamous cell carcinoma, rat mammary adenocarcinoma, murine melanoma and murine colon adenocarcinoma. However, questions as to which
step of multiple overall blood-borne metastases is suppressed by NDPK/nm23 and how NDPK/nm23 suppresses metastatic
properties within cancer cells remain largely uncertain. We have addressed these issues using a rat mammary adenocarcinoma
cell line, MTLn3, and a human squamous cell carcinoma cell line, LMF4. MTLn3 cells metastasize to the lung at a high frequency when implanted into mammary fat pads of syngeneic female F344 rats. Transfection of NDPK- cDNA, not NDPK-
cDNA, into MTLn3 cell diminished the pulmonary metastatic potential with no essential changes in lymph nodes metastasis.
The metastasis suppression was verified by counting lung foci after intravenous injection of transfected clones into nude mice.
Hence, the anti-metastatic action of NDPK- is likely to take place during the latter part of the entire metastatic process, presumably, from extravasation through secondary tumor formation. The highly metastatic MTLn3 cells displayed chemotactic
cell motility towards the lung extract, which was markedly decreased by NDPK- transfection. The altered metastatic ability
of the NDPK- - transfected clones was not attributed to changes in either secreted matrix metalloproteinases (MMP2 and
MMP9) or adhesion potential to endothelial cells. On the other hand, transfection of nm23-H2 into a metastatic human oral
squamous cell caricinoma (SCC) cell line, LMF4, caused reduction in the lung metastasis in an experimental metastasis assay.
The control and transfected cells were morphologically indistinguishable but differed each other in the proliferative potential,
serum-dependency and responsiveness to growth factors such as PDGF, IGF-1 and insulin. These findings suggest that
NDPK/nm23 is likely to alter the responsiveness of metastatic tumor cells to extracellular ligands, resulting in modification of
metastatic cell phenotype(s) and suppression of their distant metastases.
EFFECT OF HYPOTHYREOSIS
ON THE STRUCTURAL STATE OF F-ACTIN
IN SLOW (SOL) AND FAST (EDL) RAT SKELETAL MUSCLE
V.P. Kirillina1, A. Jakubiec-Puka2, I. Ciechomska2 and Yu.S. Borovikov1
1Institute
of Cytology, Russian Academy of Sciences, 194064, St.Petersburg, Russia;
Institute of Experimental Biology, Polish Academy of Sciences,
02093, Warszawa, Poland
2Nencki
It is well known that thyroid hormones affect the structural and functional properties of skeletal muscle. They accelerate muscle contraction and enhance tension development. Furthermore, thyroid hormones change the physiologic properties of
both the skeletal and cardiac muscle (Salviati et al., 1985, Muscle & Nerve, v. 8, p. 363). These changes have been correlated
with the genes expression of specific isoforms of myosin molecule under hormones stimulus (Li, Larson, 1997, J. Muscle Res.
Cell Motil., v. 18, p. 335). Thyroid hormones have profound effects on a variety of metabolic functions, including O 2 consumption, heat production, glycolysis and proteolysis (Zeman et al., 1986, Biochem. J., v. 240, p. 269). However, there is no
sufficient evidence of the influence of thyroid hormones on the interaction between the basis contractile proteins of myosin and
actin or of their effect on thick and thin filaments structure. The consequence and molecular mechanism of their interaction are
still poorly understood.
We studied the effect of hypothyreosis on the structural state of F-actin from slow (SOL) and fast (EDL) single ghost
skeletal muscle fibers by polarized microfluorimetry techniques. Female albino Wistar rats 3 months old were used. Hypothyreosis was generated by surgical thyrectomy and then propilthyouracil was given in potable water (0.04%) for 2 and 5 weeks.
Subfragment-1 (S1) from rabbit m. psoas was obtained according to Okamoto, Sekine (1985, J. Biochem. v. 98, p. 1143). Thin
filaments of ghost fibers were labeled with fluorescent probes attached to different areas of actin. These sites were: Cys-374
labelled with 1,5-IAEDANS (N-iodoacetyl-N’ (5-sulpho-1-naphtyl-ethylenediamine), TRITC-phalloidin was located in filament groove.
The conformational changes in F-actin were monitored by calculating the values of ФE and angular distribution 1/2.
The changes in the value of ФE were interpreted in terms of structure alterations of F-actin. The value of 1/2 reflects of the
flexibility of actin filament (Szczepanowska et al., 1987, Eur, J. Cell Biol., v. 43, p. 394). Binding S1 to F-actin from control
SOL and EDL muscle fibers induces marked changes in these parameters for fluorescent probes located at different regions of
actin. Thus, the orientation and mobility of fluorescent probes were changed significantly when actin and myosin interacted,
depending on fluorophore location. For the TRITC-phalloidin ФE decreased upon S1 binding. Opposite the changes were
found for 1,5-IAEDANS probes, located peripherally where ФE was increased at S1 binding.
These effects were inhibited significantly by hypothyreosis. It should be noted that in fast (EDL) muscles the maximum changes in F-actin structure are observed after 14 days. In contrast, in slow (SOL) muscles maximum effect was shown
after 34 days. Besides, it was found out that hypothyreosis increased the flexibility in thin filaments in both SOL and EDL
muscle fibers. The data obtained suggest that the changes in the conformation of F-actin molecule, induced by hypothyreosis,
modulate the character of actin-myosin interaction in both fast and slow skeletal muscles.
This work was supported by Russian Foundation for Basic Research, grant № 01-04-49310 (Y.B.).
THE PK CELL SPREADING IN NORMAL CONDITIONS AND IN THE PRESENCE OF THE BREFELDIN A
O.P. Kisurina-Evgenieva, I.N. Nifontova, G.E. Onishchenko
30
Department of Cytology and Histology, Biology Faculty,
Moscow State University, Moscow, Russia
We investigated the dynamics of the cell spreading on a solid substratum in normal conditions and in the presence of
the brefeldin A in order to analyze the role of normal function of the Golgi apparatus. The monolayer of cells was treated with
brefeldin A in concentration of 0,5 mkm/ml for 18 hours while a short-term treatment caused disassembly of the Golgi apparatus in single cells, but not in a monolayer. Disassembly of the Golgi apparatus was checked up by the staining of cells with
the C6-NBD-Cer (0,01mM).
Ultrastructural studies of suspended cells have shown that brefeldin A induced the disappearance of the Golgi apparatus and the extension of the endoplasmic reticulum. The endoplasmic reticulum forms both the swelled and spiral channels,
which are deprived of the ribosomes. Two types of vesicular structures, localized distantly from the endoplasmic reticulum,
were detected in the cells: the vesicles around the centrioles and the vesicle with the dense content. We suppose, that the first
type of the vesicles can be a part of the endosome compartment, while the second type – of the pre-Golgi compartment.
The investigation of the cell spreading dynamics has been performed by computer image analysis of these cells. In the
range from the cells plating to 24 hours, three stages of the cell spreading could be distinguished according to the dynamical
change of the average projected cell area: spreading rapidly at the 1st stage (0-1 hours; 128-274 mkm²), slowing down of the
spreading at the 2nd stage (1-6 hours; 273-312 mkm²) and pronounced but very slow spreading at the 3d stage (6-24 hours; 312753 mkm²). Brefeldin A slows down the process of cell spreading at the1st stage (0-3 hours; 180 mkm²; 3-4 hours; 180-328
mkm²) and at the 2nd stage (4-6 hours; 328-273 mkm²), and inhibited the cell spreading at the 3d stage (6-24 hours; 237-360
mkm²). In the presence of brefeldin A at all stages of spreading, the cells with the area in the range from 100-300 mkm² dominated.
Thus, in the present work it was shown, that attachment and initial spreading of the PK cells do not depend on the
state of the Golgi apparatus while normal function of the Golgi apparatus is necessary for the following spreading.
Supported by RFBR grant № 00-04-48718.
RYANODINE RECEPTOR-SPECIFIC DRUGS MODIFY CONTRACTILE AUTOOSCILLATIONS IN PHYSARUM
PLASMODIUM
A.A. Klueva, N.B. Matveeva, V.A. Teplov, S.I. Beylina
Institute of Theoretical and Experimental Biophysics RAS,
Pushchino, Moscow region, 142290 Russia
E-mail: beylina@venus.iteb.serpukhov.su
Cells have two intracellular channels for regulating the calcium release from internal stores, the ryanodine receptor
(RyR), first discovered in muscle but now known to exist in other cell types, and the inositol 1,4,5 trisphosphate (IP 3) receptor
[1]. A possible role of these channels in the contractile auto-oscillations of the Physarum plasmodium has not been yet established but some evidence for their existence in this large multi-nuclear low-eukaryotic cell has been already obtained. In particular, a caffeine-induced decrease of Ca2+ level in the plasmodial calcium stores detected as a drop of chlortetracycline fluorescence [2] suggests the presence of RyR, which is the intracellular channel modulated by ryanodine and activated by caffeine. In this work we investigated the effects of agents, commonly used for studying RyR, on the contractile activity oscillations in the plasmodium.
Physarum polycephalum was grown in a liquid shaken culture as a suspension of the microplasmodia, multinucleate
cells 100-200 µm in diameter [3]. Microplasmodia were harvested at the logarithmic phase of growth, resuspended in a nonnutritional medium and used for assays during first 6 hours of starvation. Cyclic contractions of microplasmodia and pulsating
movement of its leading edge were registered optically [2].
Caffeine-induced changes in the contractile and motile activities were relatively complex: at concentrations of 10-20
mM, caffeine caused a transitory increase in the oscillation frequency and velocity of protoplasmic streaming. This was often
followed by the disappearance of oscillations for 10-30 min accompanied by the contraction of microplasmodium and its detachment from the substrate. After renewal of the oscillations, their frequency was usually lower than that in the control. At 50
mM caffeine, the contractile activity rapidly and irreversibly stopped. The effects of caffeine on the contractile activity were
abolished in the presence of 50 mM procaine, a RyR inhibitor. Caffeine effects were totally or partially suppressed also after
pretreatment of the microplasmodia with 50-100 µM ryanodine, an agent capable of both opening the closed channels of RyRs
and closing the open ones [4].
In contrast to sea urchin eggs, where no change in the pattern of calcium waves was observed when Ca 2+ channels of
each type were inhibited (the waves were abolished only when both RyR and IP 3 receptor were knocked out) [5], in the
Physarum plasmodium procaine and ryanodine had an influence on the auto-oscillatory behavior. In about 50% of microplasmodia, a temporal strong decrease of the amplitude and a loss of the oscillation regularity were observed under the action of
procaine. The application of ryanodine caused a change of the oscillation frequency in about the same fraction of the microplasmodial population.
The results obtained in this study support the suggestion on the existence of RyR in Physarum plasmodium. The active state of this type of intracellular channel is not obligatory for the contractile oscillations, but ryanodine receptors are most
probably engaged in their regulation, at least under certain physiological conditions.
This work was supported by The Russian Foundation for Basic Research.
References
1. Berrige M. 1993, Nature 365, 388-389.
2. Kochegarov A. A., Beylina S. I., Matveeva N. B., Leontieva G. A., Zinchenko V. P. 2000, Biochemistry (Moscow) 65, 662-671
3. Daniel J. W., Baldwin H. H. 1964, in Methods of Cell Physiology (ed. Prescott J.) Academic Press, New York, 1, 9-41.
4. Chen T. H., Lee B., Yang C., Hsu W. H. 1996, Life Sci. 58, 983-990.
5. Galione A., McDougall A., Busa W. B., Willmott N., Gillot I., Whitaker M. 1993. Science 261, 348-352.
31
MATHEMATICAL DETAILS OF PASSIVE STRESS-STRAIN MODELING OF PAPILLARY MUSCLE
R.M. Kobeleva, A.V. Kobelev, Yu.L. Protsenko
Ekaterinburg Branch of the RAS Institute for Ecology and Microorganism Genetics
The problem of passive stress-strain curves is important in biomechanics of myocardium. The great deal of experimental data shows non-linearity of these dependencies, with the stiffness increase at loading. The previous investigations using
Maxwell-Foigt models to describe these features use some artificial assumptions to separate ranges in which elements with rigid or soft elements work. As computation methods in mathematical modeling concern, the expansion in powers of deformation
was used, which is not adequate to reveal some features, the more so, as the transversal deformation in many cases is not small.
The purpose of the present study is to find exact solutions of the elastic equations in static regime neglecting viscosity
for a set of 2D topological structures consisting of 3 kinds of primary Hook elements: longitudinal, transversal, inclined, or
cross-connected. In accordance with experimental conditions of stress-strain curves measuring on separated muscle sample
with firmed ends, the stretching force is applied to rigid bar of diameter h, to the edges of which the primary elements are connected. All these and other junctions of primary elements are assumed to be frictionless. Despite the linearity of primary Hook
elements in which the force is a linear function of stretching, it is obvious that the structure as a whole may be non-linear due
to its geometry.
The transversal dimension in the center of 2D structure may be either greater, or smaller than h, that does not change
the topology of the model and the resulting computational formulae. So we consider consequently 13 models, with 4 of them
topologically equivalent to the others. For computational convenience, h0 in some models. In every, but one, of the models
the explicit exact expressions for the force as a function of deformations of all elements and dependencies of deformations on
each other were derived. In one case no explicit solution was found.
The simplest models with two cross-connected elements have slightly non-linear stress-strain dependence with steeper
run at loading, caused by trigonometric function. More complicated models with springs of three kinds: inclined, longitudinal
and transversal have more steep characteristics. It was disclosed that the origin of the stiffness increase at loading is the “collapse” effect when inclined elements gain longitudinal direction. The ranges of elastic parameters and geometry dimensions in
which this effect of non-linearity is most pronounced are analyzed for all models.
The results obtained can be generalized on the 3D case, which is more close to real objects, with the help of rotation
of the structure along the longitudinal axis. After this procedure the deformation picture become clearer and give possibility of
animation in real dimensions.
PASSIVE FORCE – DEFORMATION NON-LINEAR STATIONARY DEPENDENCIES FOR MYOCARDIUM
R.M. Kobeleva, Yu.L. Protsenko, A.V. Kobelev, S.M. Routkevich
Ekaterinburg Branch of the RAS Institute for Ecology and Microorganism Genetics
The relation between passive force and length in the physiological range of deformations (0.3) is the determining
feature in the studying of contractility of myocardium. The primary elements of muscle connective tissue structure such as collagen filaments and titin in the frame of connective tissue can be treated mechanically as Hook elements in which passive force
linearly depends on extension. The morphological structure of both cardiomyocites and its environment connective frame is a
lattice with rather sophisticated topology. This is possibly the reason for elastic non-linearity of isolated myocardium tissue
and a heart in total.
At present there is a lack of the functional rheological model that describes experimental stress-strain data in the
whole range of deformations. The existing models are simply the 1D combinations of elastic and viscous elements, or at least
are processed in trivial linear range for determining elastic module.
The aim of the study focuses on the development of 2D topological structure to describe passive force – length dependencies for myocardium. The primary Hook elements are specified by their lengths and elasticity coefficients. Neglecting
viscosity, it is assumed, that the variety of elastic structures can be deduced from three types of primary elements. The nonlinearity of the model is due to its geometry. Nine topologically different kinds of such models are considered and corresponding stress-strain curves are obtained. Two asymptotic linear parts of these dependencies are characteristic to these curves in
definite range of parameters.
The measurements of applied stretching force as the function of sample length on rabbit and cat papillary muscle are
conducted in the stationary regime by method of opto-electron device with TV monitoring, described elsewhere. The nonlinearity of these dependencies was revealed, which demonstrated the increase of stiffness at loading.
The most appropriate model was chosen to describe all experimental data. In the framework of this model Young
modules of three primary elements were estimated in the assumption that their transverse - longitudinal length ratio equals 0.1.
The "local" Young modules responding to initial and final parts of stress-strain curve are not equal to the values of elastic constants of "soft" an "rigid" elements, however depending on them and geometry of the model.
As an example we present here best-fit values of elastic parameters for the sample of papillary muscle of cat 3.25 mm
long and 0.9 mm in diameter: longitudinal deformation =0.22; "local" initial and final Young modules Esta=2.45 G/mm2,
Efin=9.11 G/mm2; three primary elements Young modules: inclined E 1=110 G/mm2, longitudinal E2=16 G/mm2, and transversal
E3=2.7 G/mm2.
Such approach shows that geometrical factor can play significant role in understanding mechanisms of strain-stress relationships in myocardium. The range of obtained elastic modulus is in a good concordance with data obtained by another investigators. This give reasons to make identification of model elements with protein of muscle connective tissue.
PARTICIPATION OF CYTOSKELETAL ELEMENTS IN THE TRANSCELLULAR WATER TRANSPORT
Ya.Yu. Komissarchik, E.S. Snigirevskaya
Institute of Cytology RAS, St.-Petersburg, Russia
32
A series of structural investigations has shown that increase in the water flow through epithelial cells of the anura urinary bladders under antidiuretic hormone (ADH) action was connected with the insertion of water channels into the apical
membrane. The specific cytoplasmic structures are supposed to be the sources of this kind of channels (1,2). The migration
through the cell and fusion of these structures with the apical membrane depends on the state of the cytoskeleton (3). Cytoskeletal elements: microtubules, microfilaments and intermediate filaments are revealed along the whole cell without strong localization. Only the cortical layer of actin filaments is located exactly under the apical membrane. Especially well this layer is
saved with freeze-substitution method. However, these data, especially on the cortical layer of apical microfilaments are still
contradictory.
The main goal of this work is to analyse the structural changes of the apical microfilament layer of the frog urinary
bladder granular cells after stimulation of the water transport with vasopressin or repeated changes of Ringer solution at the serosal side with electron and confocal microscopy. In the granular cells of the control halves of the bladder the dense layer of
microfilaments is often localized parallel to the apical membrane. This cortical layer seems to prevent the contacts of specific
granules with the apical membrane. The microfilament bundles are well revealed in microvilli. Under the stimulation of water
flows, along with the preservation of microfilaments in microvilli, a significant decrease in the distribution density of microfilaments below the apical membrane is revealed. In this case, most granules are in a tight contact with the apical membrane.
Immunocytochemical analysis of actin distribution using monoclonal antibodies against actin (Boehrenger Mannheim) confirmed the actin nature of the thin apical filaments and evaluated the actin distribution density changes under different conditions of the water transport stimulation. In the control bladder halves the immuno-gold is localized both in microvilli and between them with a relatively high density. Under the water transport stimulation, the significant decrease of the gold particle
density between microvilli is observed. Inside microvilli the intensity of the gold labeling is actually not changed. The study of
the actin localization in confocal microscope with rhodamine-phalloidin indicates a significant redistribution of the apical microfilaments. Unlike the thin fibrillar net in the control bladder halves, the rather rough net with big meshes is revealed under
the water transport stimulation. Thus, the investigation shows involvement of the submembrane actin cytoskeleton in the regulation of the epithelial cell water permeability under both the ADH-action and the extraction of autacoids by the repeated
changes of Ringer solution at the serosal side (4).
The work is supported by the projects № 99-04-49554 and 00-04-49480 of the Russian Foundation of Fundamental
Research.
References
1. Wade JB (1985) Federation Proc 44: 2687-2692.
2. Komissarchik YaYu, Snigirevskaya ES (1990) Biol.Membr 7:895-908.
3. Ding G, Franki N, Condeelis J, Hays RM (1991) Am J Physiol 260:C9-C16.
4. Komissarchik YaYu, NatochinYuV, ShakhmatovaEI, Brudnaya M S ,
5. Snigirevskaya. ES, Korolev EV, Parnova RG (1996) Tzitologiya 38,1:14-21
STRUCTURE OF TROPOMODULIN,
ACTIN FILAMENT CAPPING PROTEIN
A.S. Kostyukova 1,2, I. Krieger 1, T. Fujisawa 1,
E.I. Tiktopulo 2, A. Yamashita 1 and Y. Maeda 1
1
Laboratory for Structural Biochemistry, RIKEN Harima Institute,
1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan;
 Institute of Protein Research, Russian Academy of Sciences,
Pushchino, Moscow Region, 142290, Russia
Tropomodulin (359 amino acids, 40 kDa) is the only capping protein for the slow growing end (P-end) of actintropomyosin filaments. It is known that the N- and C-terminal halves of tropomodulin play distinct roles; the N-terminal half
interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. In the present study,
three types of recombinant fragments of chicken E-type tropomodulin were prepared and used for experiments by methods of
circular dichroism, small angle X-ray scattering and differential scanning calorimetry. Tmod (N39) (a.a. 1-344) lacking 15 residues at the C-terminus of the authentic molecule, and the N-terminal fragment N11 (a.a.1-91) were both expressed in E.coli
with histidine (6 residues) tags at the N-termini. A C-terminal fragment, C20 (a.a.160-344), was obtained from Tmod (N39) by
limited proteolysis. It was found that the N- and C-terminal halves are also structurally distinct from each other. The Cterminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. C20 was
crystallized and its crystal structure was solved. It represents right-handed super-helix of altering -strands and -helices. In
contrast, the N-terminal half is elongated and lacks a definite tertiary structure in solution. The binding of N11, a fragment that
corresponds to the first quarter from the N-terminus, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and
that the N-terminal half acquires its tertiary structure upon binding to tropomyosin. A new model of tropomodulin – tropomyosin – actin complex formation was suggested.
QUANTITATIVE INTERPRETATION OF THE X-RAY DIFFRACTION PATTERN FROM MUSCLE
Natalia A. Koubassova1, S.Y. Bershitsky2, M.A. Ferenczi3 and A.K. Tsaturyan1
1Institute
of Mechanics, Moscow Lomonosov State University, Russia;
of Genetics and Ecology of Micro-Organisms,
Ural Branch of RAS, Yekaterinburg, Russia;
3Biomedical Sciences Division, Imperial College
of Science, Technology & Medicine, London, UK
2Institute
Low-angle X-ray diffraction has been used for studying molecular structure of muscle for half a century (H.E. Huxley, 1996). Since very bright and highly collimated synchrotron X-ray beams and modern 2D X-ray detectors became available
for muscle research, the X-ray diffraction provides higher spatial (Linari et al., 2000) and time (Dobbie et al., 1998) resolu-33
tion than other structural methods. However quantitative interpretation of the X-ray diffraction data is not straightforward. The
phases of the reflections are unknown, so direct Fourier synthesis of the electron density is impossible. On the other hand, high
resolution structures of F-actin, myosin head (S1) and their complex, actin-S1, have been determined recently using protein
crystallography, fibre diffraction and electron microscopy (Holmes et al., 1990, Rayment et al., 1993a, 1993b). These structures can be used for building 3D models and for comparison of calculated and observed X-ray diffraction intensity. This approach has been successfully used to study structure of the myosin filament in relaxed muscles of different species (Malinchik,
Lednev, 1992; Hudson et al., 1997; Malinchik et al., 1997).
We tried to expand this method on more complicated, but more interesting structures formed in muscle when myosin
heads interact with actin in rigor or during active contraction. 3D structural model was developed to calculate 2D low angle Xray diffraction pattern from skeletal muscle with the aim of quantitative interpretation of the X-ray diffraction data, particularly
to estimate the fraction n of myosin heads stereo-specifically bound to actin during isometric contraction.
Published structures of acto-S1 were used for modelling the actin-myosin super-lattice in the overlap zone. To reduce
the number of parameters, which determine the actin binding pattern, the ‘principle of minimal elastic distortion energy’ was
employed. According to this principle, a myosin head chooses the actin monomer on one of six surrounding thin filaments for
which binding requires minimal elastic distortion energy. Thus, the binding of up to 270 myosin heads to actin in a unit cell is
determined by two parameters: n and e (= the ratio of transversal and axial stiffness of a myosin cross-bridge). The model provides a good fit of the 2D X-ray diffraction pattern from bundles and single fibres from rabbit and frog muscles in rigor (n =1)
without recourse to global parameter search. The stiffness ratio e and parameters which determine axial and transversal lattice
disorder of the 1-st and 2-nd kind can be estimated from observed intensities of the individual layer lines.
The total off-meridional intensity of the 1-st actin layer line, A1, was found to be independent of lattice sampling and
tilting of the light chain domain of the myosin heads. For this reason, the A1 intensity can be used as a reference for quantitative data analysis. Model calculations show that dependence of the A1 intensity on n does not obey the square law of intensity.
The sum of the total off-meridional intensities of A1 and the ‘beating’ actin-myosin layer line 9 (LL9) at ~(24 nm)-1 is found to
be approximately proportional to n and independent of e.
General theoretical analysis demonstrates that the square law is not valid for a layer line intensity diffracted by any
partially occupied helix due to contribution of the Bessel functions which do not satisfy the helical selection rule.
The fraction of stereo-specifically bound heads, n, during isometric contraction at different temperatures was estimated from time-resolved X-ray diffraction T-jump experiments with small (3-5 fibres) bundles from rabbit psoas muscle at beam
line 16.1 of the SRS at Daresbury, UK. The T-jumps from ~6C to ~36C induced 2.6-fold tension rise. Parameter n estimated
from the A1 and LL9 intensities increased nearly proportionally with tension: from ~20% at ~6 C to ~45% at ~36C. Similar
estimates can be obtained from the T-jump X-ray diffraction experiments with single frog muscle fibres (Bershitsky et al.,
1997; Tsaturyan et al., 1999).
Work was supported by INTAS, HHMI, RFBR, MRC and Daresbury Laboratory, UK.
References
Dobbie, I, M. Linari, G. Piazzesi, M. Reconditi, N. Koubassova, M.A. Ferenczi, V. Lombardi & M. Irving. 1998. Nature 396: 384-87.
Bershitsky, S.Y., A.K. Tsaturyan, O.N. Bershitskaya, G.I. Machanov, P. Brown, R. Burns & M.A. Ferenczi. 1997. Nature 388: 186 190.
Holmes, K. C., D. Popp, W. Gebhard & W. Kabsch. 1990. Nature 347: 449.
Huxley, H. E. 1996. Annu. Rev. Physiol. 58:1-19.
Hudson L, J.J. Harford, R.C. Denny & J.M. Squire. 1997. J. Mol. Biol. 273: 440-55
Linari, M, G. Piazzesi, I. Dobbie, N. Koubassova, M. Reconditi, T. Narayanan, O. Diat, M. Irving & V. Lombardi. 2000. Proc. Natl.
Acad. Sci. USA 97: 7226-31.
Malinchik, S. B. & V. V. Lednev. 1992. J. Muscle Res. Cell Motil. 13: 406-19.
Malinchik, S., S. Xu & L.C. Yu. 1997. Biophys.J. 73: 2304-12.
Rayment, I., W. R. Rypniewski, K. Schmidt-Bäse, R. Smith, D. R. Tomchick, M. M. Benning, D. A. Winkelmann, G. Wesenberg, & H.
M. Holden. 1993a. Science 261: 5865.
Rayment, I., H. M. Holden, M. Whittaker, C. B. Yohn, M. Lorenz, K. C. Holmes & R. A. Milligan. 1993b. Science 261: 5058.
Tsaturyan, A.K., S.Y. Bershitsky, R. Burns & M.A. Ferenczi. 1999. Biophys. J. 77: 354-72.
INTERFERENCE SPLITTING OF MERIDIONAL MYOSIN REFLECTIONS ON X-RAY DIFFRACTION
PATTERN
FROM SKELETAL MUSCLE
N. Koubassova1, M. Linari2, G. Piazzesi2, M. Reconditi2,
I. Dobbie3, M. Irving3, V. Lombardi2
1Institute
of Mechanics, Moscow University, Russia;
2University of Florence, Italy;
3Randall Centre for Molecular Mechanisms of Cell Function,
King’s College London, UK
Fine splitting of the myosin meridional reflections on the X-ray diffraction pattern from muscle was observed experimentally and explained as the result of the interference between two halves of the sarcomere by Rome (1972) and Haselgrove
(1975). First quantitative simulation of the splitting was done later by Malinchik and Lednev (1992) using simplest approximation of the electron density of the myosin head.
Now highly intense and collimated beam is available at the new generation synchrotrons and crystal structures of S1
and actin-S1 complex have been obtained (Rayment et al, 1993; Mendelson & Morris, 1997). This allows to record more detailed interference diffraction patterns and to improve their interpretation.
Experiments were done on vertically mounted single intact muscle fibres from Rana temporaria on the ID-2 beamline
of
European
Synchrotron (ESRF, Grenoble, France)
34
During isometric contraction at sarcomere lengths 2.2 - 3.2 m, the M3 reflection associated with the repeat of myosin
heads along the filament axis, was resolved into two peaks of comparable intensity. The intensity of the high angle peak at
14.45 nm was ~ 80% of that of the low angle peak at 14.66 nm. The total M3 intensity decreased linearly with increasing sarcomere length and was proportional to the degree of overlap between the actin and myosin filaments, indicating that this intensity is mainly diffracted by the myosin heads in the overlap region. The relative intensity of the two peaks was independent of
the sarcomere length, showing that during contraction, the myosin filaments have the same periodicity of 14.57 nm in the nonoverlap and overlap regions.
The experimental data at sarcomere length 2.2 m can be simulated by two arrays of 49 myosin heads separated by
153 nm long bare zone. Both point diffractor model and atomic model of S1 (in the configuration suggested by Dobbie et al.
(1998)) provide equally good fit of the data with the same set of parameters. All changes in the experimental profile of M3 at
increased sarcomere length are reproduced by the models. Relative intensity of the M3 peaks appeared to be very sensitive to
the axial movement of the myosin heads on a nanometre scale. Fine splitting of the M3 reflection provides a high-resolution
tool for studying movement of the attached myosin heads (Linari et al., 2000).
To determine the structure of the myosin heads in rigor, axial X-ray diffraction pattern was recorded at two levels of
rigor force, "low-tension" corresponding to ~0.1 T 0 and "high-tension" corresponding to ~0.5 T 0, where T0 is tension level
reached at the plateau of isometric tetanus.
In low-tension rigor, the intensity of the small peak at 14.59 nm is about 25% of the main peak at 14.40 nm. When a
strain is imposed on rigor fibre, the intensity of M3 increases in proportion with the steady force and its fine structure alters.
The small peak at 14.28 nm is 10% of the main peak at 14.47 nm. The observed increase in intensity and changes in the profile
of M3 can be explained by either one- or double-headed models. Both models suggest that the light chain domain of S1 tilts by
~15 towards the M-line when tension increases from ~0.1 T0 to ~0.5 T0. The precise configuration of the myosin heads in rigor appears to be model-dependent as the length of the bare zone was estimated from the changes in the spacing of the M3 reflection and could not be extracted directly from the experimental data.
Besides, changes in the fine structure of the M2 and M6 reflections in rigor fibres with tension are different from
those in M3 suggesting their different origin.
Supported by RFBR, INTAS, HHMI, MURST, Telethon, MRC, EMBL, ESRF.
References
Dobbie I. et al, 1998, Nature, 396, 383-387.
Haselgrove J.C., 1975, J. Mol. Biol., 92, 113-143.
Linari M. et al, 2000, Proc. Natl. Acad. Sci. USA, 97, 7226-7231.
Malinchik S.B., Lednev V.V., 1992, J. Muscle Res. Cell Motil., 13, 406-419.
Mendelson R., Morris E.P., 1997, Proc. Natl. Acad. Sci. USA, 94, 8533-8538.
Rayment I. et al, 1993, Science, 261, 50-58.
Rome E., 1972, J. Mol. Biol., 65, 331-345.
MECHANISM OF ACETYLCHOLINE-INDUCED HYPERPOLARIZATION IN RAT SKELETAL MUSCLE
FIBRES
V.V. Kravtsova, A.E. Prytkov, I.I. Krivoi
A.A. Ukhtomsky Institute of Physiology, St. Petersburg State University, Russia
Acetylcholine (ACh) is a well-known neurotransmitter present at the neuromuscular junction of vertebrates. Binding
of ACh, released from motor nerve endings in quantal form (concentration in synaptic cleft about 0.1-1 mM), with acetylcholine receptor (AChR) the opening of the AChRs ion channel and the generation of short-term postsynaptic depolarization (mediatoric function of ACh). In addition, some studies suggest a trophic role of ACh, but the mechanism of such role for ACh is
yet unknown.
Particularly, in addition to quantal release, a great number of biochemical and electrophysiological studies indicate
that ACh may be released from motor nerve terminals in non-quantal form and may accumulate in the synaptic cleft in very
low concentrations (about 50-100 nM) even in the presence of intact acetylcholines terase. It is suggested that the physiological
role of non-quantal ACh is the formation, maintenance and modulation of synaptic neuromuscular communication. Particularly, several data show that non-quantal ACh as well as exogenous ACh in low concentrations produces a small hyperpolarization of the muscle fibres membrane (about 3-5 mV), abolished by ouabain an inhibitor of Na3+/K3+-ATPase. Conversely, it has
been shown that ACh in low concentrations activates the Na 3+/K3+-ATPase in skeletal muscle as well as some other tissues.
Hence, it was postulated that non-quantal ACh takes part in the regulation of skeletal muscle membrane potential (Vm) via the
Na3+/K3+-ATPase (the electrogenic pump). However, the target, the intracellular signaling pathways, and the participation of
membrane ion channels in ACh-induced activation of Na3+/K3+-ATPase and muscle fibres hyperpolarization remain unknown.
Our experiments were performed on isolated rat phrenic-diaphragm preparations. The value of V m was measured in
100-150 fibres from 3-6 muscles by means of standard intracellular microelectrode techniques.
We found that bath-applied ACh (100 nM) hyperpolarized non-synaptic regions of the muscle fibres by 4.6±0.6 mV
(from –79.1±0.6 to –83.7±0.5 mV, p<0.01). The nicotinic ACh-receptor antagonist, d-tubocurarine, in very low concentration
(10 nM) abolished the hyperpolarizing effect of ACh, whereas the muscarinic ACh-receptor antagonist, atropine (5 µM), was
ineffective. When K+ channels were blocked by tetraethylammonium (10 mM), the value of V m did not change in the presence
of ACh. In the presence of 1 mM 4-aminopyridine (a blocker of voltage-dependent K+ channels), or 10 M glibenclamide (a
blocker of ATP-dependent K+ channels) hyperpolarization was manifested completely. Low ouabain concentrations (1 nM 0.1 µM) inhibited the ACh-induced hyperpolarization, while high concentrations of ouabain (0.1 µM - 500 µM) depolarized
the muscle fibres. The apparent affinity constants of these two effects of ouabain differed by 1000-fold: 8±5 nM and 10±4 µM,
respectively. Under control conditions, inhibition of the Na 3+/K3+-ATPase isoform with high ouabain affinity induced the depo35
larization of muscle fibres only by 1-2 mV, while inhibition of the Na+/K+-ATPase with low ouabain affinity produced a depolarization of 16 mV.
The obtained data suggest that ACh-induced hyperpolarization of the muscle fibres membrane is caused by the activation of Na3+/K3+-ATPase and electrogenic pump with the participation of K + channels (more probably, inward-rectifying K+
channels and/or Ca2+-dependent K+ channels). Our data also suggest different functional roles of the different isoforms of the
Na+/K+ -ATPase in skeletal muscle. The isoform with low affinity to ouabain (likely the 1 isoform) appears to be the major active Na3+/K3+-ATPase while the highly ouabain-sensitive (likely 2) isoform of the Na3+/K3+-ATPase serves a special function
in cholinergic (via low non-quantal ACh) and hormonal regulation of muscle fibres electrogenesis.
We propose that a trophic (nonmediatoric) function of low non-quantal ACh is the selective regulation of the aaf232
isoform of the Na3+/K3+-ATPase. This cholinergic regulation is associated via nicotinic AChRs with extra-high affinity to dtubocurarine. These receptors may be either the desensitized conformation of muscle-type AChRs and/or a neuronal nicotinic
AChRs subtype recently revealed in the skeletal muscle membrane.
Supported by Russian Foundation for Basic Research (Grant № 01-04-49799).
ELASTIC PROPERTIES OF THE FIBROBLASTS
Sergey Krechetov, Alexandr Platonov1), Vladimir Smolyaninov2)
1)
Central Institute of Epidemiology, 111123 Moscow, 3a Novogireyskay str.,
E-mail: platonov@pcr.ru;
2) Mechanical Engineering Research Institute, 117334 Moscow, 4 Bardina str.,
E-mail: smolian@iitp.ru
In a tissue culture conditions the transformed mice fibroblasts of a line L (L-cells) differ from the normal embryonic
mice fibroblasts (NMF) by smaller mobility and smaller degree spreading. Earlier these distinctions were explained on the basis of a hypothesis about smaller adhesion of L-cells, which do not prove to be confirmed in our comparative researches of adhesive properties of these cells. On the other hand, it is possible to explain the same distinctions of properties of impellent behavior of L-cells and NMF differently — with the help of a tonical hypothesis, namely that the L-cell have greater tonical intensity.
For direct check of a tonical hypothesis the micropipette method was used. The spherical cells in an initial stage of an
attachment to substrate (in Igle medium + 10% of serum) were tested, at temperature 20 оС. To make a cellular withdrawal micropipettes were used with a diameter of the end d = 0.3-0.8D0. Length l of the withdrawal part of a cell for different values p
of negative pressure in micropipette was measured. The comparative data of elastic properties for two types of cells are received in two measurement methods: 1) method of the fixed measurements — the same micropipette at the fixed pressure p =
const were consistently made the withdrawals of NMF and L-cells (the cells beforehand were located in different standard experimental chambers, which it was possible to replace on a microscope table with micromanipulator); 2) method of the pressure-length characteristics — for different micropipette diameters the dependence l(p) was measured for two groups of cells.
Results of the fixed measurements: lNMF/lL  1,5-1,8 — NMF elasticity approximately in 1,5 and more time is higher
than elasticity of L-cells.
The results of the characteristics method confirm the previous results, but in addition show preservation of these relations in a wide range of pressure (1 < p < 10 cm H20), i.e. the characteristic l(p) is approximately linear.
Thus, the received results confirm a hypothesis of tonical distinctions between normal and transformed cells (NMF
and L-cells).
On the basis of the received characteristics l(p) the rating of applicability to connecting tissue cells of known models, which used for account of erythrocyte elastic parameters, is made. Non-adequacy only of envelope models is revealed and
the attempt to develop the model, which is taking into account volumetric elasticity of a crate is undertaken.
The micropipette method used in researches of influences on elastic properties of fibroblasts of the different factors. It
is established, that tonus (rigidity) of cells raises at downturn of environment temperature from 37 up to 20 оС.
The downturn tonus of cells occurs at added cytochalasin B, but at added colchicine of authentic changes of elasticity
of cells is not revealed. These data allow to conclude: the greater contribution to elastic properties of cells bring in superficial
contractile components, and the role cytoskeleton is not dominant.
36
THERMAL UNFOLDING OF ACTIN-BOUND TROPOMYOSIN: EFFECTS OF MYOSIN AND TROPONIN
E.V. Kremneva1, O.P. Nikolaeva2, N.B. Gusev3 and D.I. Levitsky1,2
1A.N.
Bach Institute of Biochemistry RAS, Moscow 117071, Russia;
Belozersky Institute of Physico-Chemical Biology,
3Department of Biochemistry, School of Biology,
Moscow State University, Moscow 119899
2A.N.
The method of differential scanning calorimetry (DSC) allows changes in the thermal unfolding of tropomyosin (TM)
resulting from its interaction with F-actin to be probed very precisely. Interaction of smooth muscle TM with F-actin was
shown to increase the TM thermal stability substantially by shifting the thermal transition of TM to higher temperature, by
about 6oC under conditions of full saturation of actin filaments with TM (Levitsky et al. (2000) Eur. J. Biochem. 267, 18691877). In the present work we applied this DSC approach to investigate the effects of myosin and regulatory muscle proteins
on the thermal stability of actin-bound TM. It has been shown that the binding to F-actin of isolated myosin heads (myosin
subfragment 1, S1) increases the thermal stability of smooth muscle TM bound to F-actin. In this case, however, the thermal
transition of actin-bound TM shifts to higher temperature and coincides in position with that of actin-bound S1, thus preventing
us from detailed analysis of the thermal unfolding of TM bound to F-actin in the presence of S1 (i.e. in the so called “open”
state). In order to separate the thermal transitions of actin-bound TM and S1 on the thermogram we used for these DSC experiments the S1 whose heavy chain was cleaved by trypsin between Arg-23 and Ile-24. Such type modified S1 was shown to
demonstrate very low thermal stability, even if it is bound to F-actin. Using this approach, we observed the thermal transition
of actin-bound TM, shifted by 2.8oC to higher temperature, from 44.5oC to 47.3oC, in comparison with the transition of actinbound TM observed in the absence of S1. We also studied by DSC the acto-TM complex in the presence of muscle regulatory
proteins. It has been shown that caldesmon, which does not demonstrate any thermal transitions, being bound to F-actin has no
effect on the thermal unfolding of actin-bound smooth muscle TM. To investigate the effects of troponin, we studied by DSC
the complexes of F-actin with skeletal muscle TM and showed that interaction of this TM with F-actin increased mainly the
thermal stability of C-terminal half of the TM molecule. It has been shown that troponin, whose thermal transition coincides in
position (~52oC) with that of the N-terminal half of actin-bound TM, has no appreciable effect on the thermal stability of actinbound TM, but it strongly increases the cooperativity of the thermal unfolding of actin-bound TM. This effect of troponin was
much more pronounced in the presence of EGTA than in the presence of Ca 2+. In conclusion, the results of these DSC studies
may provide new insights into structural changes of actin-bound TM accompanying its transitions between three different locations (“closed”, “open”, and “blocked”) on the actin surface.
This work was supported in part by grants № 00-04-48167 and 00-15-97787 from the Russian Foundation for Basic
Research (RFBR) and by INTAS-RFBR joint grant IR-97-577.
MUSCLE FIBRE CONTRACTION
IN THE FLATWORM PROCERODES LITTORALIS
N. Kreshchenko1, M. Totten2, A.G. Maule2, T.A. Day3, D.W. Halton2
1Institute of Cell Biophysics, Russian Academy of Sciences,
Pushchino, Moscow region, 142290, Russia, e-mail: nkresh@,icb.psn.ru;
2Parasitology Research Group, School of Biology & Biochemistry,
The Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, UK;
3 Department of Pharmacology and Toxicology,
Michigan State University, East Lansing, MI, USA
Flatworms can provide baseline information on excitation-contraction coupling and on the mechanisms of muscle
contraction and its regulation in a basal animal. A dispersed muscle fibre bioassay, which enables direct examination of the effects of test compounds on individual muscle fibres and their receptors without neuronal influence was used to study the effects of high K+-media, caffeine, serotonin and selected FMRFamide-related peptides (GYIRFamide and YIRFamide) in freeliving marine flatworm, Procerodes littoralis (Turbellaria, Tricladida). An inverted microscope-microperfusion system was
employed to deliver test drugs to selected muscle fibres. The actions of test substances were examined in conjunction with selected Ca2+-channel agonists/antagonists and Ca2+-pump inhibitors.
The following caused concentration-dependent contraction of isolated flatworm muscle fibres: depolarization by elevated extracellular K+ (20-100 mM); caffeine (1-10 mM); serotonin (0.01-100 μM), GYIRFamide (10-12 - 10-3 M), YIRFamide
(10-10 - 10-3M).
The depolarization-induced contraction in P. littoralis was blocked by dihydropyridine Ca2+-channel blockers
(nicardipine, nitrendipine and nifedipine). These compounds decreased the number of cells which responded to serotonin and
GYIRFamide but did not affect caffeine-induced contraction. Ryanodine, a blocker of sarcoplasmic reticulum Ca 2+-release
channels, decreased the number of GYIRFamide-, caffeine- and high K+-induced contractions, but did not block serotonininduced contraction. The effects of cyclopiazonic acid and thapsigargin, blockers of the Ca 2+-sequestering activity of the sarcoplasmic reticulum, both blunted the effects of flatworm peptides, high K + and caffeine on isolated muscle cells: they did not
alter serotonin-induced contractions.
The data support the occurrence of a number of muscle-based receptors in flatworm (P. littoralis) muscle. The results
reveal that depolarization-induced contraction as well as peptide- and serotonin-induced contractions of dispersed muscle cells
is dependent on extracellular Ca2+. The data indicate that flatworm muscle possesses of voltage-dependent Ca2+-ion channels
similar to those seen in vertebrates (dihydropyridine sensitivity).
Internal Ca2+ stores play a central role in the regulation of caffeine, peptide and depolarization-induced muscle contraction in P. littoralis. The contractions induced by GYIRFamide, YIRFamide and caffeine all rely on internal Ca 2+-stores;
serotonin contractions seem to be independent of these stores. Interestingly, even the depolarization induced contractions
caused by high K+ were partially dependent on internal Ca2+. This suggests that the depolarization induced influx of Ca 2+ is followed by Ca2+-induced Ca2+ release, possibly via an endogenous ryanodine receptor-like channels
37
This research was supported by Royal Society Fellowship programme and INTAS grant YSF 99-4029.
CORRELATION BETWEEN PHORBOL ESTER-INDUCED CONTRACTION AND PHOSPHORYLATION OF
CALDESMON AND MYOSIN LIGHT CHAIN KINASE IN SMOOTH MUSCLE
Mikhail A. Krymsky, Asker Yu. Khapchaev, Maria I. Sidirova, Zhanna D. Bespalova, Vladimir P. Shirinsky and Alexander V.
Vorotnikov
Institute of Experimental Cardiology, Cardiology Research Centre,
121552 Moscow, Russia
We find that phorbol 12,13-dibutyrate (PDBu) elicits sustained contraction in tonic smooth muscle and no contraction
in phasic muscle. We examined whether phosphorylation of actomyosin-associated regulatory proteins by PDBu-activated protein kinase C and/or MAP-kinases contributes to this difference. Phosphorylation of myosin light chain kinase (MLCK), a key
activator of smooth muscle contraction, and that of actin-associated inhibitory protein caldesmon was stimulated 2-3-fold by
PDBu in tonic muscle, while no effect of PDBu on phosphorylation of these proteins was found in phasic tissue. This is consistent with the proposed role for MAP-kinase/protein kinase C in reducing caldesmon inhibition of thin filaments and MAPkinase in MLCK activation. Two-dimensional phosphopeptide maps of MLCK and caldesmon immunoprecipitated from 32Plabelled PDBu-treated muscle strips revealed multiple sited phosphorylation of these proteins but showed only minor impact of
PKC in this process. Caldesmon was mainly phosphorylated at MAP-kinase sites in PDBu-stimulated tonic muscle. To resolve
the sites of MLCK phosphorylation we compared MLCK phosphopeptide maps with those of KRP (or telokin, an independent
smooth muscle protein homologous to the C-terminal domain of MLCK) phosphorylated in vitro by purified protein kinases.
The major of two phosphorylation sites in MLCK from phasic muscle was found within its KRP domain whereas the second
site was located in the other part of the molecule. In contrast, both phospho-residues were located in KRP domain of MLCK
from tonic muscle and the third phosphate was introduced in the same region upon PDBu challenge. This region was mapped
within sequence surrounding site B (Ser827) of MLCK, in common tryptic peptide of the kinase, thus making the site-specific
phosphorylation with MAP-kinase impossible to assess by phosphopeptide mapping. We have therefore used an alternative
technology and developed phosphospecific antibody that recognises the MAP-kinase phosphorylated Ser833 of MLCK, homologous to Ser18 of KRP. This antibody cross-reacted only with MLCK and KRP on immunoblots of smooth muscle extracts, but
not with purified unphosphorylated KRP. In vitro phosphorylation of KRP at neighbour Ser12 by protein kinase A (equivalent
to site B in MLCK) did not affect the antibody recognition of phospho-Ser18. Analysis of MLCK in extracts of unstimulated
phasic and tonic smooth muscles revealed high level of the MAP-kinase site phosphorylation in both tissues that was only
slightly enhanced upon PDBu challenge. This indicates that the major MAP-kinase site of MLCK is almost completely phosphorylated even in unstimulated tissues, while PDBu enhances phosphorylation of distinct, but closely located site of MLCK in
tonic muscle. Additional to Ser827 phosphorylated by PKA (site B) and Ser833 phosphorylated by p42/p44erk1,2 MAP-kinase,
Ser830 of MLCK is a potential phosphorylation site for proline-directed kinases. As Ser830-Pro is homologues to Ser15-Pro of
KRP, we used purified KRP to assess possibility of Ser 830 phosphorylation in vitro. We find that Ser15 of KRP is readily phosphorylated by an acidotropic, proline-directed glycogen synthase kinase 3 (GSK3) depending on primary phosphorylation of
Ser18 by MAP-kinase. The phosphopeptides of KRP sequentially phosphorylated by p44 erk1 MAP-kinase, GSK3 and protein
kinase A to different extent in vitro mapped to phosphopeptides found in MLCK isolated from PDBu-treated tonic smooth
muscle. This suggests that three sites can be simultaneously phosphorylated in KRP-domain of MLCK. However, question remains as to whether the GSK3 is stimulated by PDBu and able to phosphorylate MLCK in vivo, or phosphorylation of Ser830 is
mediated by other proline-directed kinase(s). To address the latter possibility, we analysed activation of different MAP-kinases
in tonic smooth muscle and found that PDBu equally stimulated both p42/p44 erk1,2 and p38 MAP-kinases, which are known to
phosphorylate Pro-X-Ser-Pro and simpler Ser-Pro motifs in substrate proteins. This suggests that PDBu-stimulated multiple
sited phosphorylation of MLCK in tonic smooth muscle may involve different MAP-kinases and studies are currently under
way to investigate reactivity of p38 MAP-kinase pathway to PDBu in phasic smooth muscle as well as to demonstrate capability of p38 MAP-kinase of phosphorylating Ser830 of MLCK in vitro.
Supported by Russian Foundation for Basic Research (grant № 99-04-49209), the Wellcome Trust to AVV, and the
Howard Hughes Medical Institute (grant 75195-546901) to VPS.
EXPRESSION OF T-CADHERIN IN THE RAT CAROTID
ARTERY WALL AFTER BALLOON INJURY
AND IN DIFFERENT RAT ORGANS
E.Yu. Kudryashova, P.P. Bashtrykov, D.B. Ivanov, Yu.G. Antropova,
O.P. Il’inskaya, E.M. Tararak, V.N. Bochkov, E.V. Parfyonova
Russian Cardiology Research Center,
121552, Moscow, 3-rd Cherepkovskaya str., 15a, Russia
The cadherins are cell adhesion molecules that mediate cell-cell adhesion and therefore affect cell behavior e.g. proliferation and migration. Alterations in expression of surface adhesion molecules on resident vascular and blood-derived cells
play a fundamental role in the pathogenesis of cardiovascular diseases such as atherosclerosis and restenosis. These disorders
are characterized by changes in vascular architecture associated with altered adhesion and migration properties and enhanced
proliferation of cells in the vessel wall.
T-cadherin (TC) is an “atypical” member of the cadherin superfamily of cell adhesion molecules. While possessing
the general extracellular structure of the classic type I cadherins, it lacks the transmembrane and cytoplasmic domains and
bounds to plasma membrane via a glycosylphosphatidylinositol anchor. It is still not clear whether TC functions as cell adhesion molecules in vivo. It was noted that in contrast to the classical cadherins TC was not concentrated at the sites of cell-cell
contacts but it was localized at the apical surface of epithelial cell. In vivo and in vitro studies suggested that TC functions as a
negative guidance cue for motor-axon projection by repulsion rather than by growth arrest through increased adhesion. TC
38
has been shown to mediate contact growth inhibition in breast cancer cells and to influence proliferative and invasive properties of carcinoma and neuroblastoma cells.
In contrast to classical cadherins tissue distribution of TC so far remained unknown. We examined the rat tissue distribution of TC using Western blotting and immunohystochemical method. Our results show that TC is highly expressed in all
types of muscles: cardiac, striated and smooth muscles. Also the endothelial cells of vessels were TC-positive. In the nervous
system TC was localized in the neurons. In epithelial tissue TC is selectively expressed in epithelium of integumentary and
lined types (e.g. in the stratified epithelium of skin and esophagus - in the basal part of basal layer cells; in the intestinal epithelium - at the apical pole of epithelial cells) whereas epithelium of glandular type doesn’t contains TC (e.g. the goblet cell, liver). The blood-derived and lymphoid cells as well as connective tissue were TC-negative. In the vessels of different caliber in
all investigated organs the SMCs and endothelial cells contained TC. Our data allow to suggest that TC has its functions (yet
unknown) in those cells which form unified structure-functional communities such as layers or sheets of cells (epithelium and
endothelium), cellular nets (neurons) or bundle of muscle filaments in contrast to independently working distinct cells of blood
and connective tissue.
The highest level of TC expression was revealed in cardiovascular system. Although TC has been detected in SMCs
its role in the intimal thickening and restenosis is not known. We examine TC expression 1st, 4th, 7th, 10th, 14th, 28th days after balloon injury of rat left carotid arteries. The expression of TC was valued immunohystochemically with semiquantitative
method. An interesting fact of boundary distribution of TC both into the vessel walls and in some another tissues (e.g. endothelium, basal epithelium layers of skin and esophagus, apical pole of intestinal epithelium) was pointed. In an uninjured artery
maximal TC expression level was revealed in the regions adjoining with vessel lumen such as prelumenal and periadventitial
(permeated by microvessels) layers. After deendotelialization TC was detected in internal (subintimal) layer of medial SMCs
where it was absent in uninjured artery. In the developing neointima highest level of TC expression was found in preluminal
layer. At the 10-th day after balloon injury TC distribution in media became the same as in uninjured artery (the TC expression
in subintimal layer of medial SMCs disappeared). The following dynamic of TC expression in the medial SMCs of injured artery was detected: it was increased as early as 1st day after balloon injury, amounted to maximum 4th day and was reduced to
level in uninjured artery 10th day. It is well known that just 1-7th day after injury the migration and proliferation processes
have been activated in the vessel wall in response to injury. Our results showing that TC expression increase in medial SMCs
of injured vessel during migration and proliferation of blood-derived and vessel wall cells may suggest the TC participation in
the processes of vessel remodelling after injury.
EXPLICIT EXPERIMENTAL EVIDENCE OF THREE-DIMENSIONAL SCROLL AS A MECHANISM OF
TACHISYSTOLIA IN ISOLATED GROUND SQUIRREL VENTRICLE PREPARATIONS
N.I. Kukushkin1, V.Yu. Sidorov1, A.V. Moskalenko2,
K.N. Kukushkina1 and A.B. Medvinsky2
1
2
Institute of Cell Biophysics RAS,
Institute of Theoretical and Experimental BiophysicsRAS,
Pushchino, Moscow Region, 142290 Russia
Three-dimensional (3D) vortex-like waves, in particular scroll waves, have been shown to originate in excitable media
of different nature. 3D vortex-like waves rotate around a filament [Winfree A.T., 1987]. However, investigation of such vortices in the heart muscle has run into the problems due to well known limitations of mapping techniques which do not allow a
total three-dimensional excitation wave picture to be visualized. Nevertheless, on account of three-dimensional nature of the
heart muscle, 3D vortex-like waves have been often considered as an appropriate mechanism of some heart arrhythmias.
In the last few decades, the circumstantial evidence for 3D vortex-like waves in the heart has been obtained [Medvinsky et al., 1983; 1984; Efimov et al., 1999]. However, the problem of 3D scroll waves as a mechanism of the heart arrhythmias
called for further investigation. In this work, we demonstrate experimental data giving evidence that 3d scroll waves with Ulike and straight line-shaped filaments arising in the isolated ventricle preparations can be a cause of tachisystolia.
28 experiments were carried out. The preparations were about 1 mm-thick 10x10mm strips excised from the wall of
the right ventricle of (Yakutiya) ground squirrel Citellus undulatus. Paroxysms of the tachysystolia were induced by a single
extrastimulus. The endo- and epicardial excitation wave patterns were visualized on the basis of electrograms simultaneously
recorded with the use of two 32 unipolar electrode arrays. In most (80%) cases 3D waves can be hardly identified since the
character of epicardial wave patterns differed drastically from that of endocardial ones. We have observed an inherent scroll
wave pictures on the one of preparation surfaces only; it was endocardial surface more often. An activation pictures on the other surface of the preparation was similar with pictures which can be conditioned a focal source or it can be the projection of the
activity rotated in the myocardial thickness. In the rest of the cases 3D waves were clearly distinguishable. Namely, in these
cases we observed both vortices with U-like filaments and vortices with straight line-shaped filaments, also known as scrollshaped waves. Usually, vortex with U-like filament manifested itself on one of the preparation surfaces as two neighboring 2D
waves rotating in opposite directions while on another surface a focal source was observed. Vortex with straight line-shaped
filament manifested itself as solitary 2D vortices at each of the surfaces. Both the epicardial and endocardial waves rotated at
the same direction and were virtually synchronous with each other. It is noteworthy that the core of the epicardial vortex located just under the core of the endocardial vortex, which is to say that the filament of the corresponding 3D scroll wave was
normal to each of the heart muscle surfaces. Sometimes, the orientation of the filament chaged in time, and the filament precession took place.
It is important to point out that transmural propagation provided synchronization of the both surface reentries. When
the excitation front on endocardial surface began significantly to leave behind the one on epicardial surface, the outstripping
excitation transition arose on epicard and the front's position evened out again for a certain time. And vice versa, when the activation process on epicardial surface began significantly to leave behind the one on endocardial surface, the outstripping excitation transition arose in reverse direction, i.e. from epicard to endocard. Interestingly the time it took for the synchronizing
transmural propagation from endocard to epicard (10+0,9 ms) was almost twice as the propagation time in the opposite direction (5+1,4 ms).
39
GLYCOXIDATIVE NON-ENZYMATIC MODIFICATION OF MUSCLE ACTIN
N.V. Kuleva, M.Yu. Baklanova, Z.S. Zalessova
Department of Biochemistry, St.–Petersburg State University,
St.–Petersburg, Universitetskaja nab. 7/9, 199034, Russia
The oxidative modification of proteins by reactive oxygen species is implicated in the etiology or progression of disorders and diseases. An increase in the rate of their production or decrease in their rate of scavenging induces the oxidative
modification of cellular molecules, including proteins. The elevation of concentration of reactive oxygen species at the expense
of sugar auto-oxidation under diabetes and galactosemia is a well-documented fact. Under these circumstances one more nonenzymatic process occurs. That is glycation, binding of sugar residues (aldehyde groups) to free aminogroups of proteins,
transformation them in ketoamine adducts and following formation of advanced glycation end products (AGEs) including oxidative transformation. The greater part of AGEs are protein cross-linkings, which can not be removed by proteolytic degradation. Accumulation of modified proteins disrupts cellular function either by loss of catalytic and structural integrity or by interruption of regulatory pathways.
We studied experimental models of galactosemic rats with forced production of intracellular reactive oxygen species
(Salganik et al., 1994) and diabetic rats (Gugluicci, Bendarjan, 1995). Together with specific glycoxidative modification of histones from hepatocytes (appearance of characteristic for AGEs fluorescence) we observed oxidative degradation of muscle actin (especially in red muscle fibers) as well as decrease of polymerization level and activation of myosin Mg 2+-ATPase. Glycoxidative modification of actin was modeled in vitro, at G- and F-actin incubation for 3 days with 10 mM glyceraldehyde
having both glycating and oxidative properties. We showed that incubation with glyceraldehyde resulted in oxidative degradation of actin monomers and high molecular weight products appearance. Simultaneously we observed alteration of number of
actin functional properties: DNase I inhibition, polymerizability, activation of myosin Mg 2+-ATPase. Two times increasing of
myosin ATPase activation and binding of aldolase was observed at incubation of F- actin with glyceraldehyde. At the addition
of muscle dipetide carnosine (10mM) to incubation mixture all structural and functional parameters of actin returned to original level.
The data evidence that muscle actin may be modified by glycoxidation, especially in G-form. We think that glycoxidation of actin may be revealed rather in non-muscle cells. Really, such modification of actin is known for endothelial cells in
response to variety of inflammatory mediators such as tumor necrosis factor, H2O2 and thrombin (Ferro et al., 1997).
The main intracellular source of reducing sugars for glycoxidation is ADP-ribose forming from NAD in many metabolic pathways, especially from polyADP-ribose at DNA damage (Cervantes-Laurence et al., 1996). The non-enzymatic ADPribosylation was demonstrated for /-isoforms of actin of platelets and polymorphonucleated neutrophils (Just et al., 1994).
Whether muscle actin may be ADP-ribosylated and which factors influence on this process is now under investigation.
CYTOSKELETAL PROTEINS IN SYMPLASTIC AND PHLOEM TRANSPORT PATHWAYS OF HIGHER
PLANTS
A.L. Kulikova, M.A.Krasavina, M.V. Turkina, N.A. Burmistrova
Timiryazev Institute of Plant Physiology, Moscow, Russia
Long-distance transport along the higher-plant phloem includes the intercellular transport via plasmodesmata. In the
mature phloem, the functioning sieve elements do not contain nuclei and ribosomes and are not capable of transcription and
translation. Therefore, most macromolecules occurring in the transport channel are evidently synthesized in companion cells
and transferred to the sieve elements through specialized plasmodesmata along the entire transport pathway (Thompson,
Schulz, 1999). Some proteins of the phloem exudate (including the most studied phloem protein PP1 that is specific for companion cells and sieve elements and capable of filament formation) can affect the macromolecule transport through plasmodesmata, increasing their size exclusion limit (Balachandran et al., 1997).
We studied the putative involvement of cytoskeletal proteins in transport processes. We detected the presence of actin
in the pumpkin phloem exudate and in the fraction of purified cell walls enriched in plasmodesmata. The content of actin in the
exudate was comparable with that in the cytoplasm of some other plant cells. Under the electron microscope, we observed filaments, which were capable of interaction with the myosin subfragment-1 from skeletal muscle and phalloidin conjugated with
colloidal gold. When 0.5 mM phalloidin was added to the phloem sap in the medium containing 20 mM DTT, which solubilizes PP1 filaments, the amount of F-actin filaments in the exudate increased. In the presence of 50 mM MgCl2 and DTT, dense
bundles of filaments and paracrystals were detected like those produced by muscle actin under similar conditions. Our data indicate that actin is capable of polymerization in the exudate. Actin microfilaments were never found earlier in the mature sieve
elements. In the plasmodesma-enriched fraction and in the phloem exudate, we also detected proteins cross-reacting with the
anti-tubulin antibody, although the content of these proteins was low. Cytochalasin-induced modification of actin filaments reversibly changed the plasmodesmal electric conductivity, thus affecting cell-to-cell communication, and the symplastic
transport of water and sucrose. The mechanism of the cytoskeleton involvement in the plasmodesmal transport is obscure. The
contraction of actomyosin bundles or reorganization of actin microfilaments can be supposed to change the dimensions of the
transport channel. On the other hand, it is not unconceivable that actin microfilaments participate in macromolecules targeting
and movement through the plasmodesma. The role of the actin in long-distance transport remains also to be elucidated.
INACTIVATED STATE OF ACTIN DEVELOPS
FROM NATIVE ONE VIA COMPLETELY UNFOLDING
OF PROTEIN MACROMOLECULE
Irina M. Kuznetsova1, Alexandr G. Biktashev, Ol'ga V. Stepanenko1,
Ol'ga I. Povarova1, Mikhail M. Shavlovsky2 and Konstantin K. Turoverov1
40
1Institute
of Cytology, Russian Academy of Sciences,
194064 St. Petersburg, Russia, E-mail: kkt@mail.cytspb.rssi.ru;
2Institute for Experimental Medicine, Russian Academy of Medical Science,
197376 St. Petersburg, Russia, E-mail: molgen@molgen.iem.ras.spb.ru
The kinetics of actin denaturation induced by guanidine hydrochloride (GdmCl) was studied. It was found that at 1.51.8 М GdmCl time dependencies of fluorescence intensity recorded at 320 nm (I 320), parameter А=I320/I365 and fluorescence anisotropy r have minimum at 100 - 50 s respectively. As it is known that AN>AI>AU and rN<rI>rU the results of kinetic measurement suggest that inactivated actin (I) is formed from native one (N) under the effect of GdmCl via completely unfolded state
(U). The rate constants k1, k2 and k3, of the transition N U, U I and I U and their dependence upon GdmCl concentration
were determined. At small concentration of GdmCl the value of rate constant k3 equals to zero, and k2 is much greater than that
of k1, and therefore the limiting stage of reaction is protein unfolding. With the increase of GdmCl concentration the rate constant k1 is increased and the rate constant k2 is decreased. This leads to the accumulation of completely unfolded molecules at
the beginning of denaturation at some concentrations of GdmCl (1.0-2.0 M GdmCl), and consequently to the appearance of
minimum in the kinetic curves of fluorescence characteristics. At GdmCl concentration more than 2 M k1>>k2 and k3 and the
registered fluorescence characteristics are determined by relation between k2 and k3. At the high concentration of GdmCl the
equilibrium is shifted to the appearance of completely unfolded state (k3 >> k2). The obtained data suggest that protein unfolding precedes the formation of inactivated actin and that so inactivated actin is not the intermediate between native and completely unfolded states as it was accepted before.
Acknowledgment: This research was supported by Grants № 00-04-49224, 00-04-81082 Bel 2000_a, 00-15-97824,
01-04-49308 from the Russian Foundation for Basic Research and St.-Petersburg United Research Center (Joint Use Center).
EXPRESSION OF MYOSIN LIGHT CHAIN KINASE GENETIC LOCUS PRODUCTS IN VERTEBRATE HEART
IN DEVELOPMENT AND DURING HYPERTROPHY
A.V. Lapshin, T.V. Dudnakova, A.V. Nickashin,
O.V. Stepanova, V.P. Shirinsky
Russian Cardiology Research Center, Moscow, Russia
The essential role of myosin light chain kinase (MLCK) was demonstrated in skeletal muscle development and cardiomyocyte hypertrophy indicating the importance of this key regulator in motile systems other than smooth muscle and nonmuscle cells. However, it has recently been demonstrated that MLCK is the only one of several products expressed from
MLCK locus in the higher vertebrate genome. In addition to the previously known 108 kDa MLCK this locus codes for 210
kDa MLCK and a Kinase-Related Protein (KRP or telokin). KRP is expressed in chicken embryo heart but not adult heart suggesting its involvement in cardiogenesis. In human heart KRP expression persists in adulthood implying there could be interspecies differences in regulation of its expression. The cellular distribution of MLCK-210 in the heart is not described. Therefore, the aim of the study was to investigate the comparative expression of MLCK genetic locus products during heart development in avians and mammals and during cardiac hypertrophy.
MLCKs and KRP were detected in the isolated cultured cardiomyocytes and hearts from 10-20 day chicken embryos,
1-4 day-old chickens and 1, 3 and 7 day-old neonatal rats by immunochemical techniques. Experimental heart hypertrophy was
established in 2,5 months-old male Wistar rats. Left ventricle hypertrophy developed 15 days after the coarctation of abdominal aorta between renal arteries to 0,9 mm diameter and was evaluated by the relative increase in the heart mass and protein marker expression.
In chicken heart KRP expression was stable during days 10-20 of embryonic development and decreased significantly
within 4 days after hatching. In contrast, KRP content in neonatal rat hearts increased from day 1 to day 7 after birth and KRP
was present in adult rat heart. Using indirect immunofluorescence and MLCK-210 specific antibodies we found that MLCK210 is expressed in smooth muscle cells and fibroblasts present in the cultures of chicken embryo cardiomyocytes but not in
cardyomyocytes themselves at detectable levels. MLCK-210 was also detected in neonatal rat cardiac fibroblasts.
Increased hemodynamic load as a result of abdominal aorta coarctation lead to the increase in the total heart mass in
rats by 17% after 15 days post operation. Concomitantly, tubulin expression in the hearts of experimental rats (n=6) increased
by 40% compared to sham operated animals (n=5). These results were consistent with the heart hypertrophic response described in literature and attested the validity of our model. KRP expression also increased in hypertrophying heart as revealed
by western blot analysis.
Therefore, we obtained evidence that KRP, but not MLCK-210, is expressed in avian and mammalian cardiomyocytes
and displays species-specific pattern of expression in development. KRP expression is sensitive to mechanical load which
leads to cardiomyocyte hypertrophy indicating that this protein may be an active participant of a hypertrophic response. Taking
into account myosin filament stabilizing activity of KRP and its capability to modulate MLCK activity (Shirinsky et al., 1993;
Silver et al., 1997) the role of KRP in de novo sarcomerogenesis could be envisioned. Interspecies variations in KRP expression may also be related to the patterns of hemodynamic load exerted on the heart in different animals.
Supported by RFBR grants 00-04-48782 (AVL), 99-04-48158 (VPS), «Ph.D.St.» TVD, HHMI-55000335 (VPS).
ACTIN-ACTIVATED ATPase IS AFFECTED
BY WEAK MAGNETIC FIELDS
V.V. Lednev and S.L. Malyshev
Institute of Theoretical and Experimental Biophysics, Russian Acad. Sci.,
Pushchino, Moscow Region, 142290, Russia
There is now a great deal of evidence indicating that weak combined magnetic fields (CMF) containing collinear static and alternating components, may affect metabolic and functional properties of biosystems. According to the theory of magnetic parametric resonance (MPR) in biosystems [1] the effects of CMFs are caused by their action on the rate of some Ca 2+dependent biochemical reactions playing the key regulatory functions in the eukariotic cells. We studied the influence of
41
CMFs on ATPase activity of the cell-free system containing pure F-actin and reconstituted myosin filaments with different degree of catalytic and structural Ca2+-sensitivity. In addition we studied the influence on the same test-system of the two more
types of CMF, which most probably affect the biosystems via the Ca 2+-independent mechanisms.
Three principally different types of CMFs composed of collinear static, BDC, and alternating, BAC  cos2f, components, where BDC and BAC denote the magnetic flux densities, were used 1. CMF tuned to the parametric resonance for Ca 2+
(Ca2+-CMF): BDC=46.5  0.1Т, BAC=86.0  3.0Т, fAC=35.6  0.1 Hz. 2. CMF tuned to the Larmor frequency of the nuclear
spins in the hydrogen atoms (Hspin.-CMF): BDC=46.5  0.1Т, BAC=86.0  3.0Т, fAC=1980  1.0 Hz. 3. CMF with very low
amplitude of the sinusoidal components (LA-CMF): BDC=46.5  0.1Т, BAC=1.0  0.1Т, fAC=35.6  0.1 Hz. The controls
were placed in the local geomagnetic field (BDC=46.5 Т).
Exposure of the actomyosin ATPase assay systems with Ca 2+-sensitive myosin to Ca2+-CMF is accompanied by the
inhibition of the ATPase activity by about 36% comparative to the control value while ATPase activity of Ca 2+-insensitive
preparations was not affected by Ca2+-CMF. Exposure of the same assay-systems to the Hspin and to the LA-CMF leads to the
inhibition of the ATPase activity by about 25 % comparative to the control values in both Ca 2+-sensitive and Ca2+-insensitive
preparations. These results demonstrate that the action of Ca 2+-CMF on the actin-activated ATPase activity of skeletal myosin
can be mediated by myosin light chains, the only Ca2+-binding proteins in this test-system. However the ability of Hspin- and
LA-CMFs to influence the rate of ATPase in both Ca2+-sensitive and Ca2+-insensitive myosin preparations excludes the Ca2+dependent biochemical reactions as the possible primary targets for these types of magnetic fields.
Supported by Grant from RAS (6th competition-examination, № 208).
Reference
[1] V.V. Lednev. Biophysics.1996; 41: 241-252 (translation from Russian).
STRUCTURAL AND FUNCTIONAL STUDIES
ON MYOSIN AND ACTOMYOSIN
USING DIFFERENTIAL SCANNING CALORIMETRY
Dmitrii I. Levitsky
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, and A.N. Bach Institute of Biochemistry, Russian Academy
of Sciences, Moscow 117071, Russia
Starting from 1990 the method of differential scanning calorimetry (DSC) is successfully used by our scientific group
for studying structural changes occurring in the myosin head in the course of ATPase reaction. For this purpose we use stable
analogues of the myosin ATPase intermediates - the ternary complexes of the myosin head with ADP and Pi analogues such as
orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride (AlF4-). It has been shown that the formation of these
complexes causes a global conformational change of the motor domain of the myosin head, which is expressed in a significant
increase of the protein thermal stability. More recently we have also applied the DSC method for analyzing the changes in the
thermal unfolding of isolated myosin heads induced by their binding to F-actin. We have shown that actin-myosin interaction
increases the myosin thermal stability substantially by shifting the thermal transition of the myosin head to higher temperature,
by 5-12oC depending on the source of myosin (the most pronounced effects were observed with myosin I from Dictyostelium
discoideum and with smooth muscle heavy meromyosin). The main goal of further studies is to reveal, by using both DSC approaches described above, the sites in the myosin head responsible for the spread of conformational changes from the ATPase
site and actin-binding sites throughout the structure of the entire motor domain of the myosin head. For this purpose we study
various modified preparations of the myosin head and select those modifications, which selectively prevent either only actininduced or only nucleotide-induced structural changes in the head. The following modifications were shown to prevent selectively the actin-induced structural changes with no effect on the nucleotide-induced structural changes: (i) introducing of many
additional negative charges into actin-binding loop 2 of the motor domain of D. discoideum myosin II or deletion of this loop;
(ii) N-terminal tryptic cleavage of the heavy chain of skeletal myosin subfragment 1 (S1), between Arg-23 and Ile-24. On the
other hand, the modifications selectively preventing only nucleotide-induced structural changes are as follows: (i) cross-linking
of SH1 (Cys-707) and SH2 (Cys-697) groups in S1 by pPDM; (ii) modification of both these SH-groups by various thiol reagents; (iii) replacing by site-directed mutagenesis of Phe-506 by glycine which is proposed to disrupt the communication between the converter and the relay loop region in the motor domain of D. discoideum myosin II. Thus, this DSC approach allows us to investigate long-distance communication pathways between functionally important but spatially far regions in the
myosin head.
This work was supported in part by grants № 00-04-48167 and 00-15-97787 from the Russian Fund for Basic Research (RFBR) and by INTAS-RFBR joint grant IR-97-577.
CONTROL OF THE ANAEROBIC SUCCINATE PRODUCTION
IN RAT HEART MITOCHONDRIA
Е.I. Maevsky, Е.V. Grichina, М.S. Okon
Institute of Theoretical and Experimental Biophysics RAS,
142290 Pushchino, Russia
It is shown that in rat heart mitochondria (RHM) there are several interconnected ways of anaerobic succinate production (ASP) started from different substrates, the succinate sources (SS). Various researchers prefer different SS to support the
ASP [Taegtmeyer, 1986; Pisarenko et al., 1986, 1987; Maevsky et al., 1989; Grishina, 1997], because the question of which of
ASP ways can be realized at oxygen deficiency remains arguable until now. Measurements of the kinetics of ASP on the isolated RHM by Н-NMR-spectroscopy carried out by us have shown that the ASP ways differ depending on the duration of anoxic conditions, the intensity of the energy deficiency, the extent of the oxidative phosphorylation uncoupling and the presence
of ammonium ions. According to these parameters it is possible to allocate at least several stages in deenergyzation dynamics
42
at the stopped respiratory chain of MM.
Initially at the moderate fall of the АТP/АDP ratio (2÷3), complete integrity of the oxidative phosphorylation coupling, high level of NADH reduction (supported at the expense of either endogenous or added substrates oxidation) the predominant ASP way is a reverse of Krebs cycle from oxaloacetate or malate up to succinate. Here the АTP resynthesis is effected due to ΔН+ generated on a level of complex I in the respiratory chain, with fumarate being the final acceptor of reduction
equivalents. In the absence of fall of the ATP/ADP ratio supported due to extramitochondrial ATP source this way of ASP is
inhibited.
At the further fall of the ATP/ADP ratio and the extent of oxidative phosphorylation coupling the most powerful ASP
way becomes the connected current of the reverse of Krebs cycle and 2-oxoglutarate oxidation. The ratio of these ways is equal
to 1:2. Thus, the best source for ASP is the mixture of aspartate and 2-oxoglutarate. Sources of energy rich compounds in this
case are the substrate phosphorylation at the level succinil-CоА and oxidative phosphorylation.
In conditions of significant deenergyzation, growth of concentration of inorganic phosphate and ammonium ions (due
to desamination of AMP and NAD) an anaerobic dismutation of 2-oxoglutarate prevails. The process of 2-oxoglutarate reductive amination up to glutamate plays a role of reduction equivalents acceptor providing the conditions for oxidation of another
molecule, 2-oxoglutarate, up to succinate. The dismutation of 2-oxoglutarate is a rather weak energy source.
In hypoxic conditions, when the high level of NADH inhibits the oxidation of NAD-dependent substrates, and succinate can oxidize due to preservation of high level of flavine oxidation, the ratio of different ASP ways almost completely depends on the presence of substrates - ASP sources and ammonium ions concentration. Thus the energy output of ASP and oxidation of succinate formed by ASP can be so great, that there is no fall of ΔН+.
EFFECTS OF LONG-DURATION EXPOSURE TO UNLOADING ON OXIDATIVE POTENTIAL IN HUMAN
SOLEUS MUSCLE FIBERS
O.A. Matveeva, S.L. Kuznetsov, B.S. Shenkman
SRC Institute for Biomedical Problems RAS,
Sechenov Moscow Medical Academy, Moscow, Russia
Along with atrophy development the decline of the muscle oxidative potential (mitochondrial content) is expected to
be observed during exposure to muscle unloading. But many authors found almost no changes in oxidative enzyme activities or
mitochondrial volume density in the intermyofibrillar compartment of rat soleus fibers following the exposures to spaceflight
or hindlimb suspension. The phenomenon is thought to be associated with the more pronounced decrease of fiber crosssectional area [Desplanches et al, 1993]. The subsarcolemmal mitochondrial volume density was shown to be decreased that
was interpreted as the mitochondrial redistribution [Bell et al., 1992].
The present study was purposed to analyze the effects of long-duration unloading on the oxidative enzyme activities
in intermyofibrillar and subsarcolemmal compartments of slow-twitch (ST) and fast-twitch (FT) soleus muscle fibers in man.
5 healthy men volunteered to participate in the 120 day 6 head-down tilt bed rest (BR). Before BR, after 60 and 120
days of BR muscle samples were obtained from soleus by means of Bergstrem needle biopsy. Muscle samples were processed
for quantitative histochemical analysis and stained for succinate dehydrogenase (SDH) demonstration (according to Nachlas)
[Lojda et al, 1982]. Enzyme activities were measured cytophotometrically by means the Leica microscope image analyzer
Quantimet 500M in the central and subsarcolemmal region of the fiber separately. Fiber typing was done by means of acid
myofibrillar ATPase staining.
After 60 days of BR the small (8-9% p<0.05) decline in mean fiber SDH activity was found. The more profound
changes were seen in FT fibers (12%). After 120 days BR the SDH activity continued to decline and was in ST fibers 14% and
in FT fibers 16% lower as compared to the baseline levels.
The subsarcolemmal SDH activity at the 60th day of BR was found to decrease more pronouncedly in ST fibers
(~10%) than in FT fibers (4-6%, ns). The further (120 days) decrease of subsarcolemmal SDH activity in ST fibers was not
sufficient (13% in comparison with the baseline level), meanwhile in FT fibers the decline was significant and reached 8%.
The intermyofibrillar SDH activity drop in ST fibers after 60 days (7%) and after 120 days (11%) of BR was not as deep as in
subsarcolemmal compartment. In FT fibers the oxidative potential decrease was more pronounced in intermyofibrillar compartment (14%) than in subsarcolemmal one and reached even 18% after 120 days of BR.
Thus the ratios of oxidative potential changes in intermyofibrillar and subsarcolemmal compartments in ST and FT
soleus fibers in the course of long-duration unloading differed substantially.
43
POSSIBLE INVOLVEMENT OF INOSITOL 1,4,5 TRISPHOSPHATE PATHWAY IN FEEDBACK LOOPS
RESPONSIBLE
FOR CONTRACTILE AUTO-OSCILLATIONS
OF PHYSARUM PLASMODIUM
N.B. Matveeva, V.A. Teplov, S.I. Beylina
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Russia, E-mail:
beylina@venus.iteb.serpukhov.su
It is well known that inositol 1,4,5 trisphosphate (IP 3) is an intracellular messenger which triggers Ca2+ release from
the internal stores due to its binding to specific Ca2+ release channel protein, the IP3-receptor. The key enzyme of the phosphoinositide turnover phospholipase C (PLC) hydrolyzes phosphotidylinositol 4,5 diphosphate (PIP 2) to IP3 and thereby mediates
a wide variety of the agonist-induced cellular functions, including the appearance of calcium oscillations and waves. However,
whether IP3 pathway involves in creation of the self-sustained oscillations of both contractile activity and intracellular Ca 2+
concentration in the Physarum polycephalum plasmodium has never been investigated. All available data on the phosphoinositide turnover are restricted by evidence on strong fluctuations of both PIP 2 [1] and IP3 [1, 2] intracellular levels in the course of
plasmodium cell cycle.
We investigated the influence of two differently acting inhibitors of PLC on the contractile oscillations in the
Physarum plasmodium. The first, neomycin, is a positively charged aminoglycoside, which forms an electroneutral complex
with PIP2 and thereby blocks the binding of PIP2 to PLC [3]. The second, 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17yl)amino)hexyl)-1H-pyrrole-2,5,-dione (U-73122), is an aminosteroid, which exerts its inhibitory action on PLC as a potent
sulfhydryl reagent [4].
Physarum polycephalum was grown in a liquid shaken culture as a suspension of the microplasmodia, multinucleate
cells 100-200 µm in diameter [5]. All nuclei in each of the microplasmodia divide with precise natural synchrony, but individual cells in the population are randomly distributed according to the cell cycle position. Microplasmodia were harvested at the
logarithmic phase of growth, resuspended in a non-nutritional medium and then used for assays. Oscillatory contractile activity
of the microplasmodia was estimated by optical technique through registration of their thickness changes.
Neomycin was shown to cause a progressive disturbance of the oscillatory contractile activity of microplasmodia. Oscillations became irregular, their amplitude gradually decreased, and then the oscillations disappeared. Within 90 min after the
inhibitor application oscillations were completely suppressed in 67% of the cells (n=27) at 0,1 mM and in 82% (n=11) at 1
mM. As the damped oscillations could last from 5 min to 2 h, the duration of oscillation decay seems to be dependent on not
only the neomycin concentration but also on the state of the microplasmodia, particularly, on their cell cycle position.
The changes of contractile oscillations induced by U-73122 were similar, but more rapid than that induced by neomycin. The only peculiarity often observed to be was a high frequency of the damped oscillations. In the range of 1-10 µM U73122, an increased number of reacting microplasmodia rather than an acceleration of the response was observed with increasing concentration of the inhibitor. In the interval from 30 to 60 min after U-73122 application, no oscillations were observed in
30 % of the cell (n =23) at 1-2 µM, in 46% (n=13) at 5µM and in 95% (n=19) at 10 µM. Dithiothreitol, a thiol-reducing agent,
effectively prevented U73122 inhibition, but was ineffective when added after U73122. As the action of U73122 was reversible, no nonspecific targets essential for the plasmodium survival were affected before the contractile oscillations stopped.
Both drugs affected the motile behaviour of microplasmodia. Cells ceased to spread and to crawl. The distinct changes
in their appearance became visible, often a long before the oscillations were completely suppressed. Neomycin strongly enhanced adhesion and induced microplasmodium flattening. Their surface smoothed down, but pseudopodia remained extended
or withdrew very slow. Unlike neomycin, U73122 decreased adhesion, provoked a rapid withdrawal of pseudopodia and induced microplasmodium rounding. Since a decrease in IP3 level has been well documented for both inhibitors of PLC, the different states of PIP2 rather than inhibition IP3-receptor (and thereby decrease of averaged Ca2+ level) are crucial for observed
changes in cell shape.
This work was supported by The Russian Foundation for Basic Research.
References
1. Makarov P. R., Rotaru V. K., Polteva N. A., Evtodienko Yu. V. 1994, FEBS Lett. 344, 47-49.
2. Belyavsky M. A., Sauer H. W. 1992, Eur. J. Cell Biol. 58, 371-376 .
3. Gabev E., Kasianovich J., Abbott T., McLaughlin S. 1989, BBA, 979, 105-112.
4. Ciapa B., Whitaker M. J. 1986, FEBS Lett. 195, 347-351.
5. Daniel J. W., Baldwin H. H. 1964, in Methods of Cell Physiology (ed. Prescott J.) Acad. Press, New York, 1, 9-41.
ROOM TEMPERATURE TRYPTOPHAN PHOSPHORESCENCE
OF NATIVE AND INACTIVATED ACTIN
Vladimir M. Mazhul1, Ekaterina M. Zaitseva1, Mikhail M. Shavlovsky2,
Anna L. Zarubina3, Irina M. Kuznetsova3 and Konstantin K. Turoverov3
1
Institute of Photobiology Nat.Acad.Sci.Belarus, Akademicheskaya Str., 27,
Minsk, 220072, Belarus, E-mail: ipb@biobel.bas-net.by;
2Institute for Experimental Medicine, Russian Academy of Medical Science,
St. Petersburg 197376, E-mail: molgen@molgen.iem.ras.spb.ru,
3Institute of Cytology, Russian Academy of Sciences,
194064 St. Petersburg, Russia, E-mail: kkt@mail.cytspb.rssi.ru
Characteristics of tryptophan phosphorescence at room temperature (spectra of phosphorescence and phosphorescence
decay curves) have been recorded for native, inactivated and F-actin. Measurements were performed in extensively deoxygenated samples using high sensitive homemade device of the Institute of Photobiology. Inactivated actin was prepared by incubation of G-actin at 70C during 30 min., by treatment with 4 M urea or 1.5 M guanidinium hydrochloride or by Ca 2+ ion removal (incubation with 5 mM EDTA). Actin concentration was 1.5-2.0 mg/ml. It has been found that not only native and F-actin,
but also inactivated actin, which fluorescence spectrum is red shifted in comparison with that of native one, had tryptophan
44phosphorescence at room temperature.
It has been found that room temperature phosphorescence (RTP) decay kinetics of actin was biexponential. The contribution of fast component (1=12.81.5 ms) is 0.76 and that of slow component (2=19422 ms) is 0.24. It has been shown
that 1 and 2 of inactivated actin were near 6 and 2 times increased in comparison with that of native actin. This result suggests
that millisecond internal dynamics of inactivated actin in the vicinity of tryptophan residues capable to RTP is substantially decrease in comparison with the native protein. This result consists with earlier obtained data concerning the peculiarities of inactivated actin structural organization, intramolecular mobility of its tryptophan residues and their microenvironments.
The analysis of microenvironment properties and peculiarities of localization of tryptophan residues in actin shows
that the main contribution to the RTP of G-actin give tryptophan residues Trp 340 and Trp 356.
Acknowledgment: This research was supported by grants from the RFBR Grant 00-04-49224, 00-04-81082 Bel
2000_a, BRFFR Grant B99R-128, ISTC Grant B-441.
NEW PATHWAYS IN THE MEDICAL MANAGEMENT
OF PATIENTS WITH CONGESTIVE HEART FAILURE (CHF) BASED ON BIOCHEMICAL-FUNCTIONAL
CORRELATION
IN THE MECHANISM OF CARDIAC DYSFUNCTION
AND ACTION OF REFRAKTERIN
A.N. Melia, Z.N. Klimiashvili, E.A. Chikobava, N.V. Salibegashvili,
I.N. Lejava, I.G. Kankava, G.V. Sukoian*, D.R. Tatulashvili*,
N.V. Karsanov*
Cardiological Clinic “GULI”, *The Republican Research Center of Medical Biophysics and Introduction of New Biomedical Technologies
(Head - Corr.member of Russian Academy of Medical Science, Prof. Karsanov NV), Tbilisi, Georgia
CHF is a major cause of morbidity and mortality in the world. Its prevalence is increasing, particularly among the elderly persons. CHF is a complex syndrome which manifestations result from underlying disturbance in cardiac function. The
multifactorial character of CHF syndromes makes it very difficult to yield to adequate therapy and in spite of all available
medical therapy in patients with refractory heart failure the damage of myocardium progresses. The primary reason of our investigation was to elucidate a structural-functional damage in the energy supply, antioxidant defense, neurohumoral and endothelial systems activities and its correlation with parameters of systolic and diastolic function of myocardium in patients with
CHF.
The study includes 10 individuals without cardiovascular diseases as controls and 89 patients (aged 41-80) with
NYHA functional class II-III CHF caused by ischemic cardiomyopathy with similar pretreatment characteristics and (i) under
treatment with a gold standard 90-th (combination of cardiac glycoside, diuretics, angiotensin converting enzyme (ACE) inhibitor and –blocker) or (ii) under the therapy with Refrakterin, diuretics and ACE inhibitor. Refrakterin is new cardiotropic drug
with the antiischemic, powerful antihypoxic and antioxidant properties (combined drug containing -acetyldigoxin, nicotinamide adenine dinucleotide (NAD), cytochrome C, oxyfederine, and inosine - patent of Russia 2035908 and USA 5,602,105).
Informed consent was obtained from each patients included in the study. Left ventricular systolic and diastolic dysfunction was
registered by the presence of dilated left ventricle (internal dimension in diastole>60 mm) in association with decrease of ejection fraction (EF<45%) and by abnormal ratio of peak velocities of the early wave (Ve) and the atrial wave (Va) according to
2D and M-mode echocardiography.
The plasma-renin activity (PRA, ng/ml per h) and endothelin-1 (mg/ml) content were determined with commercially
available RIA kits (Serino, BioChem Immunisystems, Italia, S.P.A and Human Endothelin-1 Immunoassay R&D Systems, Inc.
respectively). The content of noradrenaline, adrenaline, dopamine and DOPA were determined fluorometrically and ACE activity with N- (3-[2-furyl]acryloyl)-l-Phe-Gly-Gly by spectrophotometric method. The methods for determination of
NAD/NADH ratio and cytochrome C and activities of enzymes of antioxidant defense system were described earlier (NR Paleev et al., Klin.med., 1997, 4, 34-41 and NV Karsanov et al, Russian Medical News, 2001, 2, 31-42). A commercial statistics
software package (Microsoft Excel 7.0) was used for data analysis.
It has been found that in patients with CHF the degree of noradrenaline and adrenaline increase and the decrease of
dopamine in plasma reflect the severity (functional class) of CHF (r=0.56, p<0.01 and 0.66, p<0.05 respectively). The multifunctional correlation between both noradrenaline and adrenaline with FC CHF (r = 6.78, p<0.001) suggests an important role
of sympathoadrenal system in the CHF progression. The treatment of patients with gold standard therapy does not improve the
balance and content of the components of the sympathoadrenal system which correlates with the sharp decrease of
NAD/NADH ratio and the increase of cytochrome C in plasma (r between total noradrenaline + adrenaline + dopamine on the
one hand and NAD/NADH, cytochrome C on the other hand is 6.8, p<0.001). Moreover, the NAD/NADH and cytochrome C
content in plasma in contrast to content of ACE and renin correlates with Vcf and Ve/Va ratios.
Influence of traditional therapy even in case of treatment with ACE does not inhibit the high level of plasma activity
of renin and increased endothelin-1 (the plasma content of endothelin-1 under traditional treatment changes from 20.81.2 to
18.70.4 and after refrakterin - from 26.51.4 to 13.3  0.8 pg/ml, in the norm 9.01.4 pg/ml). Treatment with Refrakterin
permits the breakdown of refracterity to conventional therapy and decreases the functional class (FC) of CHF from 3.20.2 to
2.10.2, end diastolic dimension by 16%, mean pulmonary artery pressure by 40%, velocity of circumflex fiber shortening
(Vcf) by 25% and increases left ventricular ejection fraction (LVEF) from 25.52.5 to 35.52.3% (by 38%). These improvements in cardiac hemodynamics correlate with normalization of redox-potential (NAD/NADH ratio) and plasma cytochrome C
concentration. At the same time these two parameters of energy supply system correlate with the plasma renin activity (r=6,3,
p<0,001). Moreover, only the including of Refrakterin in the therapy of patients with CHF leads to the decrease of plasma renin activity while treatment with ACE inhibitors leads to its further increase. Such an improvement of endothelial dysfunction
may be result of pronounced effect of Refrakterin on the antioxidant activity, activation of superoxidedismutase and inhibition
of NADH-dependent endothelial NO-synthase (production of NADH also increases under traditional treatment and decreases
after Refrakterin).
45
Thus, management of CHF through the improvement of cardiac geometry and biophysical-biochemical parameters of
cardiac muscle and blood is a new therapeutic strategy in the treatment of refractory form of CHF.
QUANTITATIVE ANALYSIS OF VARIABILITY
OF THE ELECTROCARDIOGRAMS TYPICAL
OF POLYMORPHIC ARRHYTHMIAS
A.V. Moskalenko1, N.I. Kukushkin2, C.F. Starmer3 and A.B. Medvinsky1
2Institute
1Institute of Theoretical and Experimental Biophysics RAS,
of Cell Biophysics RAS, Pushchino, Moscow Region, 142290 Russia;
3Medical University of South Carolina, Charleston,
South Carolina, 29425 USA
In this work, a new approach is developed to analysis of electrocardiographic variability during polymorphic arrhythmias.
At present, electrocardiography is the most common method for differential diagnosis of cardiac arrhythmias. Moreover, many arrhytmias can be detected only by electrocardiographic analysis. There are sufficient grounds to expect that the role
of this method in differential diagnosis of cardiac arrhythmias will increase in the future.
Polymorphic ventricular tachycardias, or high-frequency arrhythmias with high variability of oscillation, pose a special problem in analysis of patient electrocardiograms. The existing methods are frequently inapplicable in the case of polymorphic arrhythmias because of the difficulties in identifying individual QRS complexes.
The approach proposed is free of the need to identify these complexes. The approach is based on quantitative comparison of two ECG fragments neighboring in the embedding space and normalization of the results obtained. Following this approach we develop the normalized-value analysis of electrocardiographic variability (the NVAEV method).
In the framework of this analysis, we calculate the distance between the current fragment and the first fragment most
closely resembling current fragment in the subsequent recording. Since each of the fragments corresponds to vector in pdimensional embedding space, the distance between the fragments is determined by the norm. For further analysis of electrocardiograms or their fragments of arbitrary lengths, we construct some integral characteristics of electrocardiographic, i.e. averaged variability, and its variation. Their changes in time (as the averaging window of a fixed duration is being shifted along
the time axis of the ECG) give us the trajectories in the parameter space, which allow us to judge the dynamics of changes in
ECGpolymorphism.
As result, every fragment of ECG or an entire electrocardiogram can be described by two parameters, one of which
one is an index of electrocardiographic variability while the other parameter characterizes how the index of electrocardiographic variability changes in time.
These two parameters are useful in assessing the polymorphism and they meet the following criteria.
(i) They make it possible to compare polymorphism of two fragments or two electrocardiograms of arbitrary lengths.
(ii) They are independent of the shape and amplitude of the QRS complexes (in fragments where the complexes are discernible).
(iii) They afford a possibility of monitoring changes in polymorphism along the arrhythmic ECG curve even if individual
QRS complexes cannot be identified.
(iiii) The information yielded by these parameters does not contradict the results obtained with the qualitative methods that
are commonly used at present in analysis of electrocardiograms.
The approach proposed is promising in diagnosing polymorphic arrhythmias and in studying their mechanisms.
THE EFFECTS OF INSULIN ON THE CONTRACTILITY OF ACTIVE GROUND SQUIRRELS MYOCARDIUM:
A POSSIBLE MECHANISM
O.V. Nakipova, L.A. Andrejeva, N.A. Chumaeva, L.S. Kosarskii,
S.G. Kolaeva, N.I. Kukushkin
Institute of Cell Biophysics, Russian Academy of Sciences,
Pushchino, Moscow Region, 142290 Russia
Insulin is widely used in cardiology in some heart disorders, still its mechanisms of action on the myocardium have
not been clarified as yet, and the data published are commonly contradictory. The investigation of the effects of reducing tissues sensitivity to insulin (insulin resistance) observed in different pathologies (diabetes-II, obesity, artificial hypothermia) and
their overcoming is of great importance. In the heart of humans and warm-blooded animals insulin resistance is usually accompanied by heart mechanical functions disorder and calcium overloading. In connection with this, hibernators are of interest,
because during the transition from the active state to hibernation and on arousal their tissues sensitivity to insulin, calciumtransporting systems activity and the mechanical properties of the heart are also changed, but these changes are usually reversible. The use of hibernators heart as a model for investigation of insulin effect on the myocardium can help to find out the
ways of natural regulation of cardiac functions in pathologies concerning with changes in cells sensitivity to the hormone.
The effect of insulin on contractility of the hibernators heart has never been documented. During the last few years
this question has been extensively studied in our laboratory (Nakipova et al., 1997, 2000). Our previous results indicated that in
ground squirrel papillary muscles insulin exerts both cardiostimulating and cardiodepressive effects at 0.5 Hz of frequency
stimulation. The direction of insulin effect depends on hormone concentration used, state of animals (active, hibernating or
arousing) and on seasonal periods (summer, autumn and winter).
The main aim of the present study was to further characterize the peculiarities of insulin action on myocardium of active ground squirrels. Papillary muscles of right ventricle of the heart in ground squirrel C. Undulatus were studied at 30±1ºC.
The following groups of squirrels were used: summer (June-July); autumn (October-November); active winter (December46February); and spring (April-May). Winter squirrels were taken between bouts.
Recently we have shown that the effect of insulin on isometric contraction of active animals depends on the frequency
of stimulation within the range from 0.1 to 1 Hz (Nakipova et al., 2001, in press). At the frequencies 0.07–0.3 Hz the low doses of insulin (0.1–0.5 nM) caused an elevation of the average amplitude of contraction on 15-25 % of control value. In the
range of frequencies between 0.3–0.5 Hz the effect of the same insulin doses was in the majority of cases biphasic: positive for
the first 7-12 min and negative after 12 min of hormone action. In this range of frequencies the relatively high doses of insulin
(1-50 nM) only reduced the contractile force. At the frequencies above 0.4–0.5 Hz insulin caused only the negative inotropic
effect at all concentrations used. Maximum effect was noted at the frequencies 0.5-0.8 Hz averaged between 40-60%.
The effect of insulin was accompanied by changes in force-frequency relationships (FFR or “staircase”). Earlier we
have shown that the character of FFR curve in active animals depends on seasonal period (Andreeva et al., 2001). In summer
and some autumn squirrels FFR is entirely negative and is similar to that in adult rats. In active autumn and winter squirrels the
force staircase contains both positive and negative components in different ranges of frequency stimulation. In the majority of
cases insulin eliminates the positive component of FFR and greatly amplifies the negative one. Thus, the seasonal-related differences in the force staircase in active squirrels are disappeared after the insulin action.
For elucidation of the role of [Ca2+]i-maintaining mechanisms in FFR changes induced by insulin, rest potentiation, an
enhance of the first contraction (F1) following a pause, has been used to reflect the level of intracellular stored calcium. In animals of autumn period the F1 value was highly augmented by insulin. In animals of summer period insulin caused slightly
changes of F1 amplitude. These findings permit us to suggest that the main mechanism of insulin action in autumn animals is
connected with an enhanced level of releasable Ca 2+ from SR.
The effect of insulin was not affected by nifedipine (inhibitor of L-type Ca2+-channel of sarcolemma), but can be reversed by agents blocking Na+–Ca2+–exchange (tetraphenylphosphony, doxorubicin). Recently the preliminary results have
been obtained which indicate that seasonal differences in the activity of tyrosine kinase and tyrosine phosphatase might be involved in divergent effect of insulin on contractility of ground squirrels heart.
This study was supported by the Russian Foundation for Basic Research, project № 01-04-48-199 and the Federal
Central Program “Integration”, project № A 0055.
References
Nakipova O.V., Gainullin R.Z., Safronova W.G. et al. Biofizika, 1997, v. 42, p.1297.
Nakipova O.V., Gainullin R.Z., Andreeva L.A. et al. Biofizika, 2000, v.45, p.344.
Andreeva L.A., Nakipova O.V., Chumaeva N.A. et al. DAN, 2001, v.377(1), p.108.
Nakipova O.V., Andreeva L.A., Chumaeva N.A. et al. DAN, 2001 (in press).
47
CALCIUM SIGNALING MECHANISMS IN MUSCLE CELLS
G.A. Nasledov
Sechenov Institute of Evolutionary Physiology and Biochemistry,
St.-Petersburg, Russia
It is known that many cellular functions are activated by intracellular calcium signaling - strong regulated brief increase of cytosolic Ca2+ concentration. In muscle cells this increase serves, first of all, as a trigger for contraction activation. In
different kinds of muscle cells the sources of Ca2+ ions are the extracellular medium and/or the intracellular stores, namely the
sarcoplasmic reticulum (SR). Further, it was established that the role of intracellular messenger in the coupling of external
stimuli to calcium release plays inositol-1,4,5-trisphosphate (IP3) (Berridge, Irvine, 1984; Vergara et al, 1985). The mechanism
of IP3 mediated Ca2+ release has been described in details for many kinds of non-muscle cells and smooth muscle cells, and can
be considered as a main basic mechanism of Ca2+ signaling in the majority of animal objects.
Another mechanism of intracellular signal transducing, highly specialized for contraction triggering pathway, has
been developed in vertebrate striated and cardiac muscles. Here the process of activation of contraction, named excitationcontraction coupling (ECC), is initiated by the depolarization of the external cell membrane and its derivative - transverse
tubular (T-tubule) membrane, which triggers the release of Ca 2+ from the sarcoplasmic reticulum (SR) by calcium channels and
associated receptors. Dihydropyridine receptors (DHPR) / L-Ca channels / voltage sensors - on the tubular membrane, and
ryanodine receptors (RyR) / Ca-release channels - on sarcoplasmic reticulum (SR) membrane, are two proteins, which face
each other on T- and SR-membranes, and functionally interact in signal transducing.
The interaction of DHPR - RyR receptors/channels in muscle cells has been developed on the basis of RyR functioning in nonmuscle cells, in which it serves, in parallel with IP3 receptors, for Ca2+-liberation from intracellular stores. In muscle
cells the Ry receptors are concentrated in SR membrane regions, which close facing the T-membrane. This facing can occur
between SR membrane and the surface of cell membrane, or between SR membrane and T-membrane (dyads and triads). The
distance between the two membranes in triads is equal to 10 nm, and in skeletal muscles the facing DHPRs and RyRs is connected by “feet” - the intramembrane (sarcoplasmic) part of RyRs. The receptors on each membrane are arranged in regular arrays. The interaction between DHPRs and RyRs undergoes the considerable development during the muscle evolution. In cardiac muscles the signal transduction is performed by Ca-induced Ca-release process (CICR). In vertebrate skeletal muscles the
direct molecular interaction is superimposed besides CICR, and is accomplished by feet. The CICR mechanism still exists in
mammalian muscles though it is not functional. Further, the sensitivity of RyRs to Ca 2+ ions in mammals has been significantly
reduced by Mg2+ binding to sites on cytoplasmic part of RyRs, probably, in order to exclude the Ca-induced modulation, which
can disturb the direct DHPRs to RyRs signal transduction.
It is generally accepted that RyRs the only are necessary here for contraction activation. The morphology of Tmembrane and SR-membrane contacts supports this opinion, because all contact regions are occupied by regularly arranged
functioning RyRs and DHPRs, named “couplons”, and feet between two membranes, visible on electron micrographs, providing the molecular interaction of these two receptor/channel proteins, acting in concert during ECC process. Meanwhile, the IP 3
gated channels on Ca2+ storage membranes do not disappear in skeletal muscles. In frog and mammalian skeletal and cardiac
muscles the IP3 receptors were found as well, but their participation in contractile activation in intact muscles remains controversial [Hidalgo, Jamovich, 1989; Posterino, Lamb, 1998]. Presumably, they can play the regulatory role, though no data are
available about it. But in some distinct cases the IP 3 gated channels can participate in contraction activation in parallel with
RyR-gated channels. This was shown for muscles of the lowest vertebrates, lamprey (Nasledov et al., 2001), and for slow rat
muscle (in opposite to fast muscle) (Talon et al., 2000). In these cases both mechanisms - based on IP3 receptors and RyRs
functioning - participate in activation of contraction. The conclusion is made, that the change of IP3 coupling mechanism for
RyR coupling occurred not suddenly in evolution, but appeared as a gradual process, during which both of them can be observed in parallel. Presumably, the higher the muscle is developed for performing the fast contractions, the more the activation
of contraction belongs to RyR-operated Ca-release channels, and that RyRs themselves change from CICR activation mechanism to direct DHPRs - RyRs molecular interaction.
Supported by Russian Foundation for Basic Research, grant № 99-04-49953.
PLASTICITY OF RAT M. SOLEUS UNDER CONDITIONS
OF LONG TERM HYPERGRAVITY
T. Nemirovskaya*, B. Shenkman**
*Moscow State University, Faculty of Fundamental Medicine,
**SSC-RF Institute for Biomedical Problems, RAS; Moscow, Russia
It is known that adaptation to physical exercise training frequently induces increase in cross-sectional area (CSA) of
muscle fibers (MF). Some authors observe transformation of MF, the increase in mitochondrial volume density, and in absolute volume of myofibrils. At the same time, only one work deals with changes in heavy chain myosin isoforms, and in CSA of
MF after 2 weeks of gravitational loading. The aim of the present work was to evaluate plasticity of skeletal muscle exposed to
chronic hypergravity.
Two groups of male Wistar rats (7 animals in each) were exposed to 19 or to 34 days of chronic +2G hypergravity
(HG), and 7 rats served as controls. The rats were rotated in peripheral cages on a 4-cage centrifuge for laboratory animals (radius - 4 m, constant speed – 21 r.p.m.). The animals received a standard vivarium rat chow and water ad libitum. After stoppage of the centrifuge the rats were dissected by decapitation. All the procedures were approved by Committee on Biomedical
Ethics, IBMP, RAS.
M. soleus was isolated immediately after the rats were scarified, and divided into 2 pieces. One piece, intended for
light microscopy, was frozen in liquid nitrogen, and another piece, intended for electron microscopic studies, was fixed in glutaral aldehyde, and in paraformaldehyde 0.1 M phosphate buffer. Morphological characteristics of m. soleus were studied by
means of light and electronic microscopy.
48
By the 34th day of HG exposure CSA of m.soleus MF was similar in accelerated and in control rats. It was found that
percentage of hybrid MF was significantly increased only by the 19 th day, but not by 34th day. Along with that, rats exposed to
19 days of HG demonstrated increased number of collagen muscle fibers, and decreased volume density of the central (but not
of the periphery) myofibrils in comparison with the controls (86.98  2.24 vs. 93.99  2.01, P< 0.05). In that group most fibers
had centrally located nucleus with highly condensed chromatin along the inner margin, probably, indicating disturbed translation and transcription.
However, volume density of both central and peripheral mitochondria was considerably increased after 19 days of acceleration in comparison with the controls (6.061.05 vs. 2.760.47, P<0.05, and 14.391.40 vs. 10.240.85, P<0.05, accordingly). Similar increase of mitochondrial volume density is frequently induced by physical training.
In general, it could be concluded that long-term +2G hypergravity induces numerous destructive processes in skeletal
muscle.
The work was supported by INTAS 99-01190 grant.
SIGNALLING ROLE OF Са2+ AND Na+ IN MUSCLES:
COMPARATIVE ASPECTS
Vladimir P. Nesterov
Sechenov Institute of Evolutionary Physiology and Biochemistry RAS,
St.Petersburg, Russia
The elucidation of transmembrane INa role in contraction activation of phasic skeletal muscle fibres (SMF) and myocardium cells (MC) of vertebrates (Nesterov 1971-2000; Leblanc & Hume, 1990; Lipp & Niggli, 1994) has opened the way to
reveal a general strategy of E-C coupling system rearrangement in the course of muscle specialization directed to the acceleration of discrete (single, twitch) contraction. The generalized scheme of regular changing of pathways and mechanisms of Ca2+
and Na+ involvement in E.-C. C. processes during muscle contractile function progressive evolution is presented, where the attention is focused on: i) natural grounds for Ca 2+ and Na+ use as intracellular trigger signal transmitters; ii) the creation of different regulator and effector proteins (membrane Na +- and Са2+-channels and pumps, Na+/Ca2+-exchanger, as well as different
Ca2+-receptor proteins - Tn C, calmodulin, etc.), their clustering and reorganization for optimal use of these cations to fit this
function; iii) mechanisms of intracellular Ca2+ mobilization (ICM) in different muscle types and increasing significance of inward INa for intensification of ICM in enlarging striated MC (INa in addition to ICa) and SMF (INa without ICa, including mechanism “INa-induced Ca2+ release from SR”). We have concluded that just INa use for supramaximal ICM has ensured acceleration
of discrete muscle contraction and the most complete dependence of SMF contractile function on the CNS impulse control.
INTERACTION BETWEEN RECOMBINANT RAT NUCLEOSIDE DIPHOSPHATE (NDP) KINASE  AND
RHODOPSIN/G-PROTEIN TRANSDUCIN COMPLEX IN BOVINE RETINAL MEMBRANES PROVIDES A
NOVEL POSSIBLE MECHANISM OF EXTREMELY RAPID TRANSDUCIN ACTIVATION IN LIVING RODS
N.Ya. Orlov 1,2), A.A. Freidin 1), D.N. Orlov 1) and N. Kimura 2)
1) Institute
of Theoretical and Experimental Biophysics RAS,
Pushchino, 142290, Russia,
2) Tokyo Metropolitan Institute of Gerontology,
35-2, Sakaecho, Itabashi-ku, Tokyo-173, Japan
It is widely accepted that G-proteins including retinal rod G-protein transducin (Gt) are activated by receptorstimulated GDP/GTP exchange [Stryer (1986) Ann. Rev. Neurosci. 9, 87-119]. There have been a lot of demonstrations supporting the exchange mechanism. Nevertheless, there are some uncertainties as to whether the exchange process is rapid
enough to provide the extremely high rate of Gt activation in living ROS. Our recent calculations and analysis of modern microcalorimetric data [Langlios et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4677-4682] demonstrate that single bleached light
receptor rhodopsin (R*) molecule consecutively activates more than 10,000 Gt molecules per 1 s, whereas time of collision between a single R* and Gt molecules was estimated to be about 60 µs [Pugh and Lamb (1993) Biochim. Biophys. Acta 1141,
111-149]. Hence, any step of GDP/GTP the exchange must occur in the time range of several tens of µs (and, thus, R* must
accelerate dissociation of stable Gt-GDP (time decay is in the order of 10 5 s) by a factor of 109-1010). Such a high rate has not
been obtained experimentally yet for any of the GDP/GTP exchange steps. Although most of the well-known kinetic studies
demonstrated high rate of Gt activation, they did not provide us with the convincing information about the precise molecular
events. Our recent direct measurements carried out by rapid filtration method with labeled GDP and GTP[S] under wide range
of experimental conditions demonstrated that R*-stimulated Gt-GDP dissociation is slow (about 5-7 s) to provide rapid generation of active Gt molecules in rods.
In the present work, an attempt of further analysis of the mechanism of rapid Gt activation was made. We formulated
a new hypothesis on the mechanism of transducin activation based on the following data: (1) the recently discovered phenomenon of high-affinity GTP[S]-dependent interaction of soluble bovine nucleoside diphosphate kinase (NDP kinase) and rat recombinant NDP kinase  (but not NDP kinase one) with the complex between R* and Gt in bovine photoreceptor membranes [Orlov et al.(1996) FEBS Lett. 389, 186-190]; (2) the existence of low-affinity interaction between NDP kinase and R*
[Orlov et al.(1996) FEBS Lett. 389, 186-190; Orlov and Kimura (1998) Biochemistry (Moscow) 63, 208-217]; (3) recent
demonstration of the ability of NDP kinase to phosphorylate GDP that was covalently attached in active site of GTP-binding
protein Rad (member of a new subfamily of Ras-related GTPases) [Zhu et al.(1999) Proc. Natl. Acad. Sci. USA 96, 1491114918], and (4) well-known common scheme of Gt activation [Stryer (1986) Ann. Rev. Neurosci. 9, 87-119]. According to the
hypothesis, NDP kinase interacts with the R*-Gt complex and phosphorylates bound GDP. Then, active Gt-GTP releases,
whereas low-affinity R*-NDP kinase complex has no time to dissociate before the rapid (1 ms) association with new GtGDP molecule. It means, that R*-NDP kinase complex once formed will interact catalytically with many Gt-GDP molecules
and provides a high rate of Gt-GTP formation even at low NDP kinase concentration (10 M) in rods. It is worth noting, that 49
NDP kinase hexamer itself is sufficiently active (turnover rate constant is as high as 10 4 s-1 per subunit) to provide the high rate
of GDP phosphorylation. The possible approaches to the further experimental checking of the hypothesis are discussed.
Acknowledgements. We are grateful to Dr. Nelly Bennett for her help in rapid filtration experiments and many fruitful discussions. Fruitful discussions with Drs C. Pfister, T.G. Orlova, S.I. Beylina, A.V. Lazarev, T. Toda, N. Ishikawa and
Prof. E.A. Burstein are gratefully acknowledged.
50
COMPARATIVE STUDY OF RECOMBINANT RAT NUCLEOSIDE DIPHOSPHATE KINASES  AND  AND
THEIR TAGGED, MUTANT AND CHIMERAS FORMS BY PROTEIN FLUORESCENCE
N.Ya. Orlov, Ya.K. Reshetnyak, T.G. Orlova, D.N. Orlov,
E.A. Burstein, Ya. Ishijima* and N. Kimura*
Institute of Theoretical and Experimental Biophysics RAS, Pushchino, 142290, Russia; *Tokyo Metropolitan Institute of Gerontology,
35-2, Sakaecho, Itabashi-ku, Tokyo-173, Japan
Nucleoside diphoshate kinases (NDP kinase) of mammals are hexamers of two sorts of randomly associated highly
homologous subunits of 152 residues each and exist as NDP kinase isoforms. The physiological significance of the existence
of the isoforms with similar catalytic and structural properties remains unclear, but our studies of recombinant rat NDP kinase
 and , each containing only one sort of subunits, revealed that NDP kinase , but not  one, is able to interact with the complex of bleached light receptor rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values [Orlov et al.
(1996) FEBS Lett. 389, 186-190]. Thus, it was found that (i) NDP kinase really interacts with G-proteins and therefore it may
be involved in cellular transduction, and, moreover, (ii) the isoforms differ in their ability to interact with receptor-bound Gprotein. The results of recent investigations of the regulation of Rad protein (a member of sub-family Ras-related GTPases)
confirmed that NDP kinase interacts with GTP-binding proteins and such interaction results in phosphorylation of GDP in its
active site [Zhu et al. (1999) Proc. Natl. Acad. Sci. USA 96, 14911-14918].
In order to reveal a molecular basis of the differences between the isoforms, we performed a comparative study of
their intrinsic fluorescence properties in a large range of solvent conditions. The NDP kinase  and  both contain tree tryptophan (Trp78, 133, and 149) and four tyrosine (Tyr52, 67, 147, and 151) residues, but exhibit at pH 8 pronounced differences in
spectral maximum positions (342 vs 335 nm), spectrum shape and quantum yields (0.42 vs 0.21), and behave differently under
pH titration. While NDP kinase  undergoes spectral changes in the pH range 5-8 with the mid-point at 6.2, no clear indication
of a structural change of NDP kinase  under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for
fluorescence of NDP kinase  resemble the pH-dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinases  and  in spectrofluorimetric pH-titration curves, and the differences in their
ability to interact with rhodopsin-transducin complex may have the same physical basis, which in its turn could be a physicochemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of threedimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and
pH-titration behavior of the isoforms  and  may associated with the difference in their global electrostatic charges, rather
than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at the deeply
buried Tyr52 in the active site can be explained by its deprotonation at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase : (i) rather long-wavelength emission of NDP kinase  (spectrum maximum at ca. 341 nm) at pH ca. 8, (ii) extremely low accessibility to external quenchers and (iii) an unusually high quantum yield value (ca. 0.42).
For further analysis chimeras NDP kinase (1-118)(119-152) and NDP kinase (1-118)(119-152), mutant proteins
in which the active site histidine 118 was replaced with alanine (NDP kinase  (His118Ala) and NDP kinase  (His118Ala))
and tagged enzymes (HA-NDP kinase , HA-NDP kinase (1-118)(119-152), HA-NDP kinase (1-118)(119-152), HANDP kinase  (His118Ala) and HA-NDP kinase  (His118Ala)) containing hemagglutinin (HA) epitope YPYDVPDYA in Nterminus have been studied. It was found that chimeras NDP kinase (1-118)(119-152), HA-NDP kinase (1-118)(119152), mutant NDP kinase  (His118Ala) and HA-NDP kinase  (His118Ala) strongly resemble in their fluorescence properties
and conformational mobility NDP kinase . NDP kinase (1-118)(119-152), HA-NDP kinase  and HA-NDP kinase (1118)(119-152) were similar in their fluorescence properties NDP kinase  at wide pH range (pH 5-9). At pH 8.0 the fluorescence properties of mutant proteins NDP kinase  (His118Ala) and HA-NDP kinase  (His118Ala) were similar to those of
NDP kinase , but lowering pH resulted in structural changes in the mutant molecules.
The nature of unusual protein fluorescence of NDP kinase  and the role of main variable regions V1 (residues 31-50)
and V2 (residues 131-152) in structural and dynamic properties of NDP kinase isoforms are discussed. The data obtained in
our work should be taken into account for analysis of results of physiological experiments in which cell lines are transfected
with expression vectors harboring cDNAs of different NDP kinase isoforms and its derivatives.
STRUCTURE OF DISULFIDE CROSS-LINKED
MUTANT YEAST ACTIN
Albina Orlova1, Vitold E. Galkin1,3, Margaret S. VanLook1, Eldar Kim2,
Alexander Shvetsov2, Emil Reisler2 and Edward H. Egelman1
1Department
of Biochemistry and Molecular Genetics, University of Virginia
Health Sciences Center, Charlottesville, VA 22908, USA;
2Department of Chemistry and Biochemistry and Molecular Biology Institute,
University Of California, Los Angeles, Ca 90095, USA;
3Institute of Cytology, RAN, St.-Petersburg, Russia
The cross-linking of adjacent protomers in the actin filament is a very useful approach for investigating the spatial arrangement of different parts of the inter-subunit contact area and for probing the relationship between actin function and its
structural dynamics. Introduction of cross-links into filaments may decrease the flexibility and perturb inter- or intra-protomer
motions in F-actin. It was been found that the in vitro motility of actomyosin could be sharply inhibited when cross-links were
placed within monomers before polymerization. Extensive cross-linking of F-actin between Cys-374 on one subunit and Gln41 on an adjacent subunit along the same long-pitch strand also resulted in an inhibition of motility.
We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds
can be formed between the Cys-41 that is introduced into subdomain-2 and Cys-374 on an adjacent protomer. We find that the
distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction.
This result reveals that disulfide formation does not cause a significant perturbation of the F-actin structure. Consistent with
this, three-dimensional reconstruction of extensively cross-linked filaments, using a new approach to helical image analysis,
show very small structural changes with respect to uncross-linked filaments. This is in conflict with refined models but in 51
agreement with the original Holmes et al. Model for F-actin. Under conditions where 94% of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide
formation makes yeast actin filaments ~ times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled by the position of
subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin. Since chemical cross-links
between subdomain-2 and Cys-374 of another protomer have been shown to inhibit acto-myosin motility, we suggest that this
inhibition does not occur by constraining the twist of the subunits, but rather by inhibition the rotation of subdomain-2.
STRUCTURE AND DYNAMICS OF F-ACTIN AND COMPLEXES
OF F-ACTIN WITH ACTIN-BINDING PROTEINS
Albina Orlova1, Vitold Galkin1, Natalya Lukoyanova1,3,
Emil Reisler2 and Edward H. Egelman1
1Dept.
of Biochemistry and Molecular Genetics,
Univ. of Virginia, Charlottesville, VA, USA;
2Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, CA, USA;
3Institute of Theoretical and Experimental Biophysics RAS,
Pushchino, Moscow region, 142290 Russia
It is clear from studies in many laboratories that F-actin can exist in different conformational states. The role of these
different states in modulating the interaction between actin and actin-binding proteins is poorly understood. However, studies
using chemical cross-linking, proteolytic modification and mutations have shown that the force generation that occurs between
myosin and actin can be greatly inhibited by changes in F-actin. Internal dynamics of F-actin are likely to be an important
component of the actomyosin power stroke, as well as important to the complexes formed between actin and more than 50 different actin-binding proteins. We suggest that the maintenance of these different internal states via the actin-actin interface
may have contributed as much to the exquisite degree of sequence conservation of actin over the course of evolution as the interactions with a large number of actin-binding proteins.
Many new insights into the structure and internal dynamics of F-actin have been obtained from the application of a
novel method of image analysis of helical filaments (Egelman, Ultramicroscopy 85, 225-234, 2000) to electron microscopic
images of F-actin and complexes of F-actin with such proteins as ADF. We have been able to show that ADF changes the
mean twist of F-actin by stabilizing a preexisting angular conformation (Galkin et al., J. Cell Biol. 153, 75-86, 2001). Analysis
of pure actin filaments (in the absence of any actin-binding proteins) has provided further support for the possibility that actin
subunits exist in one of several discrete states of twist, and that there is a cooperativity in the distribution of these states. That
is, if one subunit exists in a 158 state of twist (as opposed to an average twist of ~166), adjacent subunits are more likely to
be in the 158 state. Complexes of F-actin and ADF have shown that the tilt of subunits in F-actin is an additional mode of
freedom, and that the destabilization of F-actin by ADF may occur largely by means of a change of tilt. The variable tilt of
subunits in F-actin has previously been suggested to occur based upon observations of actin bundles.
We have extended all of these studies by characterizing filaments formed from a previously described yeast actin
Q41C mutant, where disulfide bonds can be formed between the Cys-41 that is introduced into subdomain-2 and Cys-374 on
an adjacent protomer. This covalent bond provides a strong constraint on actin dynamics, but it also constrains structural models. We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability crosslinking reaction. This result reveals that disulfide formation does not cause a significant perturbation of the F-actin structure.
Consistent with this, three-dimensional reconstructions of extensively cross-linked filaments show very small structural changes with respect to uncross-linked filaments, in conflict with refined models but in agreement with the original Holmes et al.
model for F-actin. Under conditions where 94% of the protomers are linked by disulfide bonds, the distribution of filament
twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the
disulfide, is relieved by increasing the twist of the long-pitch actin helices.
Disulfide formation makes yeast actin filaments ~ 3 times less flexible in terms of bending and similar, in this respect,
to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled
by the position of subdomain-2 (Orlova and Egelman, J. Mol. Biol. 232, 334-341, 1993), and that this region is more flexible in
yeast F-actin than in skeletal muscle F-actin (Kim et al., Biochemistry 35, 16566-16572, 1996). Since chemical cross-links between subdomain-2 and Cys-374 of another protomer have been shown to inhibit acto-myosin motility, we suggest that this inhibition does not occur by constraining the twist of the subunits, but rather by inhibiting the rotation of subdomain-2.
KINETICS AND DYNAMICS OF FIBROBLASTS INITIAL MOBILITY
Alexandr Platonov, Vladimir Smolyaninov
Central Institute of Epidemiology, 111123 Moscow, 3a Novogireyskay st.,
E-mail: platonov@pcr.ru
Mechanical Engineering Research Institute, 117334 Moscow, 4 Bardina st.,
E-mail: smolian@iitp.ru
The initial motile behavior of fibroblast is focused on active formation of adhesion contacts with substrate. This process includes three stages: 1) radial vibrations superficial folds, 2) formation of local contact zones with substrate (microcontacts), 3) transformation of contact zones into lamella. The experimental methods for quantitative testing of the cells initial attachment kinetics are developed. The method of a dynamic characteristic gives a rating of change in time of cell attachment
threshold force. The methods are used with the purpose of a quantitative rating of influence on initial mobility of cells of the
different factors: concentration of serum, albumin, ATP, and also different variants of substrate additional coverings. Is shown,
that at adding in medium or at preliminary adsorption on substrate of serum or albumin, there is a delay of an initial attachment
52
of cells in 3-4 times. The downturn of intercellular concentration ATP up to 20 % from normal too slows down an initial attachment of cells in 3-4 times.
The mathematical model of an initial active attachment of spherical cells to substrate is developed. Kinetics of an attachment it is represented as random multistep-by-step process of consecutive formation of common contact as set of microcontacts. Parameters of multidot model are: x — strength of microcontact, t — average duration of formation of new microcontact, p — difference of probabilities of formation and destruction of contacts, N — maximal number of microcontacts.
The model allows to approximate the experimental dynamic characteristics of cell attachment process, and also to estimate the
directly not measured parameters t and p. For fibroblast of a line L in Igle medium containing 10% of serum, the following
values are received:
p = 0,1-0,2; t = 10-30 s, N = 25-35, x = 3,61,8 nN
The independent dynamic researches of fibroblasts were made with the purpose of a rating of adhesion forces of local
contacts. The measurements of total force of an attachment of cells to substrate were made with the micropipette. Involved in
micropipette the single cell was delayed from substrate at the different fixed negative pressure in a capillary. In parallel the reflective interference microscope method is used to measure the contact area, which size uses then for a rating of specific
strength of contact. The measurements are executed in cultural medium of different factors: the specific strength of contact in
Igle medium is equal 2,51 nN/2, at adding to Igle medium of 10% serum the strength of contact decreases on 25 %, at adding 1% albumin — on 50%.
As have shown experiments, the measurement of adhesion force is possible only early stages of development of contact, when there are multidot contacts of a cell to substrate volume. At late stages of contacts of the large area at procrastination
of a cell form common strand, which does not detach from substrate, and pull apart on some distance from a place of contact.
The strength of strand does not depend on composition of medium (in the listed above variants) and is equal 12040 nN.
INDUCTION OF CORTICAL OSCILLATIONS IN SPREADING CELLS BY DEPOLYMERIZATION OF
MICROTUBULES
*
O.J. Pletjushkina, Z. Rujfur, P. Pomorski, Ju.M. Vasiliev, K. Jacobson
*A.N.
Belozersky Institute of Physico-Chemical Biology,
Moscow State University, Moscow 119899, Russia
Actomyosin-based cortical contractility is a common feature of eukaryotic cells but the capability to produce rhythmic
contractions is found only in a few cell types such as cardiomyocytes. Mechanisms responsible for the acquisition of this capability remain largely unknown.
We found that rhythmic contractility can be induced in non-muscle cells by microtubule depolymerization. Spreading
epithelial cells and fibroblasts in which microtubules were depolymerized with nocodazole or colcemid underwent rhythmic
oscillations of the body that lasted for several hours before the cells acquired a stable, flattened shape. By contrast, control cells
spread and flattened into discoid shapes in a smooth and regular manner. Quantitative analysis of the oscillations showed that
they have a period of about 50 seconds. The Rho kinase inhibitor, HA 1077, caused the oscillations to immediately cease and
the cells to become flat. The transient increases in cytoplasmic calcium preceded the contractile phase of the oscillations.
Wrinkle formation by cells plated on elastic substrata indicated that the contractility of colcemid-treated cells increased in
comparison with controls but was drastically decreased after HA 1077 addition.
These data suggest that an intact microtubular system normally prevents pulsations by moderating excessive rhomediated actin myosin contractility.
ASSAY OF POTASSIUM ACTIVE TRANSPORT IN MYOCELL OF ISOLATED HEART DURING CARDIAC
ISCHAEMIA
1, 2
2
1
Alexander Pogorelov , Ivan Demin , Valentina Pogorelova ,
2
Ekaterina Khrenova
(1)- Institute of Theoretical & Experimental Biophysics, RAS, Pushchino;
(2)- Puschino State University, Department of Physiology & Biophysics
The adaptation of myocardium to ischaemia is provided by the new steady state of cardiomyocyte resulting from the
+
+
+
+
+
activation of specific membrane mechanisms: ATP- and Na dependent K channels, the Na -H exchanger, K -lactate co- and
+
К -Сl antiports. Since then the reversible efflux of potassium from myocell has been shown the contribution of Na-K-ATPase
in intracellular ionic imbalance at early ischaema has been discussed. It is well evidenced that the ATP and CrP level in cytoplasm does not cause the inhibition of the active transport of ions through the cellular membrane. Then, Na-K-ATPase was indirectly shown to be activated during anoxia. However, the potassium uptake is masked by potassium efflux via specific non
active mechanisms mentioned above. The principal objective of this work is to determine weather extracellular potassium is
pumped in myocyte at ischaemic conditions. For that extracellular K + was exchanged for Rb+ as a potassium marker.
Langendorf`s perfused heart isolated from rat (Wistar) was employed as a model of cardiac ischaemia. Ischaemic circumstances were obtained by the anoxic perfusion without glucose. Tirode`s solution was degassed by vacuum pumping and
KCl was exchanged for RbCl. While the perfusion was stopped papillary muscles were excised and frozen in liquid propane
(85K). Cryosections of 20 m thick were cut in the cryomicrotome (320K) and freeze dried in a high vacuum. Intracellular
elemental concentrations (K, Rb, Na) of cardiac myocell were examined on dried cryosection with Electron Probe X-ray Microanalysis (EPMA) using JEOL-JSMU3 electron microscope.
Data obtained for normoxic conditions are shown in Table 1 and in Table 2 (with rubidium replacement). Table 3 represents elemental concentration in cardiac myocyte at ischaemic conditions.
53
Table 1. Intracellular elemental concentration (mM/litre)
in cardiac myocell of papillary muscle of isolated heart
at normoxia measured with Electron Probe Microanalysis *
Normoxic perfusion of
isolated
heart with potassium in
Tirode`s solution
Perfusion
K
Na
15 min
30 min
60 min
1143
934
1025
1045
885
704
+
with Rb (n=80)
(*) — presented as mean  sem; n = number of cells measured for single sample of papillary muscle; 8 animals were
studied simultaneously at every interval of perfusion.
Table 2. Intracellular elemental concentration (mM/litre)
in cardiac myocell of papillary muscle of isolated heart
at normoxia measured with Electron Probe Microanalysis *
Normoxic perfusion
of isolated
heart with Tirode`s
solution
Perfusion
K
Rb
Na
15 min
822
463
819
30 min
453
557
9112
+
where K was replaced
+
60 min
574
716
889
with Rb (n=36)
(*) — presented as mean  sem; n = number of cells measured for single sample of papillary muscle; 3 animals were
studied simultaneously at every interval of perfusion.
Table 3. Intracellular elemental concentration (mM/litre)
in cardiac myocell of papillary muscle of isolated heart
at ischaemia measured with Electron Probe Microanalysis *
Ischaemic perfusion of isolated
heart with glucose
free Tirode`s
solution where K
was replaced
+
with Rb (n=36)
Perfusion
K
Rb
Na
15 min
61  4
56  6
90  10
30 min
43  2
46  4
69  8
+
60 min
43  2 36  3
54  4
(*) — presented as mean  sem; n = number of cells measured for single sample of papillary muscle; 3 animals were
studied simultaneously at every interval of perfusion.
The analysis of obtained results indicates that at least the part of intracellular potassium content is replaced by rubidium. Rubidium concentration in myocell is much higher than that in Tirode`s solution (4 mM/litre). These findings suggest the
appearance of potassium active transport at both normoxic and ischaemic conditions. It should be noted the decrease of intra+
+
cellular sodium content during ischaemia. It allows us to conclude that Na /K -ATPase is activated for early ischaemia.
CELLS, GELS AND THE ENGINES OF LIFE:
A NEW APPROACH TO CELL FUNCTION
Gerald H. Pollack
Dept. of Bioengineering, University of Washington, Seattle WA 98195
E-mail: ghp@u.washington.edu
It is broadly recognized that the cytoplasm is a gel. Yet, virtually all cell biological mechanisms build on the principle
of free diffusion in aqueous solution. Practically every intracellular process in the textbook involves a cascade of rapid diffusional steps—in spite of the cytoplasm’s character as a gel, where diffusion can be extremely slow.
Because of the presumptive aqueous nature of the cytoplasm, the cell is necessarily endowed with an impermeant
membrane barrier. The barrier permits flow of ions and other solutes largely through channels and pumps that span the barrier.
Well over 100 solute-specific channels traverse the membrane, with additional ones being identified regularly. The same goes
for pumps: Since ion concentrations inside and outside the cell are rarely in electrochemical equilibrium, observed intracellular
concentrations are maintained by active pumping. These concepts are familiar, and practically axiomatic (cf. Alberts et al.,
1994).
If a continuous barrier were required for the cell to function, violating the barrier should prompt transmembrane ion
gradients to collapse. Metabolic processes should grind to a halt, enzymes and fuel should dissipate as they diffuse out, and the
cell should be brought quickly to the edge of death. Yet, large holes can be practically without effect. Holes left by withdrawn
microelecrodes, patch clamp pipettes, and electroporation pulses do not necessarily kill the cell. In the case of electroporation,
even when macromolecules are added to the bathing solution hours after electroporation, they enter the cell (Prausnitz et al.,
1994; Serpersu et al., 1985) this implies that macromolecular-scale holes remain open for extended periods. Indeed, there are
many published examples of survival after the cell has been sliced like a tomato (Maniotis et al., 1991); the fraction lacking the
proper complement of intracellular organelles eventually dies, but the fraction with the proper complement of organelles
54
goes on to live and produce daughter cells. Resealing of the membrane does not take place, as demonstrated by posttransection electron micrographic studies in nerve and muscle cells (Cameron, 1988; Krause et al., 1994).
It appears we are stuck on the horns of a dilemma. If a continuous barrier envelops the cell and is consequential for
cell function, one needs to explain why breaching the barrier is not more consequential than it seems to be. On the other hand,
if we entertain the possibility that the barrier may not be continuous, so that creating yet another opening makes little difference, we then challenge the dogma on which all mechanisms of cell biological function are based.
The standard paradigm arose on the presumption that the cytoplasm is an aqueous solution. Solutes needed to be constrained within the cell, and the constraining element is the continuous barrier. If the cytoplasm is not an aqueous solution, on
the other hand, then the need for a barrier (and pumps and channels) is less acute. This does not necessarily imply the absence
of a membrane; only that membrane continuity is not essential. This would be the case, for example, if the cytoplasm were a
gel, for gels can be sliced with no special consequence; a continuous barrier is not required.
The approach taken here, and presented in a new book (Pollack, 2001), is that the cell is indeed a gel. The cell, after
all, is densely packed with charged polymers (proteins, nucleic acids, sugars), and invested with solvent, which is retained (like
gelatin) even when the membrane is removed. That the cell is a gel has been clear from even before the time of Frey-Wyssling
(1953), whose book offered pivotal evidence for this point of view. Further, the concept of structured water (Ling, 1965) provided a way of understanding how charged proteins could retain water in a simple way, and the classical work of Troshin
(1966) showed how such constructs could partition solutes.
If the cell is a gel, then, the issue of determining how cells work reduces to the question of determining how gels
work. Gels do “work.” When prompted by a change of environment around a critical value, they undergo phase-transition to a
new physical state. The polymer-gel phase-transition is much like the water phase-transition that occurs at 0°. A small shift of
environment triggers a major shift of structure, the transformation capable of doing work.
It is argued that the cell does the same. Or, to be more precise, each organelle of the cell does the same. In different
organelles, polymer type and organization differ. In some instances (e.g., muscle filaments) a long, linear polymer is transformed into a shorter one; in other instances (e.g., secretory granules), a tangled, condensed, polymeric network imbibes water
and is thereby expanded almost explosively. Various examples ranging from those involved in communication, transport,
movement, division, action potentials, etc. are considered in the above-mentioned book (Pollack, 2001); in each case, it can be
demonstrated the observed action involves one or another type of phase-transition.
The suggestion, then, is that the phase-transition is a common denominator of cell function—a mechanism central to
all that the cell does.
References
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. (1994). Molecular Biology of the Cell, Third edition, Garland,
N.Y.
Cameron, I. L. (1988). Ultrastructural observations on the transectioned end of frog skeletal muscles. Physiol Chem. Phys. Med. NMR
20: 221-225.
Frey-Wyssling, A. Submicroscopic Morphology of Protoplasm Elsevier, Amsterdam, 1953.
Krause, T.L., Fishman, H.M. et al. (1984). Extent and mechanism of sealing in transected giant axons of squid and earthworms. J. Neurosci. 14: 6638-6651.
Ling, G.N. (1965). The physical state of water in living cell and model systems. Proc. NY Acad Sci. 125: 401-417.
Maniotis, A., and Schliwa, M. (1991). Microsurgical removal of centrosomes blocks cell reproduction and centriole generation in BSC1 cells. Cell. 67: 495-504.
Pollack, G.H.: Cells, Gels and the Engines of Life: A New, Unifying Approach to Cell Function. Ebner and Sons, Seattle, 2001
(www.cellsandgels.com).
Prausnitz, M.R., Milano, C.D., et al. (1994). Quantitative study of molecular transport due to electroporation: uptake of bovine serum
albumin by erythrocyte ghosts. Biophys. J. 66(5) 1522-1530.
Serpersu, E.H., Kinosita, K., Jr. and Tsong, T.Y. (1985). Reversible and irreversible modification of erythrocyte membrane permeability by electric field. Biochim. Biophys. Acta. 812: 779-785.
Troshin, A. Problems of Cell Permeability Pergamon Press, Oxford.
STEPWISE SHORTENING AND THE NATURE OF CONTRACTION
Gerald H. Pollack*, Felix A. Blyakhman° and Olga Yakovenko*
*Dept. of Bioengineering, University of Washington, Seattle WA, 98195;
°Dept. of Physics, Ural State University, Ekaterinburg, Russia
This presentation extends the one by Blyakhman et al., in which single cardiac sarcomeres were shown to shorten in
steps. Steps have a characteristic size paradigm for activated specimens: all steps are integer multiples of 2.7 nm—i.e., 2.7 nm,
5.4 nm, 8.1 nm, etc. The size paradigm is the same whether the sarcomere is shortening actively, or is being forcibly extended.
Consistently, step size is an integer multiple of 2.7 nm. Here we consider the implication of this distinctive finding.
The first issue is whether the finding is genuine, rather than some kind of instrument artifact. Many controls have been
carried out—all seemingly negative for artifact (Blyakhman et al., 1999; 2001; Yakovenko et al., submitted). One important
control is the imposition of the same stretch-release protocol in the absence of calcium. With unactivated specimens, step size
is a multiple of 2.3 nm, not 2.7 nm. Higher multiples of this value are clearly distinguishable from multiples of 2.7 nm (Yakovenko et al., submitted). Hence, the 2.7-nm step does not arise out of some instrument artifact.
A second question is whether the finding is general. We have repeated the protocols on a common invertebrate specimen, bumblebee flight muscle. Within the limits of experimental error, the step-size paradigm is identical to that of heart muscle (Blyakhman et al., 1999; Yakovenko et al., 2001). Hence, the integer-multiple paradigm appears to be a general feature of
striated muscle.
The third, and central issue is what the paradigm might imply for the mechanism of contraction. Here, relevance is attached to several considerations.
55
First, the steps occur synchronously over huge domains. In the single sarcomere, the number of parallel filaments is
on the order of hundreds, and bee myofibrils perhaps closer to 1000. The number of cross-bridges is perhaps 105. Yet, the step
occurs synchronously among all these elements; for otherwise, it would not be detected. The underlying process cannot, therefore, be random; it must have a deterministic element of some kind.
In fact, the extent of the synchronous domain is by no means limited to the single sarcomere. Earlier measurements on
larger specimens have consistently shown steps. Measurements have been carried out on small myofibrillar bundles using the
phase-locked loop method (Jacobson et al., 1983), larger regions using the optical diffraction method (Pollack et al., 1977), and
whole cell segments using the surface-marker method (Granzier et al., 1987). All show steps. Further, all three methods indicate that the steps are integer multiples of 2.7 nm (Pollack, 1990, p. 156). Hence the steps occur synchronously over what, by
molecular standards, is a virtually infinite domain. Clearly, any stochastic mechanism, such as randomly swinging crossbridges, cannot explain this phenomenon in a simple way. The mechanism must contain a deterministic element.
Another point of relevance is that the size paradigm for shortening and lengthening is the same. In both cases, steps
are integer multiples of 2.7 nm. The simplest explanation is that the mechanism must be reversible, at least its central core. Reversibility is difficult to fit into a rotating cross-bridge framework because of the difference between mechanisms for shortening (bridge rotation) and stretch (“give”). To arrive at the same size paradigm would require a fairly remarkable coincidence.
To explain these highly consistent step-size paradigms, we propose two complementary mechanisms, each of which
generate the 2.7-nm steps for shortening and stretch, and both of which may operate in complementary fashion. The first involves inchworm-like translation of the thin filament past the thick; the second involves shortening of the thick filament,
which, despite textbook views, is consistently reported (Pollack, 1983; 1990). We briefly consider each of these mechanisms.
The inchworm model is based on the commonly seen reptation of the actin filament. Several reports indicate that this
snake-like action propagates along the filament (deBeer et al., 1998; Hatori et al., 1998). If so, a caterpillar-like crawling
mechanism could obtain. Because actin monomers repeat every 2.7 nm along the filament (5.4 nm along each strand, the two
strands axially shifted by half of 5.4 nm), each advance will necessarily be an integer multiple of 2.7 nm, provided actin monomers bind to a given (spatially fixed) myosin. If the load is large, the step may be only 1 x 2.7; if it is smaller, it may be 2x or
3x or even 4 or 5x 2.7 nm. Similarly, as the sarcomere is forcibly lengthened, the step will necessarily be a multiple of 2.7 nm,
if actin monomers bind to myosin.
Thick filament shortening will produce steps of 2.7 nm as well (Pollack, 1990). The driving force for shortening is the
helix-coil transition along the myosin rod; this drives one rod past another, resulting in incremental shortening of the filament
(Pollack, 1990). The step-size paradigm depends on the cohesive forces that ordinarily hold together the rods. Alternating
charges on each rod create a series of quasi-stable displacements. These charges repeat every 4.1 nm along the rods—not 2.7
nm. However, the filament consists of three parallel sub-filaments. If shortening takes place in one sub-filament at a time, the
half-filament will shorten by 1/3 of 4.1 nm; and, if both halves of the filament shorten synchronously, the measured step will
be 2/3 of 4.1 nm, or 2.7 nm.
Hence, both mechanisms can generate the 2.7-nm step paradigm. Either one can produce steps that are integer multiples of 2.7 nm, and both can produce these steps during shortening or lengthening. Possibly, the two mechanisms work in a
complementary way to give rise to contraction.
References
Blyakhman, F., Shklyar, T., and Pollack, G. H. (1999). Quantal length changes in single contracting sarcomeres. J. Mus. Res. Cell Motil. 20: 529-538.
Blyakhman, F., Tourovskaya, A., and Pollack, G. H. (2001). Quantal sarcomere-length changes in relaxed single myofibrils. Biophys.
J. in press.
DeBeer, E., Sontrop, A., Kellermayer, M.S.Z. and Pollack, G. H. (1998). Actin-filament motion in the in vitro motility assay is periodic. Cell Motil. and Cytoskel. 38: 341-350.
Granzier, H. L. M., Myers, J. A., and Pollack, G. H. (1987). Stepwise shortening of muscle fiber segments. J. Mus. Res. Cell Motil. 8:
242-251.
Hatori, K., Honda, H., Shimada, K., and Matsuno, K. (1998). Propagation of a signal coordinating force generation along an actin filament in actomyosin complexes. Biophys. Chem. 75: 81-85
Jacobson, R. C., Tirosh, R., Delay, M. J., and Pollack, G. H. (1983). Quantized nature of sarcomere shortening steps. J. Mus. Res. Cell
Motil. 4: 529-542.
Pollack, G. H., Iwazumi, T., ter Keurs, H. E., and Shibata, E. F. (1977). Sarcomere shortening in striated muscle occurs in stepwise
fashion. Nature. 268: 757-759.
Pollack, G. H. (1983). The sliding filament / cross-bridge theory. Physiol. Rev. 63: 1049-1113.
Pollack, G. H. (1990). Muscles & Molecules: Uncovering the Principles of Biological Motion, Ebner and Sons, Seattle, WA.
Yakovenkov, O., Blyakhman, F., and Pollack, G. H. (submitted). Sarcomere contraction occurs in reversible 2.7-nm steps.
CHANGES IN THE NUCLEASE CHROMATIN FRAGMENTATION PATTERNS UNDER GENE ACTIVATION
MIGHT BE
A CONSEQUENCE OF NUCLEOSOME LINEARIZATION
T.N. Priyatkina, N.E. Voinova, E.Yu. Pavlova, M.M. Naumova, I.N. Sokolov
Department of Biochemistry of Saint-Petersburg State University, Russia
Interphase chromatin contains the structural information, realizing in a series of conformational transitions and also
several type motions of nucleohistone threads during the processes of compactization and decompactization of DNA. According to nucleosome concept, 9 histone molecules, represented by 5 forms, being repeated along the DNA every 200 b.p., divide
it into the units of compactization (the structural units of chromatin – nucleosomes). The process of compactization includes at
least 2 main steps – the formation of a discrete nucleosome chain and its winding in 30 nm fiber, with cycle complex formed
by 6 nucleosomes as a repeating element. The structural unit of chromatin at this level consists of the compact particle and two
56linker segments, one of them being represented by free DNA. Such organization ensures the specific pattern of the nuclease
chromatin fragmentation: as the 200 b.p. regularity in the distribution of cleavages as the ending of reaction after digestion of
linker DNA, which accounts about 15% of total DNA.
The fate of compact particles during genes activation and transcription remains obscure. The DNAse1 action resulted
in the cleavage of 50-80% DNA of active genes up to the acid-soluble products. This suggests that their DNA seems not to be
organized in canonical nucleosomes arrays. Data on the cleavage pattern of active chromatin DNA by micrococcus nuclease
(MNase) are extremely controversial. Multiple states of every derepressed gene and its regions, different conditions used for
MNase treatment, the influence of endogenous enzymes on the fragmentation of DNA, etc might be considered as a reason of
apparently conflicting results obtained.
We have studied the MNase fragmentation patterns of the coding regions of tryptophanoxygenase (TO) and tyrosinaminotranferase (TAT), the inducible genes in rat liver nucleus, where these genes are transcriptionally active. Nuclei at a DNA
concentration of about 1 mg/ml were digested with 10 to 360 U/ml MNase for 15 min in the medium of low ionic strength
(0,01 M tris-HCl, pH 8,0, containing 1,5 mM CaCl2) at temperature rising during incubation from 4 to 20 oC. Unexpected results were obtained under treatment of nuclei with high (more than 120 U/ml) concentrations of MNase. Bulk chromatin was
digested mainly up to mono-and dinucleosomes (170-200 b.p. fragments of DNA scale), while DNA of TO and TAT genes
was retained as long 1500 - 20000 b.p. fragments. The maximal length of components approached the size of their coding regions. Under similar conditions Dnase1 action resulted predominantly in DNA digestion of both genes up to acid soluble products.
The analysis of TO- and TAT gene fragments produced with various dilutions of MNase revealed the existence of
several forms of chromatin, differing in MNase accessibility to DNA within their coding regions. After low digestion the resulting fragments of TO and TAT genes corresponded to their full-scale transcriptional units. With increasing enzyme concentration to 20U/ml the MNase resistant fragments 1500-20000 b.p. long appeared simultaneously with the minor fraction of the
low-molecular DNA. The latter was not found in the hydrolyzates under the high MNase concentrations.
TO and TAT genes transcribe at a moderate level characterized by low density of polymerase molecules across transcriptional units. Moreover, only about 30% from overall number of their copies in cell population presented are involved in
transcription process.
We suppose, that specific resistance of DNA to MNase is inherent in the competent state of chromatin. Transcription
induces the appearance of the MNase sensitive areas, which probably flank RNA polymerases. After their cleaving the MNase
resistant different in length fragments remain corresponding to the nontranscribe at the moment segments of coding regions.
DNAase1 introduces single-strand cuts and splits the free DNA at the sites of their coincidence. The MNase action is
resulted in double-strand breaks. The absence of the accessible for MNase sites and the high probability of realizing of doublestrand cleavage DNA by DNAse1 along the whole length of coding regions, suggest that their DNA is devoid of the histone
protection typical for the nucleosome chromatin organization.
The model of chromatin organization is discussed , according to which nucleosome is composed of two symmetric
DNA folds, which are stabilized by histone-histone interaction. The transition of the compressed state DNA up to the expensed
loop is coupled with the dissociation of histone dimers and unfolding of their polypeptide chains. The double-strand linear histone octamers formed are adjacent to DNA in the nontranscribed sections of the active genes and detach from it during of transcription. Such a structure is in agreement with the existing data on the DNAse1 and MNase cleavage patterns of DNA of active and competent genes and their morphology visible through electron microscope.
REORGANIZATION OF CYTOSKELETON AND ORGANELLE TRANSPORT DURING THE APICAL GROWTH
Elena Rudanova, Olga Gavrilova
Biological Research Institute of Sant Petersburg University,
198504, Sant Petersburg, Stariy Peterhof, Oranienbaumskoye sch., 2,
E-mail: Elena.Rudanova@paloma.spbu.ru
Reorganization of cytoskeleton during the germination of aplanospores of cenocyte apical-growing alga Vaucheria
sessilis was studied. Apical character of growth was shown to remain at all stages of germination. Architecture of cytoskeleton
transforms appreciably during morphogenesis. At initial stages the treatment with anticytoskeletal agents (colchicine, cytochalasine D, phalloidin) blocks the process of germination. In this period actin represents cortical strands and short cytoplasmic
filaments which have no preferred direction. The factors determining the area of growth were not revealed by us, presumably,
this process involves the formation of a tight local network of thin microfilaments. With the appearance of a primary germ the
microfilaments in the whole volume of aplanospore aggregate in bundles and orientate along the axis of growth. Microtubules
at this stage are not visualized by the methods of electron microscopy and indirect immunofluorescence, however, the centrioles are permanently migrating around nuclei. At later stages of germination nuclei migrate into the forming germ; just in this
the period the short bundles of microtubules elongated along the axis of growth assembles around the centrioles. Later on, the
cortical layers of microtubules and actin microfilaments begin to organize de novo in germ; cytoplasmic bundles of microtubules elongate further. In apical area actin filaments at first are arranged in the form of a ring limiting the area of growth and
then as a coarse (-cellular) network (sieve). Microtubules are not observed in the apical area. Microtubular bundles are also absent in nuclei congestion located at the filament terminus. Using h3 thymidine it was shown the absence of mitotic activity in
this area. Incorporation of the label took place in aplanospore at early stages of germination and in the center of the germ at
later stages.
The role of microtubules and actin filaments in morphogenesis and nuclei transport is discussed.
CA2+ TRANSPORT IN MITOCHONDRIA
IN HEART MUSCLE HOMOGENATE
I.R. Saakyan, M.N.Kondrashova
57
Institute of Theoretical and Experimental Biophysics, RAS, Pushchino, 142290,
E-mail: kondrashova@pbc.iteb.serpukhov.su
Isolated in sucrose, washed mitochondria serve as a standard tissue preparation for study of energy processes. However isolation introduces some artifacts, such as damage of mitochondria with sucrose and dilution related with dissipation of
their native structure organization in assemblies and loss of part of total population of organelles in the cell. Besides procedure
of isolation is long and cannot be satisfactory with small amount of tissue.
Moreover recently investigations of mitochondrial processes rose for medical purposes, particularly diagnosis of mitochondrial diseases.
The method of preparation of liver homogenate was elaborated which allows to avoid artifacts during isolation, it is
rapid (about 10 min) and can use small pieces of tissue (about 100 mg) [Kondrashova et al. Biochemistry, Moscow 1997, 62,
129-137; Europ.Journ. Med.Res. 2000, 5/Sup.1, 30]. Usually homogenate was considered to be the most damaging step in mitochondrial isolation. This was due to sucrose and high dilution. New procedure includes KCl and low dilution. Mitochondria
in such homogenate reveal all the main energy functions and even are more native and stable.
However this is not the case with muscle or heart mitochondria in homogenate. High ATP-ase activity in muscle tissue disturbs ATP synthesis. It seems a priori that mitochondrial Ca2+ transport in homogenate is provided by endoplasmic retuculum. Meanwhile investigation of muscle and heart bioptates is most actual for medical purposes.
Below we describe conditions, which allow to investigate ATP accumulation and Ca2+ transport in heart homogenate
of rat, rabbit and pigeon. The data are compared with those in heart mitochondria and with rat liver homogenate.
Heart homogenates were prepared according to procedure cited, in KCl 125 mM, HEPES 10 mM, EGTA 1mM, pH
7.5 using tissue/medium ratio: 1/3. We additionally include glutamate in homogenization medium, which improves energy
functions. Rat liver homogenate was prepared similarly, without glutamate. Respiration was measured polarographycally and
Ca2+ transport with pH electrode.
We found that Ca2+ accumulation in heart and liver homogenates is completely abolished by inhibitor of mitochondrial Ca2+ transport, ruthenium red as well as by malonate, inhibitor of succinate dehydrogenase. Therefore Ca 2+ transport in homogenate under our conditions is completely provided by mitochondria.
The pronounced difference was revealed in the course of ADP phosphorylation in heart homogenate comparing with
heart mitochondria and liver homogenate. Proton accumulation (alkalinization) coupled to ATP formation is well pronounced
in mitochondria and liver homogenate, while this is very small in heart homogenate. It is registered as a small pulse of alkalinization, 1-5 sec duration, which is followed by prolonged acidification (60 sec). This cycle is repeated after the next ADP addition. Respectively phosphorylating respiration is lower, State 4 respiration higher and respiratory control, ADP/O, ADP/t are
lower in heart homogenate. These differences are apparently due to a higher ATPase activity in heart homogenate.
In contrast responses to Ca2+ additions are well pronounced in heart homogenate. The magnitude of pH change induced by single Ca2+ addition is 10-fold higher than that induced by ADP. This ratio is 1:1-0.5 in liver homogenate. ADP addition before Ca2+ increases H+ efflux 2-5-fold in all tissue preparations. Ca2+ capacity decreases in the row: succinate + glutamate, succinate, succinate + -ketoglutarate, glutamate or -ketoglutarate. This show that glutamate stimulates Ca2+ accumulation, while -ketoglutarate diminishes this. This phenomenon was reported [ Saakyan, in: Therapeutic action of amber acid,
Pushchino, 1976 ].
Conclusion. In heart homogenates ATP synthesis itself cannot be clearly observed because this is masked by its hydrolysis. In contrast Ca2+ induced responses are well pronounced and can serve as a sensitive parameters of mitochondrial
functions. Moreover responses to Ca 2+ allow to reveal the effect of previous addition of ADP due to ATP synthesis. Responses
to Ca2+ in heart (and probably skeletal muscle) homogenates can be proposed as a very convenient test of energy state of tissues in biochemical and clinical investigations.
CALCIUM LOCALIZATION IN CEREBELLUM MICROTUBULES EXPOSED BY MEANS OF ELECTRIC
STIMULATION
IN THE NO - PRESENCE
N.V. Samosudova, V.P. Reutov, N.P. Larionova
Institute for Information Transmission Problems RAS;
Institute of Higher Nervous Activity and Neurophysiology RAS,
Moscow, Russia
Earlier we have shown that incubation of frog cerebellum in Ringer solution in the presence of nitric oxide (NO) enhances the contrast of cytoskeletal elements thereby facilitating its visualization [1]. To determine the participation of nitric oxide in neuron interaction, namely in signal transmitting from granule cell (GrC) to Purkinje cell (PC), in present work the electric stimulation of frog cerebellum in the NO-presence was carried out.
The stimulation of cerebellum in the NO-presence revealed the appearance of the electron-dense precipitate in the
structures characterized by active metabolism: nuclei, mitochondria, synaptic contacts and microtubules (MT) of GrC-axons
and PC-dendrites. Microtubules are the main cytoskeletal component of axons and dendrites and are involved in the transport
of different proteins and non-protein substances. The electron-dense precipitate seen at longitudinal sections of MT localizes
with a period of approximately 24-25 nm. This period is characteristic for dynein, which has ATPase activity and could interact with tubulin in the presence of Ca2+. According to our data the observed electron-dense precipitate, formed by stimulation
in the presence of NO, represents the polymerized protein including calcium ions, determined by Petrunyaka method [2]. The
physiological significance of the latter is likely to be concerned with the demand of Ca 2+ for both ATPase and NO-synthase activities. Thus the suggestion could be made that electron-dense precipitate formed in the presence of exogenic NO determines
the “working” sites of endogenic nitric oxide during signal transmission from one neuron (GrC) to another – to Purkinje cell.
References
58
1. Samosudova N.V. et al., (2000). Tsitologiya. 42: 72-78.
2. Petrunyaka V. (1987). Tsitologiya. 29: 875-883.
PARACRYSTALS OF THIN FILAMENTES AND A COMMON PRINCIPLE OF PARACRYSTALLINE
STRUCTURES
T.G. Samsonidze
The Republican Research Centre of Medical Biophysics and Introduction
of New Biomedical Technologies of the Ministry of Public Health of Georgia
(Director N.B. Karsanov. Honored Scientist, Associate member of the Academy
of Sciences of Russian Federation and of the Academy of Sciences of Georgia)
By the method of electronic microscopy it was found out that a number of biological objects have the spiral symmetry. Some of them can form more complex aggregates – paracrystals. These formations have a characteristic, very stable and
diffraction picture, which indicates their rather dense packing. That is why it is very tempting to use it for more detailed analysis of spiral formations, what at the first glance seems impossible because of the specific view of the diffraction picture.
The aim of this work is to reveal principles of paracrystal formation, the possibility of the correlation between a type
of a paracrystal and the parameters of its composing spiral structures and to carry out the further conformation analysis of these
spirals on the basis of the diffraction pictures from the paracrystals.
We consider all the possible orientations of spiral structures inside the paracrystal and show that they can be brought
to three cases, which are analyzed by us using the theorem about the convolution of two functions. It is shown that a paracrystal can be presented as the convolution of the function of molecule distribution (protomere of an actin molecule) with the function of a complex grating, which on its turn is constructed from the convolution of a two-dimensional grating of a paracrystal
with a discrete spiral, parameters of which must necessarily be taken into account in the further analysis of this system.
Proceeding from the dense packing of any crystalline formation it is shown that for every case must be observed the
terms:
 = ( n0 - 2 ) r0 and
r* = n0 r0
(1)
where  - is the distance between the edges of the near-by spiral in the projection of the paracrystal, r* - is the distance between
axes of the neighbouring filaments, r0 - is the radius of the spiral structure, n0 - is the order of Bessel’s function of the main reflex. It is clear that for n0 = 1 we will get  = -r0, i.e. in the projection on the plane of observation neighbouring filaments in the
paracrystal must overlap each other.
Diffraction from the spiral with the dispersing density (r,,z) and the period c is determined by Furie-Bessel’s transformant /1-3/:
F(R, , Z =
 /c) = 
Gn  exp in(2
(2)
n
where
 2 c
Gn  =
 
 (r, , z) Jn(2rR) exp ( - i ( n + 2  z/c)) r dr d dz (3)
0 0 0
Here 2  z/c - is a displacement in the phase for the molecule chain (of each subsequent with respect to previous). F(R, , Z
=  /c) – is the sum of harmonics Gn  on the layer line  multiplied by a phase multiplier
 = exp in ( + /2)
(4)
for the azimuthal direction with the angle .
Finally Furie-Bessel’s transformant of the first and second main reflexes of a paracrystal instead of (1) will have the
following expression:
2(R, , Z=  /c) = Fn=0(R, , Z=  /c) {1 + exp (-i   sin )}
(5)
2(R, , Z=  /c) = {Fn=1(R, , Z=  /c)}2 exp (-i   sin ) / 2
(6)
Here the function sin  describes arbitrary disposition of thin filaments in a paracrystal. The expression (5) represents the superposition of two transformants corresponding to a solitary filament, which is a part of the paracrystal of the structure and a
certain constant component, which determines the structure of the paracrystal. i.e. this transformant is multiplied by itself and
such operation results in appearing new functions of Bessel what is revealed in receiving a diffraction picture of a new type,
which differs from solitary filaments. What concerns the above mentioned constant component, in exponent there exists a
number of a layer lines multiplied by , what means that pair reflexes with the same arrangements of Bessel’s function, which
are placed on the neighbouring layer lines under conditions n =1, will annihilate each other and form new solitary reflexes,
which are characteristic to paracrystalline structures. Besides, under conditions of the constant  a type of a transformant of a
paracrystal practically does not depend on the mode of its organization, i.e. on the argument sin in the exponential function.
It is natural that the transformants describing reflexes (diffraction maximums), which are characterized by the subsequent order of Bessel’s function, have more complex view, but nevertheless it can be written in analogy to (5). The common
transformant, which characterizes a paracrystal, may be received by summing up all received transformants. For instance, if
confined to the reflexes with the order of Bessel’s function from 0 to 1, the common transformant will be written as the sum of
the expressions (5) and (6).
Thus, we can conclude that the view of the diffraction picture of the paracrystal structure is determined first of all by
the transformant of the composing spiral structure and does not depend on the number of the composing filaments. It depends a
bit on the mode of the crystal construction and greatly depends on the distance between the spiral structures. Besides, it must
be mentioned that all constructions were divided into by us in case of maintaining the term (1). This means that the structure of
the paracrystal first of all depends on the parameters of the composing spiral. i.e. in order the paracrystal to be formed from the
spiral structure the order of Bessel’s function of the main reflex has to provide more or less dense packing of spirals in the
59
paracrystal and this is possible only when it is less or equal to two (n2). Thus for thin filaments n =1, that is why the formation of paracrystals is possible.
As it has already been mentioned, on the basis of the analysis of the afro-cited formulas the analogous conclusions can
be made according to the received diffraction pictures.
References
W. Cohran, F.H.C. Crick, V. Vand, Acta Cristallogr., 5, 581-586, 1952
A. Klug, F.H.C. Crick, H.W. Wyckoff, Acta Cristallogr., 11, 199-213, 1958
B.K. Vainshtein, X-ray diffraction on the link macromolecules, AC USSR, M. 1963 (in Russian).
TESEUS’S PHENOMENON AND MOVEMENT
OF THE NEUTROPHILIC LEUKOCYTE
V.P. Saprykin
Sechenov Medical Academy, Moscow, Russia
Although motility of the neutrophils is studied intensively, the results of investigations in vitro do not possess extrapolations on conditions in vivo. Now, many molecular mechanisms of the neutrophil adhesion are deciphered, but subsequent
neutrophil migration in tissues remains without sufficient attention. It is known that in vitro, the moving neutrophil acquires
the polarity: it becomes triangle in shape, in their back pole a small cell fragment connected by a cytoplasmic cord with the cell
body appears. There are no explanations of the significance of this cell transformation for the neutrophil movement in vivo.
The character of neutrophil migration between the bloodstream and tissues is pendulum-like: blood → tissues →
blood. Mechnikov (1903) was the first to describe the neutrophil recurrence to the periphery blood after phagocytosis in tissues. This fact was subsequently corroborated by other researches (Bukoemsky 1904; Nikiphorov 1909; Boyd 1941; Galankin
1994; Vorobyev 1999; etc.). However, the modern theoretical constructions of the neutrophilic vital cycle do not take into account this peculiarity of the neutrophil migration.
According to our conception of nonflogogenic neutrophil functioning, these leukocytes function in the state of natural
bacterial surrounding when inflammation does not develop. The necessary conditions of nonflogogenic neutrophil functioning
are as follows: the neutrophil migration from the blood to tissues, phagocytosis of bacteria, the neutrophil return to the bloodstream, and the removal of bacteria from tissues.
There are two recognized mechanisms of the neutrophil directed movements: chemotaxis and haptotaxis. We suppose
that Teseus’s phenomenon is a basis of these two mechanisms and pose a hypothetical model of the neutrophil movements in
vivo on basis of this phenomenon.
Tesseus’s phenomenon is compound, multistage, and dynamic process including interactions of neutrophils with endothelial cells, connective tissue components, and sources of chemoattractants. After the neutrophil adhesion to endothelial
cells of postcapillary venules, endothelial intercellular junctions are separated from each other under influence of neutrophilic
leukotriene (LTA4). The neutrophil uses PECAM-1 molecules to be tightly built into the endothelial layer, then penetrates into
the perivascular space, and begins its purposive movement towards a tissue source of chemoattractants. Despite the progressive
migration in tissues, the neutrophil retains its contact with the vessel. A distal part of neutrophil cytoplasm called the uropod
remains to be bound with the endothelial layer. A cytoplasmic cord called the fiber retraction extends from the uropod to the
cell body and terminates in a knob-like basal portion of the neutrophil.
Spatially deformed neutrophils are more sensitive to the gradient of chemoattractant concentration then rounded neutrophils (W.S. Ramsey, 1974). Formation of the uropod and the fiber retraction is a mechanism facilitating neutrophils to distinguish the gradient of chemoattractant concentration. The longer distance between the uropod in the blood vessel (a region of
stable chemoattractant concentration) and the anterior pole of the moving neutrophil (a region of variable chemoattractant concentration) exists, the less gradient of chemoattractant concentration is discerned by neutrophil receptors, and the more exactly
the neutrophil movement direction is defined. Thus, the mechanism of Teseus’s phenomenon allows neutrophils to reach the
chemoattractant source faster, more economically, and by the shortest way.
After phagocytosis of bacteria, the stimulus for further neutrophil migration disappears. Re-distribution of the neutrophil cytoskeleton occurs, and the neutrophil changes the direction of its movement: it returns into the bloodstream. The fibril of
retraction connecting the cell with the blood vessel facilitates this return.
The proposed model logically explains changes of the cell movement direction and the cell structural transformations
in nonflogogenic functioning of neutrophils.
TOPOLOGY OF ACTIN – MYOSIN MOSAICS
OF SKELETAL AND CARDIAC MUSCLES
AND PARAMETERS FOR THEIR CHARACTERISTICS
G.A. Savostyanov and E.G. Savostyanova
Sechenov Institute for Evolutionary Physiology and Biochemistry RAS,
44 M. Thorez Av., 194223, Saint Petersburg, Russia; E-mail: gensav@iephb.ru
As it is known, a main functional part of a sarcomere is the A-disk area, which consist of actin (A) and myosin (M)
myofilaments. Regular AM mosaics having a translation symmetry can be revealed on transversal sections of the A–disk area.
The composition and structure of these mosaics are different in various animals and change during ontogenesis.
At the moment, only metric parameters are used to describe the AM mosaics. These are either numerical ratio of A/M,
having relations from 1/2 up to 1/6, or amount of A fibers surrounding M fibers. The last ratio can be from 1/6 up to 1/12. Such
approach is unable to estimate the possible variability of AM mosaics and to predict their evolution.
To overcome the methodological problem, we suggest the new approach to describe the AM mosaics. It is based on
the results of our studies on a simple epithelia cell mosaics (Savostyanov 1991, 2001). The approach is able to describe the set
60of all possible variants of AM mosaic and to predict their changes in norm and pathology. Using the approach we show that
a complex analysis of metric and topological parameters are needed to reveal the characteristics of AM mosaics. It is necessary
to take in account a contiguity of both myosin and actin myofilaments.
The applied approach let us to create the set of topological models of mosaic, which have a various ratio of AM filaments. The concept of an elementary morpho-functional unit of a mosaic is suggested. Composition and structure of the units
are established like it used to be done in crystallography. The new parameter for the unit functional abilities is offered. The parameter is determined as a relation of the amount of heterogenic contacts between A and M filaments to the total amount of the
contacts of both heterogenic (AM) and homogenic (AA and/or MM) in the units. The comparison of created models with our
own experimental data and data available from a literature are in a good agreement. As a major advantage we are able to create
all the possible AM mosaics to describe both real and potential diversity of muscle tissues.
References
1. Savostyanov G. A. The theory of cell mosaics of simple epithelia // Arch. Anat. Histol. Embryol., 1991, V. 100, N 6, c. 5 - 27.
2. Savostyanov G. A. Principles of spatial organization of cell sheets // Biophysics. 2001, V. 46, (in press).
CREATINE KINASE PHOSPHOTRANSFER AND KATP CHANNEL GATING: A NEW PARADIGM IN
METABOLIC SENSING
Vitaly A. Selivanov, M. Roselle Abraham, Denice Hodgson,
Darko Pucar, Leonid V. Zingman, Be Wieringa,
Petras P. Dzeja, Alexey E. Alekseev and Andre Terzic
Mayo Clinic, Mayo Foundation, Rochester MN, USA
Inhibition by intracellular ATP and activation by MgADP render ATP-sensitive K+ (KATP) channels unique nucleotide
sensors capable of adjusting membrane potential in response to metabolic signals. In the heart, channel opening serves as an
alarm system associated with myocardial protection in ischemia. Channel opening has implied dramatic changes in intracellular ATP/ADP levels, which are however incompatible with cell survival. The mechanism of K ATP channel regulation has,
therefore, remained an enigma.
Cellular signaling is conventionally based on steep changes in the concentration of primary ligands (e.g., hormones,
neurotransmitters) followed with amplification by second messengers (e.g., intracellular cyclic nucleotides, calcium ions).
However, the KATP channel complex does not comply with such common mechanism of signaling as intracellular ATP is tightly “buffered” well above the concentration required for half-maximal inhibition (IC50 30 M versus millimolar levels of intracellular ATP) of the Kir6.2 channel pore. The metabolic messenger, ADP, a product of cellular ATPases, including the ATPase
activity intrinsic to the regulatory channel SUR subunit, reduces the ability of ATP to inhibit K ATP channels. Although the
MgADP-bound state, favored by the ATPase activity of SUR, increases the IC 50 for ATP inhibition from 30 to 300 M, such
expanded range of allosteric nucleotide-dependent channel regulation still lies far below the intracellular levels of ATP. Thus,
in the cell, MgADP-dependent KATP channel gating is not sufficient per se to account for channel opening. Rather, for MgADP
to serve as a messenger responsible for channel activation, the ATP concentration at the channel site has to be lower than the
millimolar levels present within the cytosol. In this way, membrane nucleotide sensors, such as K ATP channels, would need to
sense local rather than cytosolic pools of nucleotides.
It is conceivable that due to membrane ATPases, including hydrolysis of ATP by the K ATP channel complex itself, as
well as due to the presence of diffusional limitations near-membrane (“subsarcolemmal”) nucleotide levels may indeed differ
from cytosolic “bulk” concentrations. Here, ATP consumption by membrane ATPases, measured using 18O-phosphoryl labeling, revealed a maximal rate of 1800 nmol/min/g w wt. ATPase flux was modeled as a Michaelis-Menten reaction, and diffusional nucleotide fluxes approximated by Fick’s law. Solving a system of equations, which integrates membrane ATPase activity and nucleotide diffusion, predicts that 4 out of 7 mM of “bulk” ATP need to be converted into subsarcolemmal ADP to
produce opening of 1% of the K ATP channel population, a percentage required for significant shortening of the cardiac action
potential. This gradient between the cytosol and subsarcolemmal space implies a strong diffusional restriction characterized by
a coefficient D = 2.310-11cm2/s, that far exceeds values (~10-6 cm2/s) accepted for nucleotide diffusion. Thus, nucleotide diffusion appears limited by a steric barrier, and coordination of channel activity with cellular metabolism therefore requires an efficient mechanism transferring nucleotides over such barrier. This metabolism-driven shunting mechanism would allow local
ATP/ADP ratios to be equilibrated with the cytosol keeping K ATP channels closed under non-stress conditions, while allowing
channels to open synchronously with the onset of metabolic stress.
Creatine kinase catalyzes the majority of transcellular high energy phosphoryl transfer. Here, in permeabilized cardiomyocytes and excised membrane patches from wild-type mice, creatine kinase (CK) activated by creatine phosphate shunted
the subsarcolemmal restricted diffusion space promoting K ATP channel closure. Genetic knockout of the major CK isoform (MCK), abolished creatine phosphate-induced channel closure indicating a critical role for CK in KATP channel regulation. Under
metabolic stress induced by hypoxia, CK flux, measured using the 18O-assisted 31P NMR, decreased by ~75%. With reduced
CK flux, the ATPase-induced drop in subsarcolemmal ATP/ADP ratio was unmasked promoting K ATP channel opening. Indeed, models describing nucleotide diffusion and allosteric K ATP channel regulation predict that such drop in CK flux would be
sufficient to allow channel opening despite millimolar levels of bulk ATP. This is supported with the significant shortening of
action potential observed in the whole heart shortly following induction of metabolic stress. Thus, the dynamics of creatine kinase flux, rather than cytosolic ATP/ADP levels, govern K ATP channel behavior. The alternative phosphotransfer pathway, catalyzed by the glycolytic system, was capable to maintain K ATP channel closed in CK-deficient cardiomyocytes. However, despite such plasticity in phosphotransfer-dependent channel regulation, KATP channels readily opened under suppressed glycolysis leading to action potential shortening in CK-deficient but not wildtype cardiomyocytes, indicating a decisive role for creatine kinase phosphotransfer in coupling metabolic signals with membrane electrical events. This novel paradigm in the regulation of a metabolism-sensing ion channel may, therefore, have general implications on the understanding of cellular excitability.
61
COMBINED EPR AND DSC STUDIES OF MYOSIN SUBFRAGMENT 1
MODIFIED AT BOTH REACTIVE SH-GROUPS, SH1 AND SH2
L.I. Shakirova1, E.I. Siletskaya1, V.P. Timofeev2,
V.V. Mikhailova1 and D.I. Levitsky1,3
1A.N.Bach
Institute of Biochemistry RAS, Moscow 117071, Russia;
Institute of Molecular Biology RAS, Moscow 117984, Russia;
3A.N.Belozersky Institute of Physico-Chemical Biology,
Moscow State University, Moscow 119899, Russia
2V.A.Engelhardt
This work has been undertaken with a goal to investigate the ability of myosin subfragment 1 (S1) modified at both
reactive SH-groups, SH1 (Cys-707) and SH2 (Cys-697), to undergo structural changes induced by nucleotides and actin. The
SH1 group on S1 was spin-labeled with 4-iodoacetamido-2,2,6,6-tetramethylpiperidinooxyl (IASL), and the SH2 group on the
SH1-labeled S1 was modified with N-[[(iodoacetyl)amino]ethyl]1-sulfo-5-naphthylamine (IAEDANS). We used the EPR
method to investigate the ability of the SH1-SH2-modified S1 to undergo local conformational changes upon formation of the
S1 ternary complexes with ADP and Pi analogues such as orthovanadate (Vi), beryllium fluoride (BeFx), or aluminum fluoride
(AlF4-). It has been shown that SH2 modification has no effect on the changes induced by formation of these ternary complexes
in EPR spectra of S1 spin-labeled at SH1-group. This means that modification of both SH-groups does not affect the local conformational changes induced by nucleotides around the SH1 group. We also used the method of differential scanning calorimetry (DSC) to investigate the ability of the SH1-SH2-modified S1 to undergo global nucleotide-induced and actin-induced structural changes expressed in a pronounced increase of the protein thermal stability. It has been shown that modification of both
SH-groups (but not the modification of the SH1 alone) almost fully prevents the changes in S1 thermal unfolding induced by
the formation of the ternary complexes of S1 with ADP and P i analogues, whereas this modification has almost no influence on
the actin-induced structural changes. Thus, combination of EPR and DSC approaches shows that modification of both reactive
SH-groups on S1 has no influence on the actin-induced global structural changes of S1 and on the local nucleotide-induced
conformational changes in the region of the SH1-group, but it strongly prevents the global structural changes induced by nucleotides in the entire S1 molecule. These results suggest that modification of both reactive SH-groups, SH1 and SH2, impair
the spread of nucleotide-induced conformational changes from the ATPase site throughout the structure of the entire S1 molecule, thus disturbing a coupling between functionally important sites in the myosin head.
This work was supported in part by grants 00-04-48167 and 00-15-97787 from the Russian Foundation for Basic Research (RFBR) and by INTAS-RFBR joint grant IR-97-577.
PROPERTIES OF MYOROD, A THICK FILAMENT PROTEIN
IN MOLLUSCAN SMOOTH MUSCLES
N. Shelud’ko, T. Permyakova, K. Tuturova, O. Tyurina,
G. Matusovskaya and O. Matusovsky
Institute of Marine Biology, Far East Division of RAS, Vladivostok, Russia
Myorod, water-insoluble, heat-stable, resistant to organic solvents, susceptible to proteolytic digestion protein whose
content in muscles is 5-6% has been found in smooth muscles of bivalve molluscs [Shelud’ko et al. (1998) Biophys. J., 74,
268]. It is represented by one or two components in SDS PAGE ( and -chains) with close molecular masses (113 and 106
kDa) and approximately equal intensity. Myorod is localized on the surface of paramyosin core of thick filaments together with
myosin [Shelud’ko et al. (1999) Comp. Biochem. Physiol., 122, 277]. This protein is an alternatively spliced product of the
myosin heavy chain gene. It contains the C-terminal rod part of myosin and a unique N-terminal domain (Yamada et al. (2000)
J. Mol. Biol., 295, 169).
Myorod at physiological ionic strength possesses far lesser aggregation capacity than myosin and myosin rod. It is
much more soluble at intermediate ionic strength. The critical monomer concentration for polymerization of myorod is many
times higher. The size of polymer particles of myorod is considerably smaller than that of myosin and myosin rod. Since the
rod parts of myosin and myorod are identical, and thence the assembly competent regions are identical too, one may suppose
that assembly properties of myorod are strongly modified by its unique N-terminal domain.
Polymerization of myorod is affected by Mg2+ and Zn2+ ions, ATP and N-ethylmaleimide. Mg2+ greatly stimulates
polymerization of myorod, but Zn2+ at very low concentration causes its aggregation. ATP at physiological concentrations
causes dissolution of polymeric myorod. Modification of SH group at Cys-722 by N-ethylmaleimide inhibits the assembly
ability of myorod.
The properties of polymeric myorod are unusual. On the one hand, it appears as suspensions that are easily precipitated by low-speed centrifugation. On the other hand, it forms a viscous solution with low turbidity and volume concentration.
The low-shear intrinsic viscosity of myorod is an order of magnitude higher than that of myosin or myosin rod and is close to
that of F-actin. Ensemble of solution-suspension properties of polymeric myorod suggests that this protein forms threedimensional network of small polymers.
Myorod co-polymerizes with myosin. The copolymer’s properties differ greatly from those of myorod polymers and
are close to those of myosin polymers. A trace admixture of myosin in myorod preparations or a small addition of myosin (0.21.0 %) to myorod drastically alters the myorod polymerization. The suspensions of polymeric myorod nucleated by myosin
have a high turbidity and low viscosity and consist of large particles.
Myorod does not affect actin-activated ATPase activity of molluscan myosin in the presence and absence of Ca 2+ and
molluscan tropomyosin.
It seems likely that specific properties of myorod are associated with its unique N-terminus. This suggestion is supported by the result of myorod digestion with papain. It is cleaved by papain at the vicinity of unique domain/rod domain junction and thence the resulting 100 kDa insoluble fragment can be referred to as the rod of myorod. Suspensions of polymeric
myorod rod have much higher reduced optical density and much lower viscosity.
62
The function of myorod is unknown. Whereas myorod is present in catch muscles, twitchin, but not myorod is a target
protein for catch regulation [Butler et al. (1998) Biophys. J., 75, 1904]. If myorod also participates in catch-contraction, the
following transference path of the regulatory signal after twitchin phosphorylation/dephosphorylation should be: twitchin 
paramyosin core  myosin and myorod  actin-myosin interaction. In accordance with this scheme, myosin and myorod Nterminus are finite acceptors of the signal and they can modify the myosin-actin interaction. The direct interaction of myorod
N-terminus (“head”) with thin filaments is unlikely, since it is small compared to the myosin heads. Moreover, we failed to
find any interaction of myorod with skeletal muscle F-actin in the absence or presence of molluscan tropomyosin. Another hypothetical possibility is that myorod can indirectly influence cross-bridge attachment to actin through a change of viscosity of
the medium surrounding the myofilaments.
This study was supported by the RFBR (project № 01-04-49462).
63
GRAVITY-DEPENDENT SKELETAL MUSCLE PLASTICITY
IN MAMMALS: FROM STRUCTURAL PHENOMENA
TO MECHANISMS
B.S. Shenkman, I.N. Belozerova, *T.L. Nemirovskaya
SSC-RF Institute for Biomedical Problems, RAS;
*Moscow State University, Faculty of Fundamental Medicine, Moscow, Russia
Usually the muscle disuse (in other words, diminishing of its mechanical activities) is considered to be the most important cause for the numerous alterations in the structure and function of skeletal muscles under microgravity.
But less is known about the physiological nature of this disuse, differences in responses of tonic and phasic muscle
systems and its functional and structural consequences in the time-course of the adaptation of the motor system to the microgravity conditions. According to our data during the first weeks of weightlessness the significant decline of the voluntary
strength-velocity characteristics of ankle and knee extensors was accompanied with the profound drop of muscle stiffness and
some structural changes of the muscle tissue. Some signs of muscle fiber atrophy became evident after 3 days of dry immersion. We revealed in these studies other structural changes in muscle as well (such as tendency to capillary loss, myofibrillar
damages etc). At the same time these changes are much less profound than changes of functional characteristics during this
functional period.
Later on during the next stage, the fiber atrophy in tonic and phasic muscles reaches the significant values. But the
levels of ST and FT-fiber atrophy in the same muscle are close to similar. The atrophy development is accompanied with the
capillary loss and decrease of muscle oxidative enzyme activities, fast myosin expression in slow-twitch fibers. The strength
characteristics continue to decrease, the decreased endurance and increased physiological cost of the standard submaximal exercise are observed during this stage.
We call this stage the stage of general atrophy.
During the next stage the atrophy development becomes the fiber-type-specific. According to our data, during this distant period, we see the predominant atrophy of ST-fibers in m.soleus, and FT fibers in m.vastus lateralis. The capillary number,
the mitochondrial volume density and the physiological cost of the submaximal exercise are stabilized during this period. But
the «substitution» of the fiber volume by the non-contractile elements becomes more profound during this stabilization stage.
By the cessation of this stage the muscle structural features is seemed to meet the functional requirements at the diminished
level of its contractile activities.
Evidently the main characteristics of muscle plasticity at each of these stages are determined by the action of some
biomechanical triggers. The faster development of the atrophic processes under conditions of dry immersion as compared to
head-down tilt bedrest and prevention from atrophy by means of artificial support application allows to suppose that support
deprivation may play a trigger role in the atrophy development in weightlessness. It is believed that the support stimulus onground maintaining the activity of postural muscles which is evidently necessary for maintaining in turn the volume of the fiber myofibrillar apparatus and consequently their size. The support deprivation leads to inactivation of the tonic system followed by the fiber atrophy development. The hypothetical cellular mechanisms involved in hypogravity-induced muscle fiber
atrophy are worth being discussed. It is supposed that documented loss of muscle stiffness may lead to slack state of the sarcolemma and inhibition of the dystrophin cytoskeleton turnover. Since even the short exposure to hypergravity induced the alterations of the dystrophin layer integrity. Dystrophin cytoskeleton changes are thought to be associated with the level of anabolic
processes.
Thus in microgravity the changes in muscle contractile activities, biomechanically triggered are thought to be followed by the numerous alterations in muscle fiber elastic properties and then changes in intracellular signal transduction led to
changes in muscle structural and metabolic characteristics.
CRYSTALLOGRAPHIC RECONSTRUCTION OF THE ACROSOMAL PROCESS FROM LIMULUS
POLYPHEMUS SPERM
M.B. Sherman* #, P. Matsudaira**, W. Chiu* and M.F. Schmid*
*National Center for Macromolecular Imaging and Verna and Marrs McLean Dept.
of Biochemistry, Baylor College of Medicine, Houston, TX, USA;
**Whitehead Institute for Biomedical Research and Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA, USA
#
Current address: Department of Biological Sciences, Purdue University,
West Lafayette, IN, USA
Limulus sperm contains a dynamic macromolecular structure the acrosome. The core of this structure is a bundle
composed of a complex of actin, scruin and calmodulin. In an unactivated sperm, the acrosome - a bundle of up to 120 actin
filaments cross-linked by scruin - coils around the base of the nucleus and extends through a tunnel in the nucleus to the anterior tip of the sperm head. Within seconds after the sperm contacts the egg jelly coat, the acrosome straightens into a 60 µm
long crystalline bundle, the true discharge. Extension of the actin bundle does not depend on an ATP reaction; instead, the unusual twist of actin filaments in the coil may be a source of the energy that drives the bundle through the nucleus to contact the
egg. An alternative possibility is that binding of calcium could induce a conformation change that converts the coil to the true
discharge. The transition from coil to true discharge probably involves breaking some filament crosslinks in the coil and forming new crosslinks in the true discharge. This change in interactions is presumably mediated by scruin. Scruin is a 102-kDa
protein and is present with a 16-kDa calmodulin, both in a 1:1 complex with actin. Earlier helical reconstructions of a filament
show scruin to be a bilobed molecule which decorates the outside of actin filaments. The true discharge is well ordered and it
diffracts beyond 7 Å resolution. If scruin influences filament twist and forms the crosslinks between filaments in the true discharge, then a plausible model of the acrosome reaction may involve a change in conformation of scruin. Furthermore, because
calmodulin is present along with scruin, calcium may be the intracellular signal that either triggers or drives the acrosome
64
reaction. The acrosome reaction may involve a conformation change in scruin and altered interactions with neighboring scruin
subunits. A detailed three-dimensional structural analysis is needed to elucidate these interactions. In this study, we considered
the organization of the scruin crosslinks in the true discharge because the previous helical reconstructions were not able to resolve the question of the filament-filament interactions. We have used electron crystallography to determine the first structure
of a native actin bundle, the true discharge of the acrosome. We initially used electron tomography to reconstruct the bundle
structure at 45 Å. In that reconstruction we observed that the helical form of the actin-scruin filament is maintained in the bundle, but is modulated by the crystallographic environment. The interfilament interactions were found to be surprisingly diverse:
one or both of scruin’s domains are involved in contacts, the contact angle for scruin-scruin interactions is variable, and several
adjacent filaments can be cross-linked. These multiple types of interactions maintain the bundle as a stable and highly regular
assembly in the true discharge but also allow flexibility for the switch in types of interactions that must occur in other functional states of the bundle. Although our first tomographic reconstruction at 45 Å gave valuable insights into the structure, the
details of the structure, assembly and dynamics of the acrosomal bundle must await a higher resolution reconstruction. We are
collecting and processing data to about 10 Å resolution. We have imaged several hundred individual bundles that diffract to
that resolution. We treated short bundle segments, corresponding to two unit cells in length, as single crystals and developed an
orientation search procedure and merging scheme that is different from two-dimensional crystalline data processing. We are
extracting three-dimensional hkl reflection amplitudes and phases from diffraction patterns of individual segments. The 45 Å
reconstruction was used as an initial model to merge higher resolution data. Our preliminary reconstruction at 13 Å confirms
the helical nature of the filaments in the bundle. However, it shows that there is a systematic variability in the actin-scruin helix
that favors scruin molecules facing toward neighboring filaments rather than toward the spaces between them. Despite this accommodation, scruin crosslinks make a variety of non-identical contacts between filaments. This variety of interactions permits filaments to remain bundled whilst undergoing slippage and changes in twist relative to one another during the acrosome
reaction. Our structure indicates that promiscuous scruin-scruin contacts are the major determinants of bundle stability in the
true discharge. It also suggests that rearrangements in these interactions are permitted, which can facilitate changes in filament
twist and slippage between filaments.
Supported by grants NIH-RR02250, NSF-BIR9412521, NSF-BIR9500098 and NIH-GM52703.
References
1. Tilney, L.G. J. Cell Biol. 64, 289-310 (1975).
2. Sanders, M., et al. J. Biol. Chem. 271, 2651-2657 (1996).
3. Schmid, M.F., et al. J. Mol. Biol. 221, 711-725 (1991).
4. Schmid, M.F., et al. J. Cell Biol. 124, 341-350 (1994).
5. Sherman, M.B., et al. J. Struct. Biol. 120, 245-256 (1997).
6. Sherman, M. B., et al. J. Mol. Biol. 294: 139-149 (1999)
MODELLING THE EFFECT OF COMPLIANCE
OF THE THIN AND THICK FILAMENTS ON TENSION
APPROACH TO ISOMETRIC STEADY-STATE LEVEL
D.A. Shestakov and A.K. Tsaturyan
Institute of Mechanics, Moscow M.V. Lomonosov State University, Russia
We used kinetic cross-bridge models to simulate the effect of compliance of the actin and myosin filaments on the
time course of tension approach to its steady-state isometric level. Several different experiments, namely flash-photolysis of
caged compound and temperature jumps (T-jumps) were reproduced in computer simulations. The models are based on the
formalism developed by Ford, A.F. Huxley and Simmons (1981) and kinetic equations which take into account that strain of an
cross-bridge depends on its position within the overlap zone. The models are reduced to a system of non-linear integrodifferential equations with boundary conditions depending on tension which itself is a variable that will be found from the
equations. These equations were solved numerically using a combination of the parametric 'progonka' and iterations. The estimates of the filament compliance were taken from experimental data (H.E. Huxley et al., 1994; Wakabayashi et al., 1994; Higuchi et al., 1995; Linari et al., 1998).
It was found that strain distribution within the overlap zone affects calculated time course of tension responses which
differs from that obtained using a more simple approach used by A.F. Huxley and Tideswell (1996, 1997). Redistribution of
the compliance between the thin and thick filaments does not affect calculated tension transients, if the total filament compliance is kept constant. Generally, filament compliance leads to slowing down the late slow component of the tension transients
as myosin cross-bridges have to detach and to reattach to 'new' actin monomers to stretch compliant filaments. Therefore, tension approach to its steady-state isometric level is unavoidably accompanied by cycling reattachment of the cross-bridges
which becomes the rate-limiting step of the tension response.
The models explain the delay of the tension transient with respect to structural changes in the myosin cross-bridges
which is observed in the X-ray diffraction experiments with the T-jumps (Bershitsky et al., 1997; Tsaturyan et al. 1999a) and
flash-photilysis of caged-ATP (Tsaturyan et al., 1999b).
Even a simple model where the working stroke of a cross-bridge is described by a one-step transition from a preforce-generating to a force-generating step explains complex non-exponential kinetics of tension transients induced by the Tjumps and independence of this kinetics on preceding length step changes (Bershitsky, Tsaturyan, 1999). A model which includes detailed biochemical cycle of the cross-bridges explains the results of the experiments with photo-liberation of different
amounts of MgATP, particularly the fact that the kinetics of the early phases of tension transients is independent on concentration of ATP (Tsaturyan et al., 1999b).
The work was supported by grants from RFBR, INTAS and HHMI.
References
65
Bershitsky, S.Y. et al. 1997. Nature 388: 186-190.
Bershitsky, S.Y., Tsaturyan A.K. 1999. J. Muscle Res. Cell Motility 20: 819-820.
Ford, L.E., A.F. Huxley, R.M. Simmons. 1981. J. Physiol. 311: 219-249.
Higuchi, H., T. Yanagida, Y.E. Goldman. 1995. Biophys. J. 69: 1000-1010.
Huxley, A.F., S. Tideswell. 1996. J. Muscle Res. Cell Motility 17: 507-511.
Huxley, A.F., S. Tideswell. 1997. J. Muscle Res. Cell Motility 18: 111-114.
Tsaturyan, A.K. et al. 1999a. Biophys. J. 77: 354-72.
Tsaturyan, A.K. et al. 1999b. J. Physiol. 520: 681-696.
Huxley H.E. et al. 1994. Biophys. J. 67: 2411-2421.
Linari et al. 1998. Biophys. J. 74: 2459-2473.
Wakabayashi et al. 1994. Biophys. J. 67: 2422-2435.
CELL LOCOMOTION AND MORPHOGENETIC EVENTS
IN HUMAN KERATINOCYTE CULTURES
V.V. Shinin, O.G. Chernaya, E.A. Vorotelyak,
A.V. Vasiliev, A.A. Ivanov, V.V. Terskikh
Koltzov Institute of Developmental Biology RAS,
Sechenov Moscow Medical Academy RMAS, Moscow, Russia
The main aim of the present study was to find relationship between keratinocyte movements under different culture
conditions. Locomotion of keratinocytes in culture is indispensable in morphogenetic events. When plated on plastic surface
keratinocytes form asymmetric migrating colonies with convex leading edge and concave trailing edge. A colony moves as an
entire structure and every basal cell protrudes lamellipodium in the direction of migration of the colony. Migrating colonies
can change the direction of migration, divide into two parts and two colonies can fuse to give rise to a bigger colony. In calcium-free medium keratinocyte colonies fall apart into separate cells and directional migration of colonies was suppressed. Inhibition of proliferation also resulted in the cessation of migration. The leading edge is fored with rather differentiated cells,
while in the center of the colony, patches of cells with ‘primitive morphology’ are seen. At the later stages of cultivation
keratinocyte colonies enlarge and form confluent multilayered epithelial sheet. Similar colonies were formed on collagen gel.
But when embryo fibroblasts were embedded in the gel behavior of the colonies was changed. Migration of keratinocyte
tongues in several directions was observed.
When cultured in high density atop the gel containing embryo fibroblasts, keratinocytes organized into stratified epithelial sheets and invade collagen matrix. We also observed the formation of invasive foci when keratinocytes were incubated
with embryo fibroblast conditioned medium. To determine the architecture of oci of invasion we have reconstructed threedimensional models from serial histological sections. The analysis of reconstructions showed that keratinocyte invasion has
much in common with epithelial tubulogenesis. When keratinocytes were cocultured with dermal fibroblasts or incubated in
the presence of dermal fibroblast conditioned medium epithelial invasion was not observed. However under this conditions
keratinocytes also tend to organize stratified epithelial sheets.
When cultured in three-dimensional matrix (3D-collagen gel), keratinocytes form cysts (spheroids). The typical primary cyst consists of one layer of interconnected flattened keratinocytes organized into spheroids. We also observed small lumens inside spheroids filled with destroyed and terminally differentiated keratinocytes. Addition of embryo fibroblast conditioned medium induced epithelial tubulogenesis, a multiphase dynamic morphogenetic process. The first phase is the formation
of extension, which protrudes into the collagen matrix and might be considered as analog of lammelipodia on flat surface. Further migration of extended cells resulted in appearance of cells outside the cyst without a loss of cell-cell contact with neighboring cells. During the second phase migrating interconnected cells are sequentially rearranged into small chains. The third
phase (3-5th day of incubation) is the formation of epithelial cord which consists of 2-3 layers of cells. The last phase (the 7 th
day of incubation) is the formation of epithelial tube, which consists of 2-6 cell layers which stratify towards the tube lumen.
The latter structure appears optically dense and is surrounded with a thin and less dense basal layer. Cells of tube basal layer
form contacts with surrounding collagen matrix; formation ofdaughter tubes was frequently observed.
Mitomycin C decreased keratinocyte proliferation 7,5 fold and blocked formation of epithelial cords. Reversible metabolic blockade with excess of cold thymidine in conditioned medium also blocked formation of tubes, but in both cases we
observed the formation of extensions and chains. These results suggest that the formation of chains is independent of proliferation. Removal of metabolic blockade leads to the formation of tubes and epithelial cords. Epidermal growth actor, a factor
which stimulates keratinocyte proliferation, was not able to induce tubulogenesis, but significantly enhanced the growth of
tubes and tube precursors. The effect of EGF stimulation was observed on the 5 th day of incubation with embryo fibroblast
conditioned medium.
Results obtained suggest that proliferation is necessary for the formation of cords and tubes. Fibronectin does not induce tubulogenesis, but significantly enhances the formation of extensions and chains. Radioautography assay showed that after induction of tubulogenesis the rate and distribution of proliferatively active keratinocytes was differentially regulated in
tubes and tube precursors. Marked incorporation of 3H-thymidine was observed in chains and early cords (63,6±11,74 and,
25,3±2,63, % of labeled cells respectively). Cells that actively proliferate were organized in clusters. During later stages proliferative index decreased to 6,4±0,25% and cell proliferation was restricted to the basal layer of tubes.
To elucidate whether tubes are derived from stem or transit amplifying keratinocyte subpopulations we performed a
clonal-like assay. We suggest the clonal origin of epithelial tubulogenesis. Cells with high replicative potential, presumably
stem cells, give rise to epithlial tubes.
Taking together all results we propose parallelism in keratinocyte behavior on flat substrata and 3D matrix. When
seeded on flat substrate keratinocytes form colonies while within collagen gel they form cysts. Migrating colonies produce
broad fronts but in 3D matrix cells migrate as chains and cords, and undergo more complex morphogenetic rearrangements.
HIGH MOLECULAR WEIGHT MYOSIN LIGHT CHAIN KINASE
66
AS A POSSIBLE CYTOSKELETON INTEGRATOR
V.P. Shirinsky, D.S. Kudryashov, E.Yu. Kudryashova, E.A. Goncharova,
E.S. Nadezhdina#, N.A. Shanina^, L.J. Van Eldik*, D.M. Watterson*
Russian Cardiology Research Center, Moscow, Russia;
# Institute of Protein Research RAS, Pushchino, Russia;
^Moscow State University, School of Biology, Moscow, Russia;
*Northwestern University, Medical School, Chicago, IL, USA
Myosin light chain kinase (MLCK) was first identified in smooth muscle as an enzyme catalysing the phosphorylation
of myosin regulatory light chains (RLC). This modification was essential for the activation of myosin ATPase and myosin filament assembly in the presence of physiological ATP concentrations. Later it was found that MLCK also regulates non-muscle
myosin II and supports various cellular motile events such as amoeboid movement, secretion, endocytosis, cytokinesis, etc. To
interact with the microfilament bundles in non-muscle cells MLCK has actin-binding and myosin-binding sites located in its
N-terminal and C-terminal ends, correspondingly.
Molecular genetic studies have established that smooth muscle-type MLCK (108-130 kDa MLCK) is not the only
product expressed from the corresponding gene. Besides this isoform MLCK locus in the higher vertebrate genome codes for
at least two size classes of proteins: 210 kDa MLCK and a Kinase-Related Protein (KRP). KRP is the myosin-binding protein
with no protein kinase activity. High molecular weight MLCK isoform is predominantly expressed in non-muscle cells. Its
primary structure includes complete amino acid sequence of MLCK-108 and KRP. Consequently, MLCK-210 possesses functional features of these molecules such as catalytic activity toward myosin RLC and the ability to bind actomyosin. In addition,
MLCK-210 has a unique N-terminal tail not present in MLCK-108. Functional activities associated with this part of MLCK210 are the subject of our investigation.
We first demonstarted that the unique MLCK-210 domain interacts with F-actin in vitro and with the microfilaments
in cells and provides for the tighter interaction of MLCK-210 than MLCK-108 with the cytoskeleton of cultured cells. These
results are consistent with our observations that microfilament bundles in cells, predominantly expressing MLCK-210, are
more resistant to dismantling upon the inhibition of MLCK catalytic activity than the actin cytoskeleton of MLCK-108 containing cells. Thus, in addition to indirect stabilization of actin bundles mediated by phosphorylated myosin MLCK-210 is apparently directly involved in this process as a cross-linking protein. Further mapping of actin-binding sites within the unique
MLCK-210 tail localized them in its N-terminal and C-terminal ends.
Moreover, expressed unique domain of MLCK-210 and the enzyme purified from chicken aorta were able to interact
with tubulin and keratin in vitro. The sites of interaction with the cytoskeletal components were present within the expressed
fragment N459 corresponding to the N-terminal half of MLCK-210 unique sequence. N459 was able to aggregate monomeric
tubulin, microtubules and keratin filaments. Phosphorylation of N459 by cAMP-dependent protein kinase attenuated its interaction with the cytoskeletal proteins.
Based on our data and evidence from the literature we propose that functionally MLCK-210 is more versatile molecule than MLCK-108. Along with the regulation of the non-muscle myosin ATPase and filament assembly MLCK-210 may
reversibly integrate the main filamentous systems of the cytoskeleton. These capacities allow MLCK-210 to support dynamic
and local rearrangements of the cytoskeleton in non-muscle cells and organize myosin II-dependent cell motility.
Suported by HHMI grant 55000335 (VPS).
SOME ASPECTS OF MORPHOLOGY AND MOLECULAR
BIOLOGY OF CARDIAC MYXOMAS
V.I. Shumakov, A.S. Khubutiya, M.L. Semenovsky, D.V. Shumakov,
I.M. Iljinsky, Ya.N. Khalina1, Z.A. Podlubnaya1
Institute of Transplantology and Artificial organs, Moscow, Russia;
of Theoretical and Experimental Biophysics RAS, Pushchino, Russia,
E-mail: khalina@venus.iteb.serpukhov.su
1Institute
Cardiac myxosomas are benign tumors developing in different segments of the heart cavities and having malignant
clinical progress. Improvement of the diagnostic methods allowed the lifetime identification of cardiac tumors, particularly
myxomas, and successful surgical treatment of this category of patients.
During 1987-2000 at the Institute of Transplantology and Artificial organs 54 patients had been operated on account
of cardiac myxoma of left (51 cases) and right (3 cases) atrium. There had been 39 females and 15 males among them. The average age of the patients was 45 years. Histological and electron microscopic studying of excised myxomas was carried out.
Furthermore, 2 myxomas were studied using SDS-PAGE.
Histologically myxomas were found to be polymorphic structures differing in quantitative ratio of the basal mucoid
substance and, properly, of the tumor cells. The latter were sprouted or spindle-shaped. Electron microscopic study visualized
the vacuoles in the tumor cell cytoplasm, containing mucin, the product of extracellular secretion. Tumors were found to have
rather large blood vessels. Moreover, proliferation of cell elements resembling tumor cells was observed in the walls of some
vessels. Some tumors had of a lot of thin-walled plethoric vessels making the tumors to look like angiomas. Myxomas were often complicated with the secondary thrombosis. The precipitates of thrombotic masses are seen on the surface of the tumor.
The areas of macro- and micro hemorrhages were present in tumors relatively often. In the hemorrhagic zones the resolution of
the products of erythrocyte dissociation takes places (by) due to the macrophages. Zones of calcinosis were also found.
Hence the polymorphism is histologically and ultrastructurally inherent to the cardiac myxomas. Aetiology as well as
the general cause of tumor development is not elucidated. Histogenesis of these neoplasms remains arguable. Most of investigators consider the tumors to originate from the remainder of the embryonic mesenchymal tissue or from the endocardial endothelium. Our experience of histological and electron-microscopic studying of cardiac myxomas allowed us to suggest about
mesodermal origin of tumor from the cardiomyocytes precursors. To test this hypothesis we have carried out SDS-PAGE
67
analysis of 2 myxomas. In both cases proteins with molecular mass similar to that of heavy chains and essential and regulatory
light chains of atrial myosin were revealed. Further study is needed to clarify the hypothesis about the genesis of cardiac myxomas from myocardium.
The work was in part supported by RFBR grant № 01-04-48195.
ACTIN FILAMENT FORMATION REQUIRES
A CONFORMATIONAL CHANGE IN LOOP 262-274
Alexander Shvetsov*, Runa Musib‡, Peter Rubenstein‡ and Emil Reisler*
*Department of Chemistry and Biochemistry and Molecular Biology Institute,
UCLA, Los Angeles, CA 90095, USA
‡Department of Biochemistry, University of Iowa College of Medicine,
Iowa City, IA 52242, USA
Models of F-actin structure predict the importance of hydrophobic loop 262-274 at the interface of subdomains 3 and
4 to interstrand interactions in filaments. If this premise is correct, prevention of the loop conformational change—its swinging
motion—should abort filament formation. To test this hypothesis, we used site-directed mutagenesis to create yeast actin triple
mutant L180C/L269C/C374A. This mutation places two cysteine residues in positions potentially enabling the locking of loop
262-274 to the monomer surface via disulfide formation. Exposure of the purified mutant to oxidation catalysts resulted in an
increased electrophoretic mobility of actin on SDS PAGE and a loss of two cysteines by DTNB titrations, consistent with disulfide formation. The polymerization of uncross-linked mutant actin by MgCl2 was inhibited strongly but could be restored to
wild type actin levels by phalloidin. Light scattering measurements revealed non-specific aggregation of the cross-linked actin
under the same conditions. Electron microscopy confirmed the absence of filaments and the presence of amorphous aggregates
in the cross-linked actin samples. Reduction of the disulfide bond by DTT restored normal actin polymerization in the presence
of MgCl2 and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop
262-274 in the polymerization of actin into filaments.
NONCENTROSOMAL MICROTUBULES IN THE EPITHELIOCYTES UNDER WATER PERMEABILITY
CHANGES
E.S. Snigirevskaya, Ya.Yu. Komissarchik
Institute of Cytology of RAS, St. Petersburg
Recently, it was shown that transmembrane water permeability is performed through the water channels, aquaporins.
The delivering and insertion of channel proteins into the apical membrane is dependent on the cytoskeleton function, particularly microtubules.
In the present work some peculiarities of microtubule distribution and organization in the cells of tight epithelia at the
basal (low) and induced water permeability with different electron microscopic (EM) techniques have been studied. It has been
shown that during vasopressin stimulation of water flows across the granular cells of the frog urinary bladder the quantity o f
microtubules is increased. In these cells two types of microtubules with different diameter and stability have been revealed.
Thick microtubules (35 nm) are supposed to function as a transport vehicle for the migration of granules and vacuoles. Usual
microtubules (25 nm) seem to maintain the cell shape.
The big variety of pictures of single and bundle microtubules has been also observed in the epitheliocytes of frog large
intestine. Besides the usual microtubules with diameter 25 nm, the tight packed of microtubule bundles with associated osmiophilic globules have been revealed.
The microtubules of the cells studied are not connected with centrosomes, have unusual distribution in the cell, and
are mainly packed in the tight bundles oriented along the apico-basal cell axis. Sometimes they are reached the apical membrane by their minus ends and the intercellular junctions by their plus ends. All these features indicate that the microtubules described are the noncentrosomal ones. Their tubulin nature is demonstrated with aid of anti-tubulin antibodies and colloidal gold
labeling.
We suppose to discuss the problem of microtubule dynamics (assembly and disassembly) at different functional states
of cells.
The work is supported by the RFBR (projects № 99-04-49554 and 00-04-49480).
A POSSIBLE ROLE OF BACTERIAL ACTIN
IN PLANT-MICROBE INTERACTIONS
O.I. Sokolov, V.A. Bogatyrev, L.A. Dykman
Institute of Biochemistry and Physiology of Plants and Microorganisms RAS,
13 Prospect Entuziastov, Saratov 410015, Russia, E-mail: dykman@ibppm.saratov.su
A few types of biological motility are known nowadays, one of which is bacterial motility by means of flagella. This
system of motility is principally different from other well-investigated systems such as actin-myosin, tubulin-dynein and tubulin-kinesin. Yet a question arises whether such systems are evident in a prokaryotic cell.
Their role may lie both in organization of intracellular transport, protein synthesis which is evident for eukaryotic
cells, and in securing early stages of signal propagation in clasterisation of membrane receptors.
The purpose of our investigation was to prove the presence of actin in cells of Azospirillum brasilence and develop a
test system for complex estimation of its behavior at early stages of interaction with plant roots.
We suppose that soil bacteria-plant root interactions may manifest in state of changes in the actin component of the
microbe cell and complex of actin-associated proteins with it that comprises the notion of cytoskeleton.
68
So far a few number of publications are known devoted to isolation and identification of the actin in prokaryotic cells.
Our work is an attempt to isolate an actin-like protein from A. brasilense Sp 245. The method is based on principals valid for
actins from muscle and other sources.
We have proved the presence of actin in A. brasilense Sp 245 both by means of biochemical methods of its isolation,
and by newly synthesized markers on the basis of phalloidin and anti-actin antibodies, conjugated with colloidal gold particles.
It is supposed that an application of the above-described approaches for study on the role and state of bacterial actin
skeleton will be useful for understanding plant-microbe interactions.
This work was supported in part by CRDF (grant № REC-006) and RFBR (projects № 01-04-48736 and 99-0448833a).
SURFACE TOPOGRAPHY AND CYTOMATRIX ELEMENTS
IN PROTOPLASTS FROM PLANT CELLS
O.I. Sokolov*, Yu.V. Krivopalov*, M.K. Sokolova*,
V.V. Il'chukov* and A.V. Nosov**
*Institute of Biochemistry and Physiology of Plants and Microorganisms RAS,
Saratov, pr. Entuziastov 13, 410015 Russia; e-mail: soi@ibppm.saratov.su;
**Timiryazev Institute of Plant Physiology RAS,
Botanicheskaya ul. 35, Moscow, 127276 Russia
Cell differentiation and specialization, including the formation of the structure of its surface during transition from division to expansion, provide for morphological and physiological features of plant tissues and organs. In recent years, many efforts were made to study the surface structure of the plant cell and the intercellular systems determining this structure. It should
be noted that the results were obtained for very specialized plant cells: root hairs, root-cap cells, pollen tubes, epidermal cells
of the ovule (cotton hairs), etc. But the interconnection between morphological changes in the plasmalemma and cell wall, on
one hand, and the cytomatrix in the cortical cytoplasm layer, on the other, is not practically studied.
Isolated plant protoplasts are widely used in numerous physiological studies, the formation of the cell wall in particular. Protoplasts are also a convenient model for examination of the correlation between structural changes in the plasmalemma
and the structures outside and inside from it, viz., in the newly developing cell wall and in the cytomatrix.
Here we present the results obtained using an untraditional approach applied for studying surface topography of protoplasts from callus of Daucus sativus (Hofm.) Roehl. and from mesophyll cells of Nicotiana tabacum L. and also some data
characterizing localization of cytomatrix elements in the subcortical zone of the cytoplasm.
Protoplasts were prepared for scanning electron microscopy by a modified method without drying in the "critical
point" apparatus. After post-fixation with OsO4, carrot and tobacco protoplasts had similar topography of the surface: it was
rough and with few pores. When carrot protoplasts were not post-fixed with OsO4, their surface looked different: it was folded
and with 1.5 m pores, which sometimes were bordered with globules 0.3 m in diameter, or it consisted of conical cells varying in their depth and the size of their bases. We assume that, when protoplasts were not fixed with OsO4, they lose lipidcontaining structures from their surface, and what is left to be seen is the protein carcass of the plasmalemma and the underlying layer of the cytoplasm. The inner surface of broken carrot protoplasts had elaborate topography, apparently produced by
the elements of the cytomatrix, that is, a relatively thick layer of the cortical cytoplasm, where, using phalloidin-colloidal gold
and transmission electron microscopy, we could observe a dense network of actin filaments.
Protoplast isolation per se induces cell stress. Therefore, it is conceivably that the most part of the actin cytomatrix
can be disassembled. Nevertheless, using phalloidin-colloidal gold, we detected a relatively uniform network comprising a
large amount of F-actin in the subcortical zone of carrot callus cells. Sonobe and Shibaoka (Cortical Fine Actin Filaments in
Higher
Plant
Cell
Visualized
by
Rhodamine-Phalloidin
after
Pretreatment
with
m-Maleimidobenzoyl N-Hydroxysuccinimide Ester, Protoplasma, 1989, V.148, P.80-86) detected similar dense network of actin filaments in the subcortical cytoplasmic layer of tobacco callus cells by using rhodamine-phalloidin (a fluorescent marker).
When specimens were prepared for SEM, some protoplasts from carrot callus were broken even after their postfixation with OsO4 and their inner contents was exposed. Such preparations clearly demonstrate that the inner surface of the
subcortical zone has more elaborate topography than the outer surface. A network (more exactly a thick "fur") of interwoven
filaments and their bundles organized in rings, wavelike folds, etc., evidently forms this surface. So far, it is difficult to judge
the nature of these structures, but it is not inconceivable that they involve an actin constituent of the cytomatrix of the subcortical zone.
In general, we observed that, after post-fixation with OsO4, protoplasts from tobacco mesophyll cells and carrot callus
had similar topography of the surface: it was slightly rough and with few pores. Evidently, after such a fixation, most protoplasts from various plant tissues would appear in such a way. Without post-fixation with OsO4, carrot protoplasts had folded
surface with pores bordered with globules or the surface full of holes. The inner surface of the broken carrot protoplasts displayed a complex topography, which was possibly determined by a three-dimensional stricture of the cytomatrix components
within this zone.
The work is supported by the Russian Foundation for Basic Research, project № 99-04-48833.
MONITORING OF MYOSIN BRIDGES ORIENTATION
ON TWO-DIMENSIONAL MAPS OF BIREFRINGENCE
IN SINGLE MUSCLE FIBERS
L.K. Srebnitskaya, S.A. Neiman, * G.N. Vishnyakov, Z.E. Rogdestvenskaya
Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Russia;
*Russian Research Institute for Optophysical Measurements, Moscow, Russia
69
Two-dimensional maps of birefringence in sarcomeres from rabbit m. psoas single fibers have been obtained with
novel method of automated interference microscope. The changes in birefringence (BF) of muscle fibers reflect the movement
of myosin cross-bridges. Modifications of cross-bridges orientation were obtained by variation of pH (pH = 7.0; 6.0; 8.0) and
ionic strength ( = 0.085; 0.115; 0.235) in the bathing rigor solution. The measurements of phase shift were carried out with
two lengths of sarcomers (LS=2.5 m and LS =3.0 m) corresponding to different overlapping of actin and myosin filaments.
The maximum value of total BF in rigor state was observed at neutral pH. Total BF markedly decreased (by 40%) upon pH
changes from 7.0 to both 8.0 and 6.0. No significant changes in light phase shift were found at 1.5 fold reduction of ionic
strength in the rigor solution. The calculated values of BF were 45% higher in rigor solutions with high ( = 0.235) ionic
strength. The observed results are discussed in terms of changes in cross-bridges orientation due to movement of -helical subfragment-2 away from the filament shaft (pH=8.0) or coming closer to it ( = 0.235). The available data don’t allow to explain
the results obtained at pH=6.0.
The study was supported by the Russian Foundation for Basic Research (grant № 99-04-48265).
70
ROLE OF STRUCTURAL-CONFORMATIONAL CHANGES
OF ACTIN AND MYOSIN IN THE DECREASE
OF HEART SYSTOLIC AND DIASTOLIC FUNCTION
IN MEN WITH CHRONIC HEART FAILURE (CHF)
CAUSED BY DILATED CARDIOMYOPATHY (DCM)
G.V. Sukoian, D.R. Tatulashvili, L.T. Kuchava, N.E. Guledani, N.V. Karsanov
Republican Research Centre of Medical Biophysics and Introduction
of New Biomedical Technology, Tbilisi, Georgia
Background. DCM is a multifactorial disorder, which is considered as the final result of definitive myocardial damage caused by viral infections, metabolic abnormalities, genetic factors and others. The fact that the myocardium reacts in a
limited way to different stimuli is probably responsible for the nonspecific character of the histological findings on endomyocardial biopsy. Considering that left ventricular (LV) systolic and diastolic dysfunction may be related to general structural abnormalities affecting cardiac myocytes, it would be relevant to identify structural-conformational variables in biopsy samples,
capable of expressing the functional impairment of myocardium.
Materials and methods. Six patients with dilated cardiomyopathy were selected and studied after informed consent
obtained from all patients. All patients underwent clinical evaluation resting electrocardiogram, chest radiography, Doppler
echocardiography and cardiac catheterization, including left and right ventricular endomyocardial biopsy. The diagnose of
DCM was established according to the World Health Organization in the presence of global ventricular dilatation and diffuse
hypokinesis, LV ejection fraction (EF) 313%, end diastolic dimension > 6.7 cm and significantly decreased diastolic function
(E/A >2.0). The methods of skinned, ghost and hybrid reconstructed myocardial fiber preparation, actin and myosin extraction
were described earlier (Karsanov NV et al Exp.Clin. Pharmacol, 2000, V3, N2, 24-34). Methods of tensometric measurement,
Ca2+-Mg2+-ATPase determination and fluorescence analysis of the structural-conformational state of actin monomer and protomer in fibers see in Sukoian et al. (Bull. exp. biol. med., 1999, 2, 146-149). Obtained data were expressed as mean standard
deviation. The comparison of cardiac hemodynamic data with ability of contractile apparatus to generate force was made with
least square methods. A commercial statistics software package (Microsoft Excel 7.0.) was used for data analysis. The p value
<0.05 was considered significant.
Results. On skinned myocardial fiber from myocardium of patients with DCM has been established the sharp decrease of ability of force generation, ATP hydrolysis and the economy of energy transduction in myocardial contractile protein
system accompanied with the development of pronounced energy deficit state and disturbances in Ca-transport system those
are the key mechanism of CHF development and progression.
In contradiction to early and acute HF in case of CHF, caused by DCM, the myosin-based (as a result of damage of
hydrolytic center of myosin head) decrease of intensity of ATP hydrolysis (and as a result the energy-dependent tension generation decreases) is deposited on the actin-based decrease of the value and the velocity of generated force and the economy of
energy transduction in the contractile protein system (the ratio of tension/change of free energy falls from 2.25 to 0.8).
Described phenomena in CHF are conditioned on the molecular level by the changes of the structural-conformational
state dynamics of monomer and protomer of actin and its interaction with myosin heads of thick filament in force generation
process. These conclusions have been confirmed in experiments in which the properties of hybrid fibers, reconstructed from
failing heart thin filament (ghost fiber) and normal myosin and vice versa, are studied. The actomyosin hypothesis of the
mechanism of force generation and energy transduction disturbances in CHF has been suggested. It has been shown that at the
submolecular level in actin the distances between Lys61, Tyr69 in subdomain 2 and Cys374 and Cys10 are increased, the conformation mobility of these aminoacidic residues is decreased and microenviroment is changed. This leads to the loss of outside domain of actin protomer to generate force.
On the basis of our results and the data of atomic structure of skeletal muscle actin (Holmes KC et al., Nature. 1990;
347: 44-49; Mannherz HG. J.Biol.Chem. 1992; 267:11661-11664) and 3D reconstruction pattern of cardiac actin from normal
and failing heart (Samsonidze TG et al. Bull. Exper. Biol. Med. 1999; 1:101-105) a hypothetic models for normal myocardial
actin and actin from left ventricle of the heart from patients with DCM have been reconstructed.
The appointed faults have posttranslational character and under the creation of the optimal conditions in vitro under
the influence of the cardiac glycosides (-acetyldigoxine, -methyldigoxine and strophantine K) and under treatment of Refrakterin in vivo are able to revert.
THE ROLE OF A1 AND A2 FLAGELLINS
IN THE FORMATION OF SPIRAL FLAGELLA FILAMENT
OF HALOBACTERIUM SALINARUM
V.Y. Tarasov, M.G. Pyatibratov, I.V. Mescheriakova, O.V. Fedorov
Institute of Protein Research, Russian Academy of Science, Puschino,
Moscow Region, 142290, Russia
Halobacterium salinarum flagella are composed of a number of distinct flagellin proteins (A1, A2, B1, B2, B3) specified by genes in two separate operons (A and B). The roles of these flagellins were assessed by studying mutants of H. salinarum with insertions in either the A or B operon. It was found that only two A1 an A2 flagellin types of H.salinarum are sufficient to form a longitudinal and spiral filament. In the case of A2 flagellin minus strain of H.salinarum, when cells synthesised
only A1 flagellin, the flagella were straight. This fact confirmed the hypothesis that two different flagellins (for H.salinarum
they are A1 and A2) are necessary for the assembly of spiral archaeal flagella. According to suggested hypothetical model, arhaeal flagella filaments are arranged by several rows of protofilaments. Flagellins of two A types for H.salinarum form the two
different types of flagella protofilaments. They have different length and their association leads to formation of helical flagella.
Length differences are conditioned by different size of -helical part of N-termini of A1 and A2 flagellins.
71
MECHANISM OF COORDINATION OF LOCAL CONTRACTIONS OVER PHYSARUM POLYCEPHALUM
PLASMODIUM
V. A. Teplov
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia, E-mail:
teplov@venus.iteb.serpukhov.su
One of the most essential elements of the amoeboid cell locomotion is the endoplasmic flow, which is caused by gradients of the intracellular pressure generated by actomyosin-based contractions in the ectoplasm. In order to produce macroscopic flows, local interactions of the myosin oligomers with the actin filaments must be highly coordinated over the whole
cell body. At present mechanisms of self-organization of the spatio-temporal patterns still remains obscure. Since Turing's
classic work, theoretical modelling of such processes has been based on the idea that chemical reactions and diffusion of certain agents (activators, morphogens, etc.) initiate the formation of a chemical "pre-pattern", which then controls various components of the effector system. Recently, experimental data have become available suggesting that not only chemical, but also
mechanical processes play a part in the cellular signal transduction in biological systems.
Great scopes for investigating this problem are provided by the Physarum plasmodium, which is a single huge multinucleate cell with amoeboid type of movement. During migration the plasmodium arranges itself in a frontal protoplasmic
sheet and a network of interconnected veins (generally termed strands) in the rear. The endoplasm exhibits a regular back-andforth streaming activity throughout the plasmodium with a velocity up to 1-2 mm/s and with the period in the range of minutes.
We consider that in the plasmodium there exists some feedback loop between deformation of the ectoplasm and release of
some chemical trigger (e.g. calcium ions) controlling actin-myosin interaction. In such an event, spatial coordination of contractile activity can be achieved by hydrodynamical interactions between different regions of the plasmodium via the streaming
endoplasm. In this work we provide some experimental evidence in support of the opinion that mechanical stresses produced
by the contractile apparatus can come into play in the regulatory signal transduction pathways. Typical values of the mechanochemical parameters involved in this process will be discussed. As noted, contrary to pure reaction-diffusion systems, the regulatory systems incorporating mechanical elements have an advantage of long-range interactions, which are very essential for
pattern formation in large systems with unexcitable membranes.
This work was supported by the Russian Foundation for Basic Research.
ELECTRON MICROSCOPIC IDENTIFICATION OF SCORPION VENOM TOXINS INTERACTING WITH
ACTIN
N.R. Tiras*, S.N. Udaltsov*, S.A. Kozlov**,
I.B. Mikheeva* and D.A. Moshkov*#
*Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Russia;
**Institute of Bioorganic Chemistry RAS, Pushchino, Russia;
#Pushchino State University, Pushchino, Russia
The cytoskeletal protein actin plays a particular role in the functioning of neurons and synapses. It is involved in longterm potentiation of chemical and mixed synapses, the generation and maintenance of resistance to extremal external and internal influences. To provide further progress in this field, a search for new physiologically active substances interacting with
actin and new sources of these substances is needed. One of the sources of toxins is arachnida venoms of which scorpion venoms are the most extensively studied. The goal of the work was to identify the components of the venom of the scorpion Orthochirus scrobiculosus that specifically interact with actin. The objects of investigation were goldfish Mauthner neurons (Mcells) whose function largely depends on actin. Actin is present throughout the cytoplasm of M-cells and in specialized desmosome-like contacts (DLC) of afferent chemical synapses. The structure of DLC is easily identified in ultrathin sections by electron microscopy. The study was performed in the following order: in preliminary experiments, the ability of the crude scorpion
venom and its individual fractions to protect or inhibit the function of goldfish fry M-cells after fatigue stimulation (the effect
on the actin component of the cytoskeleton) was determined. For this purpose, either crude venom or its fractions were applied
to M-cells, and neurons were subjected to natural fatigue stimulation. Then two different approaches were used. The first approach involved studying the ultrastructure of DLC of chemical synapses after applying the venom fractions in vivo either to
intact M-cells or to M-cells whose structure and function were stabilized against fatigue by repeated stimulation. In some experiments, the application of venom fractions was combined with fatigue stimulation. The second approach consisted in studying the interaction of venom components with the muscle actin in vitro by the method of negative staining. Using the first approach, two out of nine isolated fractions were found to affect neuronal actin at the cellular level. One of these fractions, which
consisted of low-molecular peptides, was capable of polymerizing actin in the cell. The other fraction consisted of highmolecular peptides and had the property of destroying polymeric actin in the neuron. Further we studied these fractions using
the second approach and found that the interaction of the low-molecular peptide fraction with G-actin induced the formation of
filaments and bundles of F-actin. Similar filaments were observed in the control upon the interaction of G-actin with phalloidin
and 100 mM KCl. Moreover, the application of this fraction to intact M-cells also led to an increase in osmiophility and homogeneity of the perimembrane electron dense material and the area occupied by this material, as well as in the length of DLC of
afferent chemical synapses of intact M-cells. Similar alterations occurred in DLC after repeated stimulations of M-cells. These
changes are known to result from the additional actin polymerization in the region of contacts of synapses with the neuron soma surface. The effect of the high-molecular peptide fraction on F-actin was similar to that of cytochalasin D. By the action of
these peptides, actin filaments were broken into fragments, and their density in the field of vision decreased. Treating intact Mcells with this fraction led to a marked decrease in the osmiophility of the perimembrane fibrous material of DLC containing Factin. Loci of inhomogeneous electron density appeared in the structure of DLC. Many DLC became asymmetric. Upon combined action of this venom fraction and fatigue stimulation on functionally stable M-cells, only a fourth of DLC retained the
normal structure. In many DLC, either a nonuniform decrease in the osmiophility of the perimembrane fibrous material on the
cytoplasmic side occurred, or there appeared cavernae where this material was absent. A third of DLC were transformed to
72
semi-DLC, with the electron dense material being only on one side of the contact. Similar changes occurred in DLC of functionally stable M-cells treated with cytochalasin D. Thus, considering the data on the localization of F-actin in M-cells and its
involvement in maintaining various functional states of these neurons, as well as the results of the present study on the similarity of changes occurring in DLC by the action of venom fractions, phalloidin, cytochalasin D and on direct interactions of venom fractions with actin in vitro, the following conclusion can be drawn. The venom of O. scrobiculosus contains substances of
peptide nature that interact with the actin cytoskeleton. Further separation of these fractions into individual components
showed considerable promise of studies along this line.
The study was supported by a grant of the Russian Foundation for Basic Research (№ 99-04-49033) and a grant of the
Ministry of Education of the Russian Federation “Basic research in higher school in the field of natural and humanitarian sciences” (E00-6.0-290).
ADAPTATIVE CHANGES OF CREATINE KINASE OBTAINED FROM SKELETAL MUSCLE OF ONDATRA
ZIBETHIKA
Ya.L. Tolstov, I.Ye. Krasovskaya, R.V. Dyatlov,
Ye.V. Konisheva, L.V. Lislova, G.P. Dizhe
St.-Petersburg State University, St.-Petersburg, Russia
Creatine kinase (CК) (АТP: Creatine N-Phosphotransferase; EC 2.7.3.2.) is one of the major enzymes, playing a key
role in provision of energy that is necessary for muscle contraction. CК is very sensitive to changes of a physiological status in
organism that allows to consider it as a parameter of organism adaptation to stress.
Animals evolutionary adapted to oxygen deficiency, such as muskrat (Ondatra Zibethika), represent original model to
study hypoxia influence on muscles metabolism.
CК was obtained and purified from skeletal muscle of muskrat. The value of KM for phosphocreatine is 2,550,4 mM,
S05 value for АDP is 0,550,12 mM. The highest activity of creatine kinase was observed under the following conditions: temperature 30oС, рН 5,2, μ=0,02. 15 SH-groups per a mole of enzyme were discovered. These results considerably differed from
the similar characteristics of rabbit, hen and rat muscle CК. However, creatine kinase isoenzymes spectrum in cardiac and
skeletal muscles of muskrat was the same as the one of these animals.
The results allow us to assume, that the founded in this study properties of muskrat creatine kinase characteristics are
connected with changes in energetic of muscle contraction during adaptation to oxygen deficiency.
NON-STATIONARY DYNAMICS
OF BACTERIAL POPULATION WAVES
M.A. Tsyganov, G.V. Aslanidi, V.Y. Shakhbazyan,
V.N. Biktashev, G.R. Ivanitsky
Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Russia;
Institute for Mathematical Problems in Biology RAS, Pushchino, Russia;
Liverpool University, UK
Bacterial population waves may propagate symmetrically and with constant velocity, which depends on the motility
of bacteria and their chemotactic properties. This can lead to formation of the well know «Adler's rings». Such propagation can
be well described by mathematical models based on equations by Keller and Segel. In certain conditions, the bacterial population waves may propagate nonstationary, which may lead to creation of different structures, such as grain-like patterns, branching fractal-like bursts and stationary, non-propagating rings. Formation of these patterns can not be described by the KellerSegel equations and requires development of new mathematical models.
In this work we suggest a mechanism of propagation of chemotactic bacterial waves which takes into account local
changes of the environment by the bacteria. Based on this mechanism, we suggest a mathematical model of the bacterial population development, which can describe both stationary and nonstationary propagation of population waves. The nonstationary
waves leave behind stationary, spatially ordered patterns. We present experimental and numerical data of the bacterial population growth, illustrating this mechanism.
This work has been supported in part by the Russian Foundation for Basic Research (project 00-04-48144) and by the
Liverpool University.
ACTIN FOLDING. WHETHER INACTIVATED ACTIN IS INTERMEDIATE STATE OR RESULT OF
MISFOLDING?
Konstantin K. Turoverov and Irina M. Kuznetsova
Institute of Cytology, Russian Academy of Sciences,
194064 St. Petersburg, Russia, E-mail: kkt@mail.cytspb.rssi.ru
Protein folding and refolding are often accompanied by the association of partially folded intermediates. It represents
an essential problem in biotechnology because over-expression of recombinant proteins is often complicated by the formation
of associates of the misfolded protein intermediates accumulated in inclusion bodies. The association or aggregation of specific
proteins results in the development of many diseases such as neurodegenerative diseases, malignant myeloma, cataracts and
others, including the so-called prion afflictions.
All the above have generated much interest to the investigation of partially folded proteins' associates/aggregates and
the pathways of their formation in recent years.
Four stable structural states are known in actin. At low ionic strength actin exists as a monomer (G-actin), but in the
presence of neutral salts it is polymerized into a double-stranded polymer (the so-called fibrous form of actin, or F-actin). In
the solutions with high concentrations of denaturants (6 M urea and 4M GdmCl) macromolecule of actin is completely un-73
folded. The release of calcium ion by EDTA or EGTA treatment leads to the transformation of G-actin into the inactivated
form in which the protein molecule loses its capability to polymerize. Inactivated actin can be also obtained as a result of heat
denaturation, at moderate urea or GdmCl concentration, by dialysis from 8 M urea or 6 M GdmCl and spontaneously during
storage. We have shown that properties of inactivated actin are invariant to the way of denaturation and characterized this state
in details. In particular it had been shown that inactivated actin represents thermodynamically stable, monodispersed associate
of 15 monomer units. On the surface of the associate there are a large hydrophobic clusters, while the polar groups are located
in the inner regions of this formation. The tryptophan residues, located in this inner polar regions are more restricted than that
in native actin.
In all works devoted to actin denaturation without exception it was assumed that actin successively transferred from
native to inactivated and then to completely unfolded state: N  I  U, i.e. inactivated actin (I) was considered as intermediate state between native (N) and completely unfolded (U) states. And it seemed that all steady-state experiments prove this
model. At the same time the irreversibility of the transition N  I calls for kinetic investigations of the formation of inactivated
actin.
To elucidate the mechanism of the formation of inactivated actin the kinetics of actin denaturation induced by guanidine hydrochloride (GdmCl) was studied. The kinetic curves of fluorescence intensity at 320 nm were registered at different
concentrations of GdmCl. Interestingly, at 1.0 - 2.0 M of GdmCl the fluorescence intensity at first decreases, but then increases
approaching to the value characteristic to inactivated actin. These experiments along with the data showing that the lowest intensity at 320 nm is recorded for unfolded protein as compared with that of native and inactivated actin suggest that the transition from native to inactivated actin occurs via unfolding of protein structure.
The same form of the kinetic curves was found for other spectroscopic characteristics, which values for unfolded
macromolecule of actin are lower than that for native and inactivated states and which in contrast to fluorescence intensity define the structure of protein macromolecule qualitatively. Kinetic curves of parameter A=I 320/I365, that characterize the fluorescence spectrum position, fluorescence anisotropy and CD in the far UV region []222 at 1.0 - 2.0 M of GdmCl have minimum
also. On the base of all experimental data the following kinetic pathway of actin denaturation was proposed:
In the framework of this kinetic model the values of rate constants ki and the fraction of protein in native, inactivated
and unfolded states were determined on the base of experimental kinetics curves of fluorescence intensities by non-linear leastsquares method. The possibility to find best fit for the experimental kinetic curves proves the correctness of the proposed
pathway of actin denaturation. The calculation of rate constants allowed to determine the region of GdmCl concentration in
which the accumulation of completely unfolded molecules takes place and consequently minimum in the kinetic curves appears.
So all these data allowed us to conclude that inactivated actin is not the intermediate state between native and unfolded state, as it was assumed before, and that the transition from native to inactivated state passes through unfolding of protein
macromolecule. The result is essential for the search of the pathway of actin folding in vitro.
Acknowledgment. This research was supported by Grants 00-04-49224, 00-04-81082 Bel 2000_a, 01-04-49308 from
the Russian Foundation of Basic Research and St.Petersburg United Research Center (Joint Use Center).
SIGNS OF MYOGENESIS IN M.SOLEUS OF ADULT RATS
INDUCED BY MUSCLE UNLOADING (TAIL SUSPENSION), HYPERGRAVITY LOADING, IN COMBINE OF
THE BOTH FACTORS (ULTRASTRUCTURAL STUDY)
M.M. Umnova 1, I.B. Krasnov 2
1A.N.Severtzov
Institute of Ecology & Evolution, RAS,
117071 Moscow, Leninsky prospect, 33;
N
k1
k2
U
I.
k3
2Institute
of Medico-Biological Problems, State Scientific Centre RF, RAS,
123007 Moscow, Choroshevskoe shosse, 76a.
It is well known that the satellite cells are the stem cells of skeletal muscle during ontogenesis and at regeneration of
damaged muscle fibers. The experimental model of rat hindlimb suspension usually used to study the influences of muscle disuse and the simulated microgravity conditions (Бабакова, Деморжи, Краснов и др. In: abstracts of conference “Mechanisms
of structural, functional and neurochemical plasticity of brain”. Institute of the Brain, RAMN. Moscow. 1999. p. 9; Бабакова,
Деморжи. In: abstracts of the 2-nd Russian Congress of Pathophysiology. Moscow. 2000. p. 333; Desaphy et al., p. 146; Nemirovskaya, Shenkman, p.243; Praysse et al., p. 167; Saint-Pierre et al., p. 292.; Stevens et al., p. 303. In: abstracts of the International Congress of Myology. Acropolis-Nice-France. 2000.); we took advantage of the same model. The purpose of the present study was to determine satellite cells response and ultrastructural changes in m. soleus muscle fibers of adult males Wistar
rats after: 1/ hindlimbs tail suspension (freely hanging, unloading) for 24 days (3 rats, 1-st series), 2/ influence of hypergravity
conditions (+2GZ) during rotation of centrifuge for 1 hour/day/19 days (3 rats, 2-nd series), 3/ treatment by 19-day hypergravity load (+2GZ) of ultrastructural alterations in m. soleus muscle fibers at rat’s hindlimbs suspension for 1 hour/day/24 days (3
rats, 3-d series). We used also vivarium control Wistar rats (VC). The control and experimental rats were sacrificed by decapitation. Bioptates of m. soleus were fixed by 2.5% glutaraldehyde on 0.1 M cacodylate buffer, pH 7.4 and postfixed by 1% osmium tetroxide in the same buffer. Muscle samples were embedded in epon-araldite. Longitudinal and transverse ultrathin sections of m. soleus examined under JEM-100C transmission electron microscope. At VC and experimental rats number of nuclei
contained satellite cells on muscle section was counted per 100 myonuclei.
74
At VC rats, with body mass - 330,0 g, number of satellite cells was equal: 7%, 14,8%, 17,5% (preliminary data of 3-d
different animals). Sometimes two satellite cells placed a line under muscle fibres basal lamina. Muscle fibers of VC rats had
usual ultrastructure including small and large autophagosomes with dark dense substances and light lipid drops located in the
vicinity of muscle nuclei; some vacuolate mitochondria also occurred. At the experimental rats of the 1-st series, body mass 306,0 g, in disuse of m. soleus after hindlimbs suspension it occurred: 1./ 6-7% of satellite cells number was counted (diminished or equal in comparison with the VC rats; preliminary data of 2-d animals), 2./ lateral surface of satellite cells contacted
with short processes of neighboring satellite cells located on the same muscle fibre; this feature reflected proliferating activity
of satellite cells, 3./ some muscle fibres showed normal ultrastructure with formation of short processes, probably, as result of
fusion of satellite cells with fibers. At the same time in rat’s disuse m. soleus signs of destruction of neuro-muscular system
and activity of connective tissue were observed: 1./ two muscle nerve bundles with 4-10 myelin axons with destructed myelin
coat at a rat, 2./ degeneration of single axonal terminal at the neuromuscular junction, 3./ destruction of peripheral myofibrils,
4./ appearance of short muscle fibres (or part of muscle fibres) which had not any myofibrils (destroyed), 5./ penetration of
macrophages at the interstitial space among muscles fibres, 6./ fibroblasts shown intensive granular reticulum filled with substance of middle electron density that reflected collagen synthesis. Comparing of rat’s m. soleus neuromuscular system alterations under microgravity after hindlimb suspension with changes of neuromuscular apparatus of tritons sojourn stayed on
‘sputnik’ after space flight for 16 days at the microgravity it was shown similarity and differences of ultrastructural transformations. At the triton neuromuscular junctions Schwann cells occupied the site of withdrawal axonal terminals (Umnova,
Krasnov, Mitashov, 2000; Umnova, Mitashov et al., to press) in comparison with the lesser degree of destructive lesions of
rat’s neuromuscular junctions (preliminary data). It might be connected with different environmental conditions and with species of experimental animals.
Experimental rats of the 2-nd series, body mass was 300,7 g, were subjected with influence of hypergravity conditions
(2GZ). Muscle fibres of usual ultrastructure showed satellite cells number of 7% (preliminary data). Single satellite cells located in neighboring muscle fibres on the same level of the muscle section. We did not notice destruction of muscle fibers at
the studied blocks of specimens.
At the 3-d series of experimental rats, with body mass 281,0 (treatment of hindlimbs suspension by séances of hypergravity:2GZ), satellite cells number of m. soleus was equal to 9% (preliminary data). This index of increased satellite cells
number grown up compared with the both series (1-st and 2-nd). We observed the binuclear satellite cell, located under basal
membrane of mature muscle fiber of an adult rat; this satellite cell may be considered as an early developing myotube. We noticed preserved ultrastructure of motor and sensory endings in intrafusal nuclearbag dynamic and static fibers (bag1 and bag2).
We observed as destructive as regenerative features in rat’s m. soleus muscle fibers under experimental conditions reflected of muscle plasticity. The most destructive signs of muscle fibers were noted in disuse muscle fibers (at rats hindlimb
suspension).
The used treatment of rat’s m. soleus muscle fibres disuse by 2GZ conditions had made positive and effective influence on muscle atrophic process, based on the muscle ultrastructure. Authors are thankful to Dr. T.L.Nemirovskaya for the participation at organization of material fixation for electron microscopic study. This work was supported by the grant of the
RFFI: 01-04-49076 and by the grant INTAS-ESA 99-1191.
ORIENTATION TRANSIENTS OF THE REGULATORY
LIGHT CHAIN OF SKELETAL MUSCLE MYOSIN
D.S. Ushakov, *J. Borejdo
Div. of Physical Biochemistry, National Institute for Medical Research, London, UK;
*Dept of Molecular Biology and Immunology, University of North Texas,
Fort Worth, USA
Muscle contraction is believed to involve a “swing” of the C-terminal, regulatory domain of myosin head. The distal
end of this domain contains the regulatory light chain. In order to follow rotations of the regulatory domain during tension generation by skeletal muscle fibers, the time-resolved changes of the fluorescence anisotropy of probes attached to the regulatory
light chain were measured. A single Cys residue introduced in the N-terminal lobe of the regulatory light chain was covalently
labelled with fluorescent probes. The labelled light chain was exchanged with the native light chains of myosin of a skeletal
muscle fiber. The confocal microscope was modified to measure the anisotropy with a time resolution of 1.39 msec from a
sampled volume of 0.3 m3, containing ~600 labelled cross-bridges. Only the cross-bridges within the sampled volume were
activated by a precise delivery of ATP from a caged precursor, assuring high level of synchrony. Upon photogeneration of
ATP, the anisotropy changed with a half time of 17 msec, reflecting dissociation of cross-bridges from actin. After this initial
fast change, the regulatory domain underwent a small, slow reorientation with a half time about 5 times longer, suggesting
generation of force by the activated cross-bridges.
INFLUENCE OF CAFFEINE ON LOCOMOTION OF DICTYOSTELIUM DISCOIDEUM CELLS
O.O. Vasieva, S.I. Beylina
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia, E-mail:
beylina@venus.iteb.serpukhov.su
Dictyostelium discoideum is an unicellular organism feeding with bacteria, which demonstrates a co-operative behaviour and forms multi-cellular structures after the nutrition supply is exhausted. The basis for cell aggregation is the ability of
Dictyostelium cells to secret cAMP and responds to it chemotactically. This process realizes itself in waves of cell excitation
and converging chemotactic motion [1]. How the cell responds to a rising flank of the cAMP wave and appears paralysed or
randomly moving at the declining flank of the wave is still a mystery. The dominating point of view supposes that both spatial
and temporal mechanisms of gradient sensing take part in the oriented motion of cells.
75
The detection of temporal changes in cAMP gradients is, probably, based on the biochemistry of cGMP response,
which shows rapid and exact adaptation to constant stimuli (2). Stimulation of the cells with attractants leads to a ten-fold increase in the cGMP level with a maximum at ten seconds and with a 30-s seconds recovery time. Ca2+ influx is stimulated by
external cAMP with a comparable kinetics (3).
During chemotaxis, a cell not only increases the resulting speed of locomotion but changes the shape, becoming elongated (polarized) in the direction of the attractant source. Intracellular Ca2+ gradient was shown to be required for cell orientation (4).
Before aggregation and between chemotactic waves, Dictyostelium cells are supposed to move in a random manner.
The mechanisms that make cell motility random are understood even less than those underlying locomotion provoked by an attractant. It is possible that both types of cell crawling share the same regulating pathways, and the randomly moving cell, like
obviously oriented one, uses extracellular self-signalling to escape previously occupied territory (5).
Caffeine is known by its influence on cAMP pathway and as an modulator of Ca 2+ intracellular concentration in different systems (6,7). Caffeine was shown to inhibit adenylate cyclase activation in Dictyostelium (8), to enhance cGMP response (2), and to modify cell aggregation pattern (9), though the mechanism of its action is not yet clearly understood.
When studying the effect of caffeine on the wave propagation in Dictyostelium, we paid attention on that the drug influenced not only cell aggregation but cell locomotion in pre-aggregation period as well. So, the cell locomotion on caffeinecontained substrata was analysed in both of these periods.
A light microscope "Opton", equipped by the time laps set-up was used for monitoring cell fields. All measurements
were performed directly on enlarged images. Quantitative computer analysis of cell contours was applied to determine an average cell orientation in a local cell layer domain.
Our results show that, during random locomotion of Dictyostelium cells, caffeine increases cell polarization and regularity of cell extension and retraction. During aggregation, backward loops of cell motion were noticed. We discuss the possibility of that the vector of motile reaction to the attractant can be modulated in Dictyostelium cells by intracellular concentrations of Ca2+ or cGMP, as it is known for paramecia (10) or for growing neuronal cone (11).
This work was supported by The Russian Foundation for Basic Research.
References
1. Devreotes P. S., Potel M. J.,Machay S. A.,1983. Dev. Biol. 96, 405.
2. Mato J.,1978. BBA, 540, 408-411.
3. Wick U., Malchow D., Gerish G., 1978. Cell. Biol. Int. Rep.,2, 71-75.
4. Unterweger N., Schlatterer C., 1995. Cell Calcium. 17, 97-110.
5. Beylina S. I., Matveeva N. B., Priezzhev A. V., Romanovsky Yu. M., Sukhorukov A. P., Teplov V. A., 1984. In: Self-Organization,
Autowaves, and Structures far from Equilibrium (ed. Krinsky V.I.) Springer-Verlag.
6. Coronado R., Morrissette J., Sukhareva M., Vaughan D.M., 1994. Am. J. Physiol., 266, C1485-C1504.
7. Oyama M.,Kubota K.,1995. J. Biochem., 118, 117-121.
8. Brenner H.,Thoms S. D., 1984. Dev. Biol., 101, 136-146.
9. Siegert F., Weijer C., 1989. J. Cell Sci., 93, 325-335.
10. Naitoh Y., Kaneko H.,1973. J. Exp. Biol., 58, 657-676.
11. Song H-J., Ming G-L., Lehmann M., McKerracher L.,Tessier-lavigne M., Poo M-M. 1998. Science, 281,1515-1518
POSSIBLE ROLE OF OUABAIN-SENSITIVE ISOFORM
OF NA+/K+-ATPase IN RAT MOTOR NERVE ENDINGS
A.N. Vasiliev, V.V. Gromova, I.I. Krivoi
A.A. Ukhtomsky Institute of Physiology, St. Petersburg State University, Russia
The decrease of activity of Na +/K+-ATPase, major regulator of cellular functions, is one of the most general attributes
of the various forms of neuropathologies and many other diseases. However functional specialization and the mechanisms of
regulation of different isoforms of Na+/K+-ATPase in variousdepartments of nervous system, including neuro-muscular apparatus, in many respects are not investigated. It is suggested, that vertebrate skeletal muscle fibres express two isoforms of
Na+/K+-ATPase (usually referred to as 1 and 2); motor nerves express 1 and 3 isoforms of Na+/K+-ATPase. The aim of the
issue given was to investigate functional role of Na +/K+-ATPase isoforms in maintenance of efficiency of mammal skeletal
muscle.
Experiments were carried out on isolated rat phrenic-diaphragm preparations with acetylcholinesterase inhibited by
armine. The mechanographical methods and the microelectrode technique were applied. As it is expected, that the physiological importance of isoforms of Na+/K+-ATPase is exhibited at extreme loadings, the model of muscle fatigue by continuous rare
(1/s) indirect stimulation was used.
The continuous fatiguing stimulation of nerve led to weakening of muscles contractions up to 14,5±2,5 % from a reference value. The bath-applied (for 15 minutes) acetylcholine (ACh) in low concentration (100 nM), comparable with background physiological level of non-quantal ACh in a synaptic cleft, caused long-term (1-2 hours) strengthening of contractions
of fatigued muscles in 2,49±0,26 times (n3D6, р<0,01). The contractions in response to a testing direct stimulation of muscles
did not change. The aftereffect of ACh was absent in the presence of muscarinic acetylcholine receptors antagonist atropine (110 M), and was eliminated at dose-depended manner in the presence of inhibitor of Na+/K+-ATPase ouabain (0,1-10 M).
Addition of ouabain (10 M) on the background of ACh-induced restoration of muscles efficiency eliminated this aftereffect
of ACh. In conditions of intact acetylcholinesterase the aftereffect was reproduced only after application of non-hydrolyzed
analogue of ACh - carbachol (100 nM), but not ACh. The received data suggest a synaptic nature of ACh-induced restoration
of fatiguing muscles efficiency.
The frequency of extracellularly registered miniature end-plate currents (MEPC) at presence of ACh was reduced up
to 52,6±9,3 % and continued to decrease up to 35,6±9,6 % (n3D5, р<0,01) from initial level after 60 minutes of washing. The
76
ACh-induced MEPCs frequency decreasing and the muscle efficiency restoration had similar time scale. In the presence of 10
 ouabain (non-blocking concentration for 1 neuronal isoform of rat Na+/K+-ATPase) the frequency of MEPCs did not
change; the addition of ouabain during washing out of ACh increased MEPCs frequency. The mechanism of such MEPCs frequency decrease is not clear. We suggest, that this effect may reflect the hyperpolarization of nerve terminals membrane and/or
the reduction of intracellular Ca2+ concentration (via Na+/Ca2+-exchanger) by activation of Na+/K+-ATPase.
Our data allows to assume, that ACh-induced long-term restoration of fatiguing muscles efficiency has a presynaptic
nature and proceeds with involving unestablished systems of intracellular transduction. The aftereffect of ACh, presumably, is
realized through activation of ouabain-sensitive neuronal 3 isoform of Na+/K+-ATPase. The functional specialization of this
isoform, apparently, consists in maintenance of a level of evoked transmitter release upon the factors lowering the efficiency of
synaptic transmission. It is considered to apply the revealed effects of ACh to physiological testing of endogenous digitalis-like
inhibitors of Na+/K+-ATPase. The participation of muscarinic autoreceptors in cholinergic modulation of presynaptic function
by low non-quantal ACh is discussed.
Supported by Russian Foundation for Basic Research (grant № 01-04-49799).
CRYSTAL STRUCTURE OF TROPONIN C IN COMPLEX WITH
N-TERMINAL FRAGMENT OF TROPONIN I AT 2.3Å RESOLUTION
D.G. Vassylyev, S. Takeda, S. Wakatsuki, K. Maeda, Y. Maeda
RIKEN Harima Institute, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo, Japan
Troponin (Tn), he complex of three subunits (TnC, TnI, and TnT), plays key role in Ca 2+-dependent regulation of
muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we
have determined the crystal structure of TnC (two Ca 2+ bound state) in complex with the N-terminal fragment of TnI (TnI147). The structure was solved by the single isomoorphous replacement method in combination with multiple wavelength
anomalous dispersion data. The refinement converged to a crystallographic R-factor of 22.2% (R-free= 32.6%). The central
connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center and bent by
90degrees. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and Cterminal domains, in contrast to the elongated dumb-bell shaped molecule of TnCfree. The 31-residues long TnI1-47 alphahelix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC domains. The
amphiphilic C-end of the TnI1-47 alpha-helix is bound in the hydrophobic pocket of the TnC C-domain through 38 van der
Waals interactions. The results indicate the major difference between Ca 2+ receptors integrated with the other proteins (TnC in
Tn) and isolated in the cytosol (calmodulin). The TnC/TnI1-47 structure implies a mechanism of how Tn regulates the muscle
contraction and suggests a unique a-helical regulatory TnI segment, which binds to the N-domain of TnC in its Ca2+ bound
conformation.
SARCOPLASMIC RETICULUM FRACTIONS
FROM RABBIT SKELETAL MUSCLES ARE
CONTAMINATED BY MITOCHONDRIAL PARTICLES
O.M. Vekshina* , N.L. Vekshin
*Institute of Biochemical Physics, Moscow, Russia;
Institute of Cell Biophysics, Pushino, Russia
E-mail: olgavek@yandex.ru; Fax (0967)790509; Tel.(095)923-74-67, ext. 284
Sarcoplasmic retuculum fractions from rabbit skeletal muscles are contaminated by mitochondrial particles. These
particles can participate in processes of calcium transport: accumulation and releasing of Ca 2+, similarly like reticulum vesicles. Contamination by mitochondrial proteins contributes ~ 40 % to a total protein for the heavy fraction, and ~ 20 % - for
light fractions. Other proteins here are “associated” and lumenal sarcoplasmic reticular proteins. Ca 2+-ATPase is 60-80 % of
the total protein.
The content of mitochondrial particles was estimated by intensity of the flavin fluorescence and by dehydrogenase activity. The intensity of the flavin and NADH emissions of the heavy fraction was much higher, then of light fraction. About of
50 % of the heavy fraction are intact mitochondria that was estimated by comparing of optical densities at 280 nm (triptophan
absorbtion) and at 415 nm (the Soret band of cytochrom absorbtion).
Native mitochondria are absent in light fractions. Light fractions contain fragments of mitochondria and promitochondria. This fractions have not cytochroms, but are enriched by dehydrogenases. This conclusion was supported by the presence
of enzymatic activities of the mitochondrial NADH- and succinate-dehydrogenases. Dehydrogenase activities were induced by
electron acceptors – DCPIP and ferricyanid. Dehydrogenase activities were not revealed in protein-lipid preparations of Ca2+ATPase. These preparations were purified from light fractions of sarcoplasmic reticulum by choleat-contained solutions of
high ionic strength. Dehydrogenase activities were detected in supernatant after sedimentation of choleat-solubilized Ca2+ATPase. Reconstitution of calcium-transport for the preparation was obtained by the phospholipid addition in the presence of
carbonhydrate.
Our results mean that purification of light fractions of sarcoplasmic reticulum by choleat-contained solutions of high
ionic strength allows to obtain the Ca2+-transporting preparation of sarcoplasmic reticulum without mitochondrial fragments.
77
MYOSIN-ASSOCIATED PROTEIN TITIN
OF SKELETAL AND CARDIAC MUSCLES:
BEHAVIOR UPON ADAPTATION AND PATHOLOGY
I.M. Vikhlyantsev1, I.V. Makarenko1, Ya.N. Khalina1, M.D. Shpagina1,
S.N. Udaltsov1, S.L. Malyshev1, Z.I. Vishnevskaya2, Z.A. Podlubnaya1
1Institute
of Theoretical and Experimental Biophysics, 2Institute of Cell Biophysics, Russian Academy of Sciences; Pushchino, Moscow region, 142290, Russia
Hibernation is the ability of some mammals to inhibit reversibly the activity of all physiological systems, including
muscles. Upon hibernation a moving activity of skeletal muscles is suppressed completely, and a contractile ability of myocardium is considerably inhibited. In particular, during hibernation of ground squirrels the heart beat rate drops from 180-240
beats/min to 4-20 beats/min, and the cardiac output reduces by 65 times [1]. However, molecular bases of such phenomena
remain unclear. We have for the first time shown that the changes in isoform composition of light and heavy chains of myosin
and thereby in its functional properties can contribute to the impairment of contractile capacity of the above muscles [2]. The
understanding of the molecular mechanisms of hibernation requires the elucidation of the contribution of other myofibrillar
proteins to the change in physiological state of animals at this period. The myosin (thick) filaments in muscles are known to
contain along with myosin other proteins and among them such as titin (=connectin). The behavior of this protein during hibernation has not been studied. Titin is a giant elastic protein with molecular weight of about 3000 kDa. Each molecule of titin
spans the distance between the Z- and M-lines of the sarcomere connecting the ends of myosin-containing filaments with Zdisks. In the region of the A-disk the titin binds strongly to the myosin filaments while it is free in the I-disks of the sarcomere.
Its function(s) is unclear. It is supposed that titin helps to maintain sarcomeric integrity during contraction and is implicated in
myofibrillogenesis as scaffold as well as in generation of passive tension by muscle due to its extensible region in the I-disks
[3,4,5,6]. Different titin isoforms specific either for different types of muscles or for identical muscle types of different species
have been shown to exist. It has been revealed [7,8] that the length of titin isoforms determines the stiffness of cardiomyocytes
and thereby of extensible properties of myocardium. The higher stiffness was associated with shorter titin isoform and the lower stiffness was explained by high level of the longer titin isoform having the longer extensible region. In the authors’ opinion,
the variations in the levels of titin isoforms expression may be related to the variations in the filling rate and/or filling volume
of the heart. Lower myocardial stiffness would allow the faster filling and larger end-diastole volumes for the given filling
pressures, and vice versa. It was reasonable to assume that the changes in the titin isoform composition and in particular, in
their length might contribute to reduced contractile сapability of the myocardium upon hibernation by modulating the ventricular filling and emptying processes during diastole and systole, correspondingly. Using electrophoresis and immunoblotting we
have for the first time registrated the appearance of shorter titin isoforms in the skeletal and cardiac (ventricle) muscles of hibernating animals in comparison with active animals. In addition, it has been shown that shorter titin isoforms of skeletal muscles of hibernating animals affect actin-activated ATPase activity of rabbit skeletal myosin to a lesser extent. Thus, our data
suggest strongly the contribution of smaller (shorter) isoforms of titin to the suppression of contractile capacity of the cardiac
muscle of ground squirrels upon hibernation. As above noted, these changes are reversible upon arousing of the animal without
any pathological consequences.
Though unlike reversible stages upon hibernation the changes in pathology are irreversible, however the development
of cardiomyopathies also passes an adaptive stage. Our further study was directed to elucidating the contribution of titin to the
development of dilated cardiomyopathy (DCM). Cardiomyopathies - myocardium diseases, leading to distortion of its contractile function and characterizing by hypertrophy and the increase in its stiffness. This, in turn, results in the decrease of cardiac
output. Earlier the distortion in elastic properties of myocardium of patients with DCM was explained by the development of
sclerosis and fibrosis processes. However, some authors are now disposed to the importance of changes in myofibrillar proteins. The goal of this work is to elucidate the contribution titin to the distortion of contractile function of myocardium in
DCM. On the basis of the studies on adaptive changes of cardiac muscle titin of ground squirrels in hibernation which is accompanied by considerable suppression of contractile ability of myocardium, it was reasonable to assume that in DCM cardiac
titin can also undergo the changes in isoform composition contributing to the development of heart failure. Indeed, by the use
of SDS-gel electrophoresis and immunoblotting we have revealed that human cardiac biopsy samples of patients with DCM
contain the shorter titin isoforms. Moreover, the decrease of its molecular weight becomes more considerable with increase of
duration of pathological process (stage of DCM). We used as a control endocardium of left ventricles of rabbit and pig. It is
quite probably, that shorter titin molecules cannot cover the distance from M-line to Z-line. This can lead to distortion of the
integrity of sarcomere and as consequence to the impairment of contractile function of myocardium. In addition, as shown by
our preliminary tests, "pathological" forms of titin inhibit actin-activated ATPase activity of myosin that can also lower functional activity of myocardium. Taking into consideration the established dependence of cardiomyocyte stiffness from the
length of titin molecules [7] the conclusion was drawn about the contribution of shorter titin isoform to the increase of diastolic
stiffness of ventricle and thereby to the decrease of sistolic fraction output. The results obtained by us are important for creating a new prognostic test of DCM development, based on the registration of the changes in titin. Such a test allows to choose
the category of patients with negative prognosis of disease. This will favor the objective registration of potential recipients in
"waiting list" and also carrying-out of the heart transplantation at the earliest possible date and thereby the decrease of the level
of preoperative mortality.
The work is supported by RFBR grants №№ 99-04-04033, 00-04-48200, 01-04-48195, and DFG grant Be 347/10-1.
References
1. Popovic V. Amer. J. Physiol. 1964, v. 207, p. 1345.
2. Lukoyanova N.A. et al. I.M. Sechenov Russian Physiol. J. 1997, v. 83, p. 143.
3. Gregorio C. et al. Cell Biol. 1999, v. 11, p. 18.
4. Trinick J., Tskhovrebova L. Trends Cell Biol. 1999, v.9, p. 377.
5. Labeit S., Kolmerer B. Science. 1995, v. 270, p. 293.
6. Wang K. et al. Proc Natl Acad Sci USA 1991, v. 88, p. 7101.
78
7. Cazorla O. et al. Circ. Res. 2000, v. 86, p. 59.
8. Granzier H.L., Irving, T.C. Biophys. J. 1995, v. 68, p. 1027.
PHOSPHORYLATION-DEPENDENT INTRACELLULAR INTERACTIONS OF MYOSIN LIGHT CHAIN KINASE
AND KINASE-RELATED PROTEIN
Elena L. Vilitkevich, Tatyana Dudnakova, Mikhail A. Krymsky,
Asker Yu. Khapchaev, Maria I. Sidirova, Zhanna D. Bespalova,
Vladimir P. Shirinsky and Alexander V. Vorotnikov
Institute of Experimental Cardiology, Cardiology Research Centre,
Moscow 121552, Russia
The myosin light chain kinase (MLCK) related protein (KRP, also known as telokin) is smooth muscle specific myosin-binding protein abundantly expressed in phasic rather in tonic smooth muscles. The function of KRP is thought to bind the
neck region of dephosphorylated smooth muscle myosin and stabilise its filamentous structure in the presence of ATP [1]. The
major myosin-binding sequence was located in the C-terminal end of KRP, while much less contribution of its N-terminal sequence to this interaction was observed [2]. KRP has been later implicated in relaxation of smooth muscle contracted by submaximal Ca2+, and phosphorylation of KRP by protein kinases A and/or G at N-terminal Ser12 was suggested to potentiate its
relaxing effect [3]. We earlier found that KRP is multiply phosphorylated in intact smooth muscle within short N-terminal sequence at Ser12, Ser18 and, most probably, at Ser15 [4], however the phosphorylation does not apparently affect binding of KRP
to isolated myosin in vitro. Here we hypothesised that phosphorylation may affect the in vivo interactions of KRP with naturally arranged myosin filaments and/or other subcellular targets. To investigate this possibility, we developed the phosphospecific
antibodies against protein kinase A phosphorylated Ser 12 (R5) and MAP-kinase phosphorylated Ser18 (R8) of KRP. The antibodies were found specific to KRP and smooth muscle MLCK, which contains KRP sequence at the C-terminus, on the western blots of smooth muscle extracts. The phospho- and site-specificity of R5 and R8 was confirmed on the western blots of recombinant chicken and mammalian KRP phosphorylated in vitro by purified kinases, showing no alteration in recognition of
Ser12 and Ser18, correspondingly, following the complete phosphorylation of neighbour residues. Next we used phosphospecific antibodies in conjunction with polyclonal anti-KRP antibody that equally recognise the phosphorylated and unphosphorylated protein to compare extraction of phosphorylated KRP to that of the total protein from the rat ileum muscle extracted by a
stepwise increase in ionic strength. Similar results were obtained for KRP phosphorylated at Ser 12 and KRP phosphorylated at
Ser18. More than a half of the total protein was extracted at low ionic strength (25 mM) and this fraction consisted mostly of
phosphorylated KRP. The rest of phosphorylated KRP was completely extracted by intermediate (25-85 mM) ionic strength,
while the residual unphosphorylated protein that constituted approximately 30% of total KRP content in the tissue, could not
be extracted even at physiological ionic strength (150 mM). These results indicate that KRP is heavily phosphorylated in intact
smooth muscle and phosphorylation prevents KRP interaction with intracellular components under physiological ionic conditions. As myosin was characterised as the only KRP target to date, this suggests that native arrangement of myosin filaments
may be important for binding of the N-terminal domain of KRP and phosphorylation may regulate this process. Alternatively,
interaction of KRP with other targets may be also controlled by phosphorylation, although it is difficult to distinguish since intracellular distribution of KRP in smooth muscle is not yet defined. We have therefore used R5 antibody to examine whether
endogenous phosphorylation of Ser827 of MLCK (site B, homologous to Ser12 of KRP) affects the extractability of mainly actomyosin-associated MLCK. We find that minor fraction of MLCK is phosphorylated at site B in resting rat ileum, and most of
it, but not all, is solubilised under low ionic strength. This demonstrates that phosphorylation of site B decreases, but does not
abolish the MLCK interaction with cell contractile domain. The majority (>70%) of total MLCK was still associated with actomyosin under physiological ionic strength and was partially phosphorylated. The partial insensitivity of MLCK extraction to
phosphorylation of site B in the C-terminal KRP domain is consistent with a suggested actin-bound localisation of MLCK
which requires the N-terminal sequence. The quantitative distribution of phosphorylated and unphosphorylated MLCK between actin and myosin will be discussed. Altogether, these results indicate that phosphorylation of KRP and corresponding
sites in MLCK inhibits their intracellular interactions, presumably within the contractile domain, and demonstrates the potential use of the new phosphospecific antibodies for assessing the in vivo phosphorylation state of these proteins. Supported by
the grants from Russian Fund for Basic Research (99-04-49209) and the Wellcome Trust to AVV, the Howard Hughes Medical Institute grant 75195-546901 to VPS.
References
1. Shirinsky, V.P. et al. (1993) J. Biol. Chem. 268, 16578-16583.
2. Silver, D.L. et al. (1997) J. Biol. Chem. 272, 25353-25359.
3. Wu, X. et al. (1998) J.Biol.Chem. 273, 11362-11369.
4. Krymsky, M.A. et al. (2001) J. Muscle Res. Cell Motil., in press.
THE INTERACTION OF THE TRANSMEMBRANE
AND CYTOPLASMIC DOMAINS
OF THE SARCOPLASMIC RETICULUM Ca-ATPase
M.G. Vinokurov, M.N. Ivkova, V.G. Ivkov, V.A. Pechatnikov
Institute of Cell Biophysics, Russian Academy of Sciences,
Pushchino (Moscow Region), 142290 Russia
The interaction of transmembrane and cytoplasmic domains of Ca-ATPase of the sarcoplasmic reticulum (SR) has
been studied. To affect the hydrophobic transmembrane domain, the steroids having the biphilicity of molecular structure were
used. Four samples of the complex ethers of 20-oxypregnane’s dibasic acids consisting of the steroid nuclei cholesterol-like
steroid nuclei and different in the structure of the side chains were applied. Modifying effect of the steroids on the function
79
of SR Ca-ATPase was studied with pH-metric technique and the structural changes at the hydrolytic center were monitored by
the energy transfer between FITC and Nd3+ chromopores bound at the Mg-ATP substrate site.
It was shown that the rates of ATP hydrolysis and Ca 2+ transport were inhibited by the steroids, with carboxyl groups
in the side chains. The efficiency of the hydrolytic activity inhibition by different steroids increased in the following order (L145<L-122<L-128). The steroid without the carboxyl group had no marked effect on the SR Ca-ATPase function. Analysis of
the fluorometric titration curves of FITC-Ca-ATPase in SR vesicles by Nd3+ in the presence of steroids used showed that the
apparent dissociation constant for Nd3+ bound at the hydrolytic center increased in the same order the efficiency of the hydrolytic activity inhibition. These results suggest that there is a structural change in the active center.
The steroids inhibited more effectively Ca2+ transport than hydrolytic activity of the enzyme (L-145 and L-128 twice
as high and L-122 three times as much) due to in part by the increase of the passive permeability of the membrane in the addition of steroids in the row (L-145<L-128<L-122), indicating the efficiency of interaction of steroids used with the lipid bilayers. On the basis of analysis of both the own and literature data it was assumed that the inhibition of the hydrolytic and
transport Ca2+-ATPase function in SR membrane was due to the interaction of steroids used with the transmembrane -helix
segments. The effects of the steroids were largely dependent of the biphilicity of molecular structure (the availability the polar
groups at A an D regions), the structure of the side chains, and possibly the distance between the molecular polar groups making available their interaction with the transmembrane structure of SR Ca-ATPase.
THE ROLE OF APOPTOSIS IN THE FUNCTION
OF THE HUMAN NEUTROPHILS
M.G. Vinokurov, M.M. Yurinskaya, V.A. Pechatnikov
Institute of Cell Biophysics, Russia Academy of Sciences,
Pushchino, Moscow Region, 142290 Russia
The key mechanism of maintaining the structural and functional constancy of organs and tissues in the human body is
apoptosis. Of special interest is to study the molecular mechanisms of the apoptosis in the cells of the immune system, in particular, neutrophils, which play an important part in the normal and various pathological states of the human body. Neutrophils
are phagocytic cells that participate in inflammatory reactions as a first line of defense against bacteria and parasites. UV light
is known to induce the apoptosis in cells of different types. Currently, UV range C irradiation is widely used in practical medicine for treating patients with various diseases, although the molecular mechanisms of its action have hitherto remained virtually unknown.
UVC is widely used in medicine against sepsis. Human neutrophils play the important role in the host defenses
against the invasion of Gram-negative bacteria. Mature neutrophils have a circulating life span in vivo 6-10 h, after which they
undergo programmed cell death or apoptosis. Recent studies have shown that proinflammatory agents can prolong netrophil
survival. Lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, protects neutrophils from apoptosis. It was found, that UVC (254nm) in dose range 6 – 600 J/m2 in vitro accelerates apoptosis of human peripheral blood neutrophils in dose-dependent manner, with the saturation occurs under UVC doses of 250 - 300J/m2. Lipopolysaccharide
(1g/ml) suppresses UVC-induced neutrophils apoptosis. The joint action of lipopolysaccharides and UVC on apoptosis of the
human neutrophils was investigated. It is supposed, that the action of UVC on the apoptosis of neutrophils is realized through
Fas- tyrosinekinase-dependent pathway of regulation. The obtained data testify that the activation of apoptosis in the Fasdependent pathways occurs much faster, than the inhibiting action of lipopolysaccharides on apoptosis.
INVESTIGATION OF BIREFRINGENCE OF MUSCLE FIBERS
BY OPTICAL METHODS
G.N. Vishnyakov, G.G. Levin
Russian Research Institute for Optophysical Measurements, Moscow, Russia
The optical methods of muscle birefringence analysis contributed greatly to studying of molecular mechanism of force
generation during contraction. Most of them are based on measurements of light intensity passed through crossed polarizer, analyzer and a fiber (light polarized microscopy). The common disadvantages of these schemes were: 1) A necessity of making
measurement of the fiber thickness, which is non-uniform along the fiber. 2) A small number of data-points due to the absence
of automation of experiment; 3) Impossibility to use the high numeric aperture (NA) objective in order to obtain high resolution images (single sarcomeres). Our method based on automated Linnick interference microscope equipped with laser abolishes the disadvantages mentioned above. We use direct phase measurements of optical path length at parallel and perpendicular orientation of polarization plane of the probing radiation. The phase image is reconstructed from interferograms with the
use of four-frame phase-shifting algorithm. The obtained phase images of one and the same central part of a fiber at different
orientation of polarization planes represent two-dimensional numerical maps of the optical path length. Subtraction of these
images gives a two-dimensional map of phase shift, which includes information about birefringence. The formulae deduced for
birefringence measurements has a certain advantage in comparison to used earlier since it does not contain a thickness of a fiber that depends on the measurement point. We normalize a birefringence to a value of half sum of phases, which are measured
separately in the process of the experiment.
The study was supported by RFBR grant № 99-04-48265
ROLE OF MAP-KINASE ACTIVATION AND PHOSPHORYLATION OF NON-MUSCLE CALDESMON IN
UROKINASE-INDUCED HUMAN SMOOTH MUSCLE CELL MIGRATION
Alexander V. Vorotnikov, Elena A. Goncharova, Zoryana V. Grishina,
Elena O. Gracheva, Irina Beloglazova and Victoria V. Stepanova
80
Institute of Experimental Cardiology, Cardiology Research Centre,
Moscow 121552, Russia
Urokinase type plasminogen activator (urokinase, uPA) is chemoattractant for smooth muscle cells in vivo and in
vitro. We find that binding of recombinant urokinase (r-uPA) to cell surface rather that its proteolytic activity towards extracellular matrix is required to stimulate the migration of cultured human airway smooth muscle (hAWSM) cells in vitro. Using a
set of deletion and point mutants of r-uPA we show that binding of central kringle domain of uPA to a yet unidentified cell surface target, but not the binding of the N-terminal growth factor-like domain of r-uPA to the specific uPA receptor, is primarily
required to stimulate hAWSM cell motility. We hypothesised that signalling mechanisms of chemotactic effect of r-uPA may
involve activation of cell motility through the effects on contractile/cytoskeletal proteins. As actomyosin motor activity is primarily stimulated via Ca2+ and calmodulin-dependent myosin phosphorylation by myosin light chain (MLC) kinase and deinhibition of actin filament bound caldesmon by Ca2+-calmodulin, we first explored the effect of urokinase on intracellular Ca 2+
concentration ([Ca2+]in) and its role in cell migration. Using the Fura-2AM loaded hAWSM cells we failed to observe the effect
of r-uPA on [Ca2+]in suggesting that urokinase stimulates cell motility in rather Ca2+-independent fashion. Indeed, hAWSM
cells loaded with BAPTA to chelate cytosolic Ca2+ effectively migrated toward r-uPA, while chemotactic effect of thrombin,
which basically depends on the increase in Ca2+in, was abolished. This indicates that signalling mechanisms of urokinasestimulated hAWSM cell migration are largely Ca2+-independent and thus may involve other means of actomyosin activation,
such as protein phosphorylation. Consistently, we found considerable phosphorylation of MLC even in unstimulated cultured
hAWSM cells which could be further transiently stimulated 2-3-fold by r-uPA. Selective inhibition of Rho-associated kinase
with the cell-permeable drug HA1077 strongly inhibited r-uPA-induced cell motility. This suggests that urokinase likely triggers recently described Ca2+-independent signalling pathway involving subsequent activation of Rho family small GTPbinding proteins, Rho-kinase mediated phosphorylation and inhibition of MLC phosphatase and resulting in the increased
MLC phosphorylation and cell migration. However, phosphorylation of MLC in hAWSM cells was also similarly increased by
an r-uPA deletion mutant which had no effect on cell migration. This indicates that myosin phosphorylation alone is not sufficient to promote hAWSM cell migration and other signalling mechanisms are also involved. Searching for those, we found that
r-uPA transiently activates p42/p44erk1,2 and p38 mitogen-activated protein (MAP) kinases and this is critical for r-uPAinduced chemotaxis. However, as revealed by the use of r-uPA deletion mutants and inhibitory analyses, only the p38, but not
p42/p44erk1,2 MAP-kinase activation required the kringle binding and coupled it to the hAWSM cell migration response. Cytoskeleton-associated downstream targets of the r-uPA-induced p38 MAP-kinase signalling were further examined. Treatment of
hAWSM cells with r-uPA did not alter endogenous phosphorylation of the 27 kDa small heat shock protein, a known downstream target for the p38 MAP-kinase pathway suggested to modulate actin dynamics in the phosphorylation-dependent manner. In contrast, binding of r-uPA to hAWSMC caused an about 3-fold increase in endogenous phosphorylation of nonmuscle
caldesmon isoform as detected with the phospho-specific antibody against one of the MAP-kinase phosphorylation sites in
caldesmon. The increase in caldesmon phosphorylation was sustained for at least over 2 hours of stimulation, i.e. the period
comparable to the development of cell migration response. Moreover, the r-uPA-stimulated phosphorylation of caldesmon was
blocked in the cells pretreated with concentrations of p38 MAP-kinase inhibitor sufficient to inhibit chemotaxis of hAWSM
cells.
Altogether these results demonstrate that human smooth muscle cell migration stimulated by urokinase binding is essentially Ca2+-independent and apparently involves activation of Rho-related small GTP-binding proteins and triggering diverse downstream signalling pathways that target actomyosin-associated regulatory proteins and modulate their activity resulting in activation of the contractile machinery and cell motility. Specifically, the activated Rho-kinase is likely to mediate regulation of myosin phosphorylation and activation, while activation of p38 MAP-kinase pathway is required to target the actinassociated regulatory protein caldesmon. From the in vitro studies, the consequence of caldesmon phosphorylation by MAPkinases was the attenuation of its interaction with actin cytoskeleton and thereby elimination of inhibition exerted by
caldesmon upon actomyosin motor activity. The molecular mechanisms by which phosphorylation affects caldesmon interaction with actin will be also discussed.
Supported by RFBR grant № 99-04-49209 and the Wellcome Trust.
SENSORY «FREE » RECEPTORS REGENERАTION:
A STUDY OF NERVE TERMINALS MOTILITY
I.N. Zamuraev, V.G. Lukashin
I.P. Pavlov Institute of Physiology RAS, St.-Peterburg, Russia
Change of shape and sizes of neuron processes is one of unmuscle motility types. Growth of sensory neuron processes
(i.e. nerve fibres) into inner organs’ tissues is the least known at present. Morpho – electrophysiological investigation of sensory free terminals development under their regeneration has been aimed by us.
Sensory free terminals occurring in frog’s urinary bladder wall have been studied. From the morphological point of
view, these terminals are arborizations of myelinated afferent fibres 5-8 mcm in thickness. Upon loss of myelin, the fibre is divided many times to form tens of plates from 1 to 15 mcm in size which are connected in between by fine (<0,5mcm) filaments. Concerning functional viewpoint, as we have shown before, these free receptors are mechanoreceptors capable of producing spontaneous impulse activity for the most part.
For receptors’ degeneration and following regeneration have taken place we have made special operation, namely: the
nerves of plexus ischio-coccygeus, which give rise to nerves innervating bladder, have been cut and subsequently sutured (by
10/0 suture). Operated animals have been maintained at 20-220C. From 1 week to 1 year the frogs have been decapitated, their
urinary bladder have been isolated and regenerated free receptors have been investigated out.
It has been shown, that receptors regeneration in bladder wall is begun with growth and division of afferent fibres into
nerve fascicles. In what follows the motility of distal parts is connected with remyelination beginning in fascicles. Fibres leave
the fascicles and make arborizations up in surrounding tissues. Initiation of impulse activity from receptors, as has been
81
shown by us, independent either from number, shape, or from the size of terminal plates. So, there is little likelihood that plates
are involved in a bioelectric activity generation as it to be supposed previously. We have found morphological features suggesting terminal plates are to be growth cones of receptors. In some cases like-pseudopodium processes from plates have been
revealed. These pseudopodiums are to be elongated and connected neighboring plates, which is the case. What’s more morphology of the regenerated terminals is similar to one of sensory neurons growing processes in tissue culture. Availability of
many tens and hundreds of growth cones, maybe, allows to keep the functional integrity for free receptor: damage in some its
terminal part makes the neighboring parts to grow in order to level this damage.
This work is supported by Russian Fund for Basic Research (grant № 00-04-48084).
PARTICIPATION OF MICROFILAMENTS AND MICROTUBULES IN THE ROOT PRESSURE BUILDUP
V.N. Zholkevich, M.M. Puzakov, S.V. Sushchenko, I.B. Emelyanova
K.A.Timiryazev Institute of Plant Physiology, Russian Academy of Sciences,
Moscow, Russia, E-mail: ifr@ippras.ru
As is well known, roots not only absorb water consumed by plants, but also pump water into shoots, the root pressure
being the driving force. So root pressure is the lower inducer of uprising water flow in plant. It is summarized by two principally different constituents, namely metabolic and osmotic. By means of inhibitory analysis it was revealed an immediate participation of cell contractile systems in the metabolic constituent buildup [1-7]. Root pressure is in particular manifested by exudation of detached roots.
The present communication contains new data on the effect of contractile proteins inhibitors and stabilizers upon exudation rate. Experiments were performed with detached roots of Zea mays L. seedlings and Helianthus annuus L. plants as well
as with two models – “mittens” and “air” roots. “Mittens” are Zea mays L. detached roots without a stele. “Air” roots are Helianthus annuus L. detached roots without surface-water placed into humid chamber; they absorb water from outside not at all
but nonetheless excrete an exudate heavily at the expense of parenchyma cells secrete. “Air” roots constantly and “mittens”
during the initial period of exudation cannot operate like an osmometer – it is a principal advantage of these models. Exudation
of “mittens” occurs mainly and that of “air” roots exclusively at the expense of the metabolic constituent of root pressure.
It was shown that latrunculin B (LATB, a high selective inhibitor of G-actin polimerisation, blocks actomyosin) and
2,3-butanedione-monoxime (BDM, a high selective inhibitor of ATPase activity of myosin, blocks actomyosin) suppressed exudation, especially that of “mittens” and “air” roots. It is in agreement with earlier established fact of an exudation depression
by cytohalasin B (CHB), which disintegrates microfilaments. When applying LATB and BDM or LATB and CHB, BDM and
CHB together we observed some additivity of their suppressing influence on exudation. At the same time phalloidin (stabilizer
of G-actin) considerably stimulated exudation. In the presence of LATB stimulating effect was completely eliminated. Taxol (a
stabilizer of microtubules) like phalloidin considerably stimulated exudation. In the presence of colchicine (CHC, a desintegratror of microtubules) stimulating effect of taxol was absent. Under combined action of LATB and CHC practically a complete additivity was observed.
The data obtained confirm the earlier made conclusion on an immediate participation of microfilaments and microtubules in the metabolic constituent of root pressure buildup.
The work was supported by the Russian Foundation for Basic Research (grants № 98-04-48822 and 01-04-48447).
References
1. Zholkevich V.N., Sinitsina Z.A., Peisakhzon B.I. et al. (1979). Russ. J. Plant Physiol. V.26. P.978-993.
2. Zholkevich V.N., Chugunova T.V., Korolev A.V. (1990). Studia biophys. V.136, P.209-216.
3. Zholkevich V.N. (1991). In: Plant Roots: The Hidden Half. N.Y.: Marcel Dekker. P.589-603.
4. Zholkevich V.N., Chugunova T.V. (1995). Dokl. Acad. Nauk. V.341. P.122-125
5. Zholkevich V.N., Zubkova N.K., Korolev A.V. (1998). Dokl. Acad. Nauk. V.359. P.551-553.
6. Zholkevich V.N., Talat Mahmud Md., Monakhova O.F. (2000). Dokl. Acad. Nauk. V.371. P.696-699.
7. Zholkevich V.N., Puzakov M.M., Monakhova O.F. (2001). Dokl. Acad. Nauk (in press).
ANALYSIS OF IRREVERSIBLE THERMAL DENATURATION
OF PROTEINS WITHIN THE LIMITS OF KINETIC THEORY
OF DENATURATION. DOMAIN STRUCTURE OF THE MYOSIN HEAD AS STUDIED BY THIS APPROACH
Eugene O. Zubov1,3 and Dmitrii I. Levitsky2,3
2A.N.Belozersky
1Department of Biophysics, School of Physics;
Institute of Physico-Chemical Biology, Moscow State University; 3A.N.Bach Institute of Biochemistry, Russian Academy
of Sciences, Moscow; Russia
Differential scanning calorimetry (DSC) is the most effective method to study the thermal unfolding of proteins and to
obtain information about thermodynamic and kinetic parameters of the unfolding process. Thermal denaturation of many proteins is calorimetrically irreversible. The simplest model of irreversible denaturation is the “two-state” model including one
monomolecular irreversible step. There are established methods allowing to estimate the main parameters of irreversible thermal denaturation (the energy of activation, Ea, the temperature at which the rate constant equals 1 min-1, T*, and enthalpy, H)
from the excess heat capacity (Cex) vs. temperature (T) profile, as well as to verify the model of denaturation for a given protein. However, many proteins whose thermal denaturation is irreversible consist of more than one calorimetric domain (i. e. the
region in the protein molecule which unfolds cooperatively and independently from other regions). In such a case, he Cex(T)
profile is composed of a sum of the peaks of different domains. If these peaks do not overlap on the thermogram, or if they
overlap only partly, the method of “successive annealing” can be successfully used to estimate the parameters of thermal denaturation of separate domains. We propose a new method for analyzing the domain structure of irreversibly denaturing proteins
from their DSC curves, which is suitable even in the case of overlapping peaks of separate domains. An important feature of
82
this method is that the annealing is performed in opposite direction, i. e. the parameters of thermal denaturation of the most
thermostable domain are determined first. The DSC experiments are preceded by preliminary incubation of a protein at definite
temperature for a definite time. Temperature for this incubation and its duration are determined from analysis of the DSC
curves of a protein measured at different scanning rates. The parameters of thermal denaturation of separate calorimetric domains are verified by DSC experiments with such type treated protein at different scanning rates.
We have applied this method to study the domain structure of the myosin head. Only one calorimetric domain was revealed in the recombinant fragment M765 of the head of Dictyostelium discoideum myosin II corresponding to the globular
motor portion of the head that lacks the “neck” region and the light chains. On the other hand, at least two additional calorimetric domains were revealed in myosin subfragment 1 (S1) from rabbit skeletal muscles besides the main motor domain. One of
these domains was less thermostable, and another - more thermostable than the motor domain; the enthalpy values for these
two domains were ~10% and ~20-25% of the total enthalpy of the thermal unfolding of S1, respectively. Since S1, unlike
M765, contains some part of the “neck region” with associated alkali light chain (regulatory domain, “lever arm”), two small
calorimetric domains of S1, that are absent in the M765, can be assigned to thermal denaturation of the regulatory domain of
the myosin head. Preliminary results on the nucleotide-induced changes in the S1 domain structure will also be presented.
This work was supported in part by grants 00-04-48167 and 00-15-97787 from the Russian Foundation for Basic Research (RFBR) and by INTAS-RFBR joint grant IR-97-577.
83
INDEX OF AUTHORS
Abraham M.R., 132
Ader G., 12
Agadzhanyan G.M., 3
Aglintsyan T.S., 3
Akatov V.S., 5
Alekseev A.E., 6, 132
Almo S.C., 24
Andoh T., 54
Andrejev Y.A., 8
Andrejeva L.A., 8, 9, 102
Antropova Yu.G., 84
Arefieva T.I., 11
Aslanidi G.V., 156
Avrova S.V., 26
Azarova V.S., 31
Baklanova M.Yu., 87
Bashtrykov P.P., 84
Beinbrech G., 12
Beloglazova I., 17380
Beloussov L.V., 48
Belozerova I.N., 13, 137
Belyaev A.V., 33
Bennett P.M., 14
Bershitsky S.Y., 14, 15, 73
Bespalova S.V., 17
Bespalova Zh.D., 82, 169
Beylina S.I., 20, 67, 96, 162
Bienengraeber M., 6
Biktashev A.G., 89
Biktashev V.N., 156
Blyakhman F.A., 22, 119
Bobkov A.A., 24
Bochkov V.N., 84
Bogatyrev V.A., 24, 147
Borejdo J., 161
Borovikov Yu.S., 26, 65
Bozhkova V.P., 27, 29
Brown N., 45
Buckingham M., 45
Bukatina A.E., 30
Bulyakova N.V., 31
Burmistrova N.A., 90
Burstein E.A., 109
Bykov V.A., 33
Campione M., 45
Chekanov A.V., 5
Chernaya O.G., 141
Chernenko A.S., 17
Chikobava E.A., 34, 98
Chiu W., 138
Chumaeva N.A., 8, 9, 102
Ciechomska I., 65
Cusso R., 36
Day T.A., 81
de Lisi R., 45
Dedova I.V., 26
Demin I., 115
Demolombe S., 43
Dizhe G.P., 36, 156
Dobbie I., 75
Dominguez J.N., 43
Domnina L., 36
dos Remedios C.G., 26
84Dronenko G.E., 3
Dudnakova T.V., 38, 39, 90, 169
Dumaine R., 43
Dushkin I.V., 33
Dyatlov R.V., 36, 156
Dykman L.A., 24, 147
Dzeja P.P., 6, 132
Efremova T., 61
Egelman E.H., 47, 110, 111
Elizarov S.M., 40
Emelyanova I.B., 176
Escande D., 43
Fedorov O.V., 152
Feklisov S.G., 33
Ferenczi M.A., 14, 41, 73
Fetisova E., 42
Franco D., 43, 45
Freidin A.A., 107
Fujisawa T., 72
Fukuda M., 64
Galkin V.E., 47, 110, 111
Gavrilova O., 124
Gerson J.H., 48
Glagoleva N.S., 48
Golovko V.A., 49
Goncharova E.A., 50, 143, 173
Gracheva E.O., 173
Grichina Е.V., 93
Grishina Z.V., 173
Gromova V.V., 163
Guledani N.E., 57, 151
Gusev N.B., 80
Halton D.W., 81
He Zh.-H., 41
Hellmann S., 12
Higashi-Fujime S., 54
Hodgson D., 6, 132
Il’inskaya O.P., 84
Il'chukov V.V., 148
Iljinsky I.M., 62, 145
Ilyasov F.E., 52
Irving M., 75
Ishii A., 64
Ishijima Ya., 64, 109
Ishikawa N., 64
Ishikawa R., 54
Ivanitsky G.R., 156
Ivanov A.A., 141
Ivanov D.B., 84
Ivanov V.V., 55
Ivanova O., 36, 42
Ivkov V.G., 171
Ivkova M.N., 171
Jacobson K., 114
Jakubiec-Puka A., 65
Kalabukhova T.N., 56
Kankava I.G., 98
Kapelko V.I., 38
Karsanov N.V., 34, 57, 98, 151
Katina I.E., 59
Kelly R., 45
Khaitlina S., 61
Khalina Ya.N., 62, 145, 167
Khapchaev A.Yu., 82, 169
Khrenova E., 115
Khromov A., 63
Khubutiya A.S., 145
Kim A.B., 6
Kim E., 110
Kimura N., 64, 107, 109
Kirillina V.P., 65
Kisurina-Evgenieva O.P., 66
Klimiashvili Z.N., 98
Klueva A.A., 67
Kobelev A.V., 68, 69
Kobeleva R.M., 68, 69
Kochegarov A.A., 20
Kohama K., 54
Kolaeva S.G., 8, 9, 102
Komissarchik Ya.Yu., 61, 71, 146
Kondrashova M.N., 125
Konisheva Ye.V., 36, 156
Kosarskii L.S., 102
Kostyukova A.S., 72
Koubassova N.A., 14, 73, 75
Kozlov S.A., 154
Krasavina M.A., 88
Krasnikova T.L., 11
Krasnov I.B., 159
Krasovskaya I.Ye., 36, 156
Kravtsova V.V., 77
Krechetov S., 78
Kremneva E.V., 80
Kreshchenko N., 81
Krieger I., 72
Krivoi I.I., 77, 163
Krivopalov Yu.V., 148
Krymsky M.A., 82, 169
Kuchava L.T., 57, 151
Kudryashov D.S., 143
Kudryashova E.Yu., 84, 143
Kukushkin N.I., 9, 85, 100, 102
Kukushkina K.N., 85
Kuleva N.V., 87
Kulikova A.L., 88
Kuznetsov S.L., 94
Kuznetsova I.M., 89, 97, 157
Matveeva N.B., 20, 67, 96
Matveeva O.A., 94
Maule A.G., 81
Mazhul V.M., 97
Medvinsky A.B., 85, 100
Melia A.N., 98
Mescheriakova I.V., 152
Mikhailova V.V., 134
Mikheeva I.B., 154
Mishchenko A.M., 17
Moorman A.F.M., 43, 45
Morozov M.A., 52
Moshkov D.A., 154
Moskalenko A.V., 85, 100
Muhlrad A., 24
Muratov R.M., 5
Musib R., 146
Nadezhdina E.S., 143
Nakipova O.V., 8, 9, 102
Nasledov G.A., 59, 104
Naumova M.M., 122
Neiman S.A., 150
Nemirovskaya T.L., 13, 105, 137
Nesterov V.P., 106
Nickashin A.V., 90
Nifontova I.N., 66
Nikolaev D.P., 29
Nikolaeva O.P., 80
Nosov A.V., 148
Okon М.S., 93
Onishchenko G.E., 66
Orlov D.N., 107, 109
Orlov N.Ya., 107, 109
Orlov V.P., 55
Orlova A., 47, 110, 111
Orlova T.G., 109
Lakomkin V.L., 38
Lapshin A.V., 90
Larionova N.P., 126
Lednev V.V., 91
Lejava I.N., 98
Levin G.G., 173
Levine B.A., 50
Levitsky D.I., 80, 92, 134, 178
Linari M., 75
Lislova L.V., 36, 156
Lombardi V., 75
Louchinskaia N.N., 48
Lukashin V.G., 175
Lukoyanova N., 47, 111
Parfyonova E.V., 84
Pavlova E.Yu., 122
Pechatnikov V.A., 171, 172
Permyakova T., 135
Piazzesi G., 75
Piskunov V.A., 24
Platonov A., 78, 113
Pletjushkina O.J., 114
Podlubnaya Z.A., 62, 145, 167
Pogorelov A., 115
Pogorelova V., 115
Pollack G.H., 22, 117, 119
Pomorski P., 114
Potapov M.M., 29
Povarova O.I., 89
Priyatkina T.N., 122
Protsenko Yu.L., 68, 69
Prytkov A.E., 77
Pucar D., 132
Puzakov M.M., 176
Pyatibratov M.G., 152
Maeda K., 165
Maeda Y., 72, 165
Maevsky Е.I., 93
Makarenko I.V., 167
Malyshev S.L., 91, 167
Marston S.B., 50
Matsudaira P., 138
Matusovskaya G., 135
Matusovsky O., 135
Reconditi M., 75
Reisler E., 24, 48, 110, 111, 146
Reshetnyak Ya.K., 109
Reutov V.P., 126
Rogdestvenskaya Z.E., 150
Romanovsky Yu.M., 29
Routkevich S.M., 69
Rubenstein P., 146
Rudanova E., 124
85
Rujfur Z., 114
Ryabokon’ E.N., 5
Saakyan I.R., 125
Sakamoto T., 54
Salibegashvili N.V., 98
Samosudova N.V., 126
Samsonidze T.G., 127
Saprykin V.P., 129
Saunin S.A., 33
Savostyanov G.A., 131
Savostyanova E.G., 131
Schiaffino S., 45
Schmid M.F., 138
Scopin I.I., 5
Selivanov V.A., 132
Semenovsky M.L., 145
Shakhbazyan V.Y., 156
Shakirova L.I., 134
Shanina N.A., 143
Shavlovsky M.M., 89, 97
Shekchonin B.V., 38
Shelud’ko N., 135
Shenkman B.S., 13, 94, 105, 137
Sherman M.B., 138
Shestakov D.A., 14, 140
Shigayev A.S., 24
Shimada N., 64
Shinin V.V., 141
Shirinsky V.P., 38, 39, 50, 82, 90, 143, 169
Shpagina M.D., 167
Shumakov D.V., 145
Shumakov V.I., 145
Shvetsov A., 110, 146
Sidirova M.I., 82, 169
Sidorov V.Yu., 85
Sieck G.C., 30
Siletskaya E.I., 134
Smolyaninov V.V., 78, 113
Snigirevskaya E.S., 71, 146
Sokolov I.N., 122
Sokolov O.I., 24, 147, 148
Sokolova M.K., 148
Somlyo A.P., 63
Somlyo A.V., 63
Srebnitskaya L.K., 150
Starmer C.F., 100
Stepanenko O.V., 89
Stepanova O.V., 39, 90
Stepanova V.V., 173
Strazhevich A.S., 29
Sukoian G.V., 34, 57, 98, 151
Sumaroka M.V., 24
Sushchenko S.V., 176
Takagi Yo., 64
Takeda S., 165
Tararak E.M., 84
Tarasov V.Y., 152
Tatulashvili D.R., 34, 57, 98, 151
Taylor K.A., 12
Teplov V.A., 20, 52, 67, 96, 153
86
Terskikh V.V., 141
Terzic A., 6, 132
Tiktopulo E.I., 72
Timofeev V.P., 134
Tiras N.R., 154
Tolstov Ya.L., 36, 156
Totten M., 81
Tsaturyan A.K., 14, 73, 140
Tsyganov M.A., 156
Turkina M.V., 88
Turoverov K.K., 89, 97, 157
Tuturova K., 135
Tyurina O., 135
Udaltsov S.N., 154, 167
Umnova M.M., 159
Ushakov D.S., 161
Van der Meer W., 26
Van Eldik L.J., 143
VanLook M.S., 110
Vasieva O.O., 162
Vasiliev A.N., 163
Vasiliev A.V., 141
Vasiliev Ju.M., 36, 42, 114
Vassetzky S.G., 40
Vassylyev D.G., 165
Vekshin N.L., 166
Vekshina O.M., 166
Vikhlyantsev I.M., 167
Vikhorev P.G., 26
Vikhoreva N.N., 26
Vilitkevich E.L., 169
Vinokurov M.G., 171, 172
Vishnevskaya Z.I., 62, 167
Vishnyakov G.N., 150, 173
Voinova N.E., 122
Vorobiev S., 24
Vorotelyak E.A., 141
Vorotnikov A.V., 50, 82, 169, 173
Wakatsuki S., 165
Watterson D.M., 143
Wieringa B., 132
Wong W.W., 48
Wriggers W., 47
Yakovenko O., 22, 119
Yamashita A., 72
Yu T., 48
Yurinskaya M.M., 172
Zaitseva E.M., 97
Zaitseva I.S., 24
Zalessova Z.S., 87
Zamuraev I.N., 175
Zarubina A.L., 97
Zhitnikova Y.V., 59
Zholkevich V.N., 176
Ziegler C., 12
Zingman L.V., 6, 132
Zubov E.O., 178
РОССИЙСКАЯ АКАДЕМИЯ НАУК
НАУЧНЫЙ СОВЕТ ПО ПРОБЛЕМАМ БИОЛОГИЧЕСКОЙ ФИЗИКИ
ПУЩИНСКИЙ НАУЧНЫЙ ЦЕНТР БИОЛОГИЧЕСКИХ ИССЛЕДОВАНИЙ
ИНСТИТУТ ТЕОРЕТИЧЕСКОЙ И ЭКСПЕРИМЕНТАЛЬНОЙ БИОФИЗИКИ
ИНСТИТУТ БИОФИЗИКИ КЛЕТКИ
МЕЖДУНАРОДНЫЙ СИМПОЗИУМ
БИОЛОГИЧЕСКАЯ ПОДВИЖНОСТЬ:
НОВЫЕ НАПРАВЛЕНИЯ В ИССЛЕДОВАНИИ
Пущино  2001
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