Abstrak Peserta Seminar
LAMINARINASE ACTIVITY PRODUCED BY RIAU LOCAL
BIOCONTROL FUNGI
Titania Tjandrawati Nugroho*), Saryono, Ruth Sri Ulina, Asina E. R. Silitonga
Jurusan Kimia, FMIPA, Universitas Riau, Kampus Bina Widya Km 12,5, Jln. Raya
Soebrantas, Pekanbaru 28293, email: titania_nugroho@yahoo.com, fax.
(+62)(761)839631, ph. (+62)(761)7076553. *) Corresponding author.
ABSTRACT
Trichoderma asperellum TNJ63 and TNC52, and Gliocladium sp. TNC59 and
TNC73 are effective biocontrol fungi isolated from Riau soil. These strains have been
tested for their effectiveness in plant protection through antagonistic studies and plant
protection studies. The ability of T. asperellum TNJ63 and TNC52, and Gliocladium sp.
TNC73 and TNC59 (Riau biocontrol strains) to protect plants against fungal pathogens
lies among others to their ability in producing fungal Cell Wall Degrading Enzymes.
Apart from chitin, -1,3-1,6-glucans are among the major fungal cell wall
constituents.
All the Riau biocontrol strains were formerly isolated based on their ability to
produce chitinases. Here we explore the ability of these strains to produce laminarinases
t
-1,3-1,6-glucans (laminarins). Appart from its function in
biocontrol of plant fungal pathogens, laminarinases have importance in modification of
engineering.
Laminarinase from Riau biocontrol strains were produced in production media
containing 0.2% laminarin from Laminaria digitata, pH 5,5, room temperature, and
monitored over time. Activity of Laminarinase was determined by incubation of crude
extracts with 0.2% laminarin for 1 hour at 40oC, pH 5.5. One unit of activity was defined
as micromols reducing sugar released per mL extract per minute. Although maximum
activity was reached slower by both Gliocladium strains, compared to Trichoderma
strains, Gliocladium strains produced higher laminarinase activity than the Trichoderma
strains. The highest laminarinase activity was produced by Gliocladium sp. TNC73 with
an activity of 0.058 units after 7 days of production, which is significantly higher than
that produced by the other strains. Ability of the Riau biocontrol strains to produce
laminarinases does not correlate with their ability to produce chitinases. T. asperellum
TNJ63 produces significantly higher chitinase than Gliocladium sp. TNC73. These
differences may underlie the difference in specific species biocontrol.
Key words: Laminarinase, Trichoderma, Gliocladium,
glucanase
-1,3-
-1,6-
Using microarrays data for topological mapping of yeast’s genes
involved in axial and bipolar budding process
Fajar Restuhadi
Fakultas Pertanian, Universitas Riau Jl. Binawidya 30 – Pekanbaru, Indonesia
(f.restuhadi@unri.ac.id)
Abstract
During the cell cycle division process, the cells are divided along specific cleavage planes.
Polarised cell division is a fundamental process in which cells divide along specific cleavage
planes, because it can mediate appropriate cell-cell contacts and partition cytoplasmic
components asymmetrically between daughter cells.
Bud sites can be selected in either of two distinct spatial patterns, termed axial and bipolar. The
axial pattern genes are AXL1, BUD10/AXL2, BUD3, BUD4, in which their products mark the
mother-bud neck during one cell cycle as a site for budding in the next round of division. On the
other hand, several genes are required for the bipolar budding pattern of diploid yeast cells but
not for haploid axial budding. However, only two genes of this class, BUD8 and BUD9, have
been described that shift the bipolar pattern to a whereas bud9 mutants bud with high frequency
from the distal cell pole. Therefore, Bud8p and Bud9p have been proposed to act as
bipolar landmarks that might recruit components of the common budding factors, e.g. Bud2p,
Bud5p or Rsrlp/Budlp, to either of the two cell poles.
The Self-organising Map (SOM) is a type of neural network method, loosely based on how the eye
works. SOM is a non-linear projection of high dimensional data to a lower dimensional space,
typically the plane. The concept of “self-organised topological feature maps” was introduced by
Kohonen as maps that preserve the topology of multidimensional representation within the new
one- or two-dimensional array of “neurons” which can be associated as an array of clusters.
Using the yeast cell-cycle microarrays dataset from Cho et al. (1998), the topology of four genes
of the BUD family in the dataset (BUD3, BUD4, BUD8, BUD9) were successfully arranged in the
map by the SOM according to their gene expressions pattern. Three genes (BUD3, BUD4, and
BUD8) were placed in a close neighbourhood while BUD9 was spatially apart from these two
genes neighbourhood in the U-matrix map. Consistently, SOM was able to visualise the spatial
topography between the two genes, BUD8 and BUD9. Clearly, BUD8 is placed by SOM in
adjacent neighbours of BUD3 and BUD4, while BUD9 is located at a further distance away.
Several proteins, particularly Bud3p and Bud4p, presumably associated in a complex with others,
assemble in the mother-bud neck to form the spatial landmark for the next round of axial
budding. The spatial relationship in a close vicinity of neighbourhood between the BUD3/BUD4
with BUD8 is clearly visualised by the SOM, while positioning the BUD9 at other part of map
away from these clusters.
KEYWORDS :
Realtime PCR – rotary analyzer – High Resolution Melting – Mutations
THE EFFECT OF AVOCADO (Persea Americana Mill.) SEEDS IN METHANOL
EXTRACT ON DIABETIC MALE MICE (Mus musculus L.) OF
DDY STRAIN
Elsa Lisanti
(Biology Department, Faculty of Mathematics and Natural Sciences
State University of Jakarta)
Email : elsa_lisanti@yahoo.com
Indonesia is rich in plant biodiversity. Among them there are many species considered as
tradicional medicinal plant (herbals). Avocado (Persea Americana Mill.) is considered as
medicinal plants, but there is no report that avocado has been used as drug so far. The
objective research is to find out the effect of avocado seed extract on diabetic mice which
was induced by alloxan. The experiment has been conducted at the chemical and
biological labs of UNJ. The design is Randomized Block Design (RBD) with 6
treatments and 4 replications. The treatments was include A (normal control), B (diabetic
control), C (diabetic + glibenklamid), D (diabetic + extract dose 0,2 g/kg BW), E
(diabetic + extract dose 0,5 g/kg BW) and F (diabetic + extract dose 1 g/kg BW). The
blood glucose of mice was measured by using glucotest meter, that are at day 0, day-1,
day-7, day-14 and day 21. After analysis of variant at α 0.05 and then continued with
least significant different (LSD) test, the results showed that the diabetic injected by
avocado seed extract could reduce the glucose content of mice. The best rate of treatment
was 0,2 g/kg BW, which was 43,70 % reduction of glucose content. It was concluded,
avocado seed can be used as diabetic medicine.
Key words : avocado seed, methanol, mice, diabetic, blood glucose
Compounds from Jati Belanda Leaves (Guazuma ulmifolia Lamk.)
Potential in Increasing Hydrolytic Activity of Lipases
By
Yuana Nurulita1), Latifah K. Darusman2), Dyah I. Pradono2)
1)
Chemistry Dept,. Math and Science Faculty, University of Riau
2)
Chemistry Dept., Math and Science Faculty, Institute of Bogor Agriculture
Lipase catalyzes the hidrolysis of triglycerides to become glycerol and fatty acids.
This mecanism is important for reducing fat in the over weight individual. Jati Belanda
leaves is a traditional medicine that people use for reducing fat. The aim of this research
was to identify compounds from Jati Belanda leaves that could increase hidrolitic activity
of lipase.
Jati Belanda leaves were extracted by methanol soxcletation and chloroform
maceration. Secondary metabolites from both of extracts was assayed qualitatively.
Methanol extracts containeds alkaloid, saponin, flavonoid, steroid, tannin, and quinones.
Chloroform extract contained alkaloid, triterpenoids, steroid, and tannin. The result
showed that 0.01 g/ml of chloroform extract influenced in vitro lipase activity from
Rhizopus arrhizus (crude enzyme) and Sigma tipe XI L-4384 more actively compared to
methanol extracts and controls. So compounds in the chloroform extract could become
lipase’s activators to catalyze lipid hydrolytic reactions.
Keywords: Jati Belanda leaves, Lipase activity, chloroform extract.
ISOLATION and SOME PROPERTIES of XYLANASE from
SOIL-TERMITE GUTS and INFLUENCE of ENZYMES in BAKING
Anak Agung Istri Ratnadewi* , Agung Budi Santoso, and Muhammad Naqib
Department of Chemistry, Faculty of Mathematic and Natural Sciences,
The University of Jember at Kampus
Tegalboto
*To whom correspondence should be addressed. E-mail: dewi_pjw2003@yahoo.com
Abstract
Termites harbor a diverse variety of bacteria in their gut. The bacteria which has been isolated
from gut of soil-termites that captured in around of College The University of Jember can
secretes of xylanolytic enzymes. These enzyme acts as catalyst in hydrolysis of xylan
synergically. The xylanolytic enzymes showed activities toward oat-spelts xylan was 4.78 U
(referred as endo-β-1,4-D-xylanases), whereas activities toward
p-nitrophenyl
derivative substrates were 1.94 U (referred as α-L-arabinofuranosidases) and 2.64 U (referred as
α-D-glucuronidases). The xylanolytic enzymes did not showed any activities of both exo-β-1,4-Dxylosidases and acetyl-xylan esterases. Attemp to purify of enzyme endo-β-1,4-D-xylanases also
has been performed. Purification have so far been carried out were partially by ion-exchange
column chromatography. Both activity and protein contents of endo-β-1,4-D-xylanases was
determined by DNS (Miller, 1959) and Bradford (1976) method, respectively. The highest
releasing-fractions of enzyme endo-β-1,4-D-xylanase on ion-exchange column chromatography
obtained at saturation of 0.5 M NaCl with specific activity 1.62 U.mg-1. Enzyme has an optimum
pH and temperature at 5.0 and 40oC, respectively. These enzyme were stable at temperature of
400C for four hours and range of pH 5.0 -8.0. The relative molecular mass possessed of endo-β1,4-D-xylanases is around of 45,000-66,200 Dalton by SDS-PAGE, whereas analysis of
zymogram using SDS-Xylan-Page used to confirm that it contained endo-β-1,4-D-xylanase
activity at the range of 45,000-66,200 Dalton. These analyses revealed that enzymes could
degrade oat-spelts xylan as substrates. Based on their both activity and characteristics, the
potential of endo-β-1,4-D-xylanases as technological aids in baking was clearly demonstrated.
Baking trials were performed with five wheat dough prepared with 0.0 ml (control), 12.5 ml, 25.0
ml, 37.5 ml, 50.0 ml, and 62.5 ml enzyme volume against 2,500 g wheat flour. In bread baking,
endo-β-1,4-D-xylanase produce an effect that can result in many desirable benefits including
increased of physico-chemical performance (moisture, reducing-sugar contents equivalent with
glucose and xylose, mass, product volume, porosity and texture), improved crumb softness and
organoleptic aspects (shelf-life, taste and aroma). The presence of
endo-β-1,4-D-xylanase
at volume 25.0 ml in the dough led to improvements in the breadmaking quality (i.e. product
volume, porosity, and reducing-sugar content) on breads compared to control.
Keywords: activity, baking trial, bread quality, characterization, endo-β-1,4-D-xylanase
Rince S. Damanik1, Rini Puspitaningrum1, Mohamad Sadikin2,
Irsan Anwar1, Rusdi1, Nurmasari Sartono1
1. Jurusan Biologi FMIPA UNJ
2. Bagian Biokimia & Biologi Molekuler FKUI
Judul Poster :
PRODUCTION OF ANTIBODY – ANTI HEMOGLOBIN OF CHELONIA
MYDAS
WITHOUT FREUND ADJUVAN INDUCTION
Abstract
Antibody usually produced by using invasive freund adjuvant as an
immunomodulator induction technique. Therefore, the aim of this research was
produced the antibody-anti hemoglobin of Chelonia mydas without
immunomodulator induction. Research was conducted in Laboratory of
Biochemistry & Molecular Biology of FMIPA UNJ and FKUI from Pebruary until
June 2008. The concentration of antibody-anti hemoglobin Chelonia mydas was
detected by using simple titer technique in fourth of booster induction. The titer
concentration of antibody – anti hemoglobin C.mydas was …. .The result indicated
that the titer of antibody could elevated by using Hemoglobin protein antigenic of C.
mydas induction technique without adjuvant freund in more four times of booster
induction.
Keywords : freund adjuvant, antibody – anti hemoglobin C. mydas, titer technique
Dwita Oktiarni and Fida Madayanti
Biochemistry Division, Faculty of Mathematics and Natural Science
Institut Teknologi Bandung, Jl. Ganesa No. 10 Bandung 40132
E-mail: fida@chem.itb.ac.id
Abstract
Isolation, Purification and Characterization of β-amylase from Sweet potatoes (Ipomoea
batatas (L.) LAM) Lampeneng Cultivar
ABSTRACT
β-amylase (E.C 3.2.1.2) is an enzyme commonly found in plants and bacteria. The
enzyme is exo-acting carbohydrolase which hydrolyzes α-1.4-glucosidic linkages of
starch, removing maltose units from the non-reducing end of the polysaccharide chain,
producing β-maltosa and β-limit dextrin as the final product. β-amylase is widely
distributed in the higher plants such as sweet potato. This ezyme was extracted from
Lampeneng sweet potato in 0.05 M acetate buffer pH 4.8 and followed by ammonium
sulphate fractionation. The fraction containing highest spesific activity (determined by
Somogyi-Nelson and Lowry methods) was further purified by FPLC (Fast Protein Liquid
Chromatography) and Native Gel Electrophoresis. The purified Lampeneng’s β-amylase
showed increasing spesific activity as much as 37 folds compared to those of the crude
enzyme. Spesific activity of Native Gel Electrophoresis purified Lampeneng β-amylase
was 9.57 mg sugar.mg protein-1.minute-1. The molecular weight of the purified βamylase was around 150 kDa as shown by Native PAGE and Reverse Zymography data.
Further SDS-PAGE data showed that this enzyme consisted of more than one different
molecular weight sub unit (heteromer). The characterization of purified β-amylase
showed that Lampeneng derived β-amylase had optimum pH and temperature of 5.52 and
70°C respectively. Identification of its kinetic parameter, such as KMapp, Vmaxapp and
Hill constant showed the values of 0.012 mg/mL, 4.36 mg sugar.minute-1 and 1.07,
respectively. Affect of effector showed that Cl- as anion and K+ and Na+ as cation
showed the positif effect to the enzyme activity, whereas Cu2+ as cation showed as a
negative effector to the enzyme activity.
Keywords: β-amylase, FPLC, Hill constant, Lampeneng, native PAGE, purified enzyme,
reverse zymography, sweet potato.
Arry Yanuar ---, Department of Pharmacy, Faculty of Mathematics and Natural S
Anggi Sekar Tantrilestari ---, Department of Pharmacy, Faculty of Mathematics
and Natural Sciences, University of Indonesia Nurmayasari ---, Department of
Pharmacy, Faculty of Mathematics and Natural Sciences, University of Indonesia
Molecular Dynamics Simulation of disordered regions the RGK-family of small GTPase
revealed no GTPase activity Single paper presentation: 0 words, 10 minutes
Abstract
Unlike other small GTPases, RGK family members including Rad and Gem lack several
conserved residues in the GTPase domain. RGK proteins such as Rad and Gem have a
function as potent inhibitors of voltage dependent calcium channels and modulate Rhodependent remodeling of the cytoskeleton. The X-ray structures of Rad and Gem
revealed unexpected disordered structures of both switches I (residue 116-124 of Rad, 99
-102 of Gem) and II (147-153 of Rad, 137-138 of Gem). The disorder regions of both
switch I and II were then built using program MODELLER 9v3 with 2A78_A (RalA),
1JAH (Ras), 2DPX_A (Rad) as template for Rad model and 2HT6_A (Gem), 2A78_A
(RalA), 1AGP (Ras) for Gem model. The best model with switch I and II for both Rad
and Gem were had 93,0% and 90,6% residues located at most favoured regions and none
of residue located at disallowed regions in the Ramachandran plot. Ten nanoseconds of
molecular dynamics simulations were then carried out using GROMACS for both Rad
and Gem with and without ligands (GDP or GTP). The results revealed that switch I and
II had the highest flexibility for both Rad and Gem, whereas switch I was more flexible
than others based on its trajectory and RMSF. There was no interaction between gammaphosphate with residues Ala120, Ala123, Glu147 in Rad and Cys106, Leu109, Glu134 in
Gem that substitute the functionally important residues for GTP hydrolysis (Tyr32,
Thr35, and Gly60) in Ras, suggesting no intrinsic GTPase activity in RGK protein.
Abstrak Pembicara Utama
Mechanisms of Sec-mediated protein translocation in bacteria
Arnold Driessen, Department of Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute and Zernike Institute for Advanced Materials, University of
Groningen
Kerklaan 30, 9751 NN HAREN. The Netherlands
About 25 - 30 % of the bacterial proteins function in the cell envelope or outside of the
cell. These proteins are synthesized in the cytosol and the vast majority is recognized as a
ribosome-bound nascent chain by the signal recognition particle or by the secretiondedicated chaperone SecB. Subsequently, they are targeted to the Sec-translocase in the
cytoplasmic membrane, a multimeric membrane protein complex with a highly conserved
protein conducting channel, SecYEG, and peripheral bound ligands, the ribosome or the
ATP-dependent motor protein SecA. The Sec-translocase mediates the translocation of
proteins across the membrane and the insertion of membrane proteins into the
cytoplasmic membrane. Translocation requires the energy sources ATP and the proton
motive force, while membrane protein insertion is coupled to polypeptide chain
elongation at the ribosome. In this seminar I will discuss our present knowledge on the
mechanism and structure of the Sec-translocase and in particular discuss how unfolded
polypeptides are threaded through the translocation pore.
Suggested reading:
Driessen, A.J.M., Nouwen, N. (2008) Protein Translocation across the Bacterial
Cytoplasmic Membrane. Annu. Rev. Biochem. 77: 643-667.
24/6 - 16:30 - 17:00
Protein translocation mechanisms in bacteria, multiple pathways for the translocation of
unfolded and folded polypeptides
Arnold Driessen, Department of Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute and Zernike Institute for Advanced Materials, University of
Groningen, Kerklaan 30, 9751 NN HAREN. The Netherlands
In bacteria, two major pathways exist to secrete proteins across the cytoplasmic
membrane. The general Secretion route, termed Sec-pathway, catalyzes the
transmembrane translocation of proteins in their unfolded conformation, whereupon they
fold into their native structure at the trans-side of the membrane. The Twin-arginine
translocation pathway, termed Tat-pathway, catalyses the translocation of secretory
proteins in their folded state. Although the targeting signals that direct secretory proteins
to these pathways show a high degree of similarity, the translocation mechanisms and
translocases involved are vastly different.
In Gram-positive bacteria, translocation across the cytoplasmic membrane
suffices for secretion, but in Gram-negative bacteria, the outer membrane forms a further
barrier for secretion. For outer membrane secretion, a range of mechanisms exist that are
termed type I-V secretion. These systems interconnect with the translocases at the
cytoplasmic membrane or mediate the secretion of proteins across both the cytoplasmic
and outer membrane in a single step. In this seminar, I will discuss the various
mechanism of protein secretion in particular in relation to pathogenic bacteria and host
infection.
25/3 workshop: 9:00-10:00
Protein folding and translocation, a single molecule study on maltose binding protein
Arnold Driessen, Department of Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute and Zernike Institute for Advanced Materials, University of
Groningen, Kerklaan 30, 9751 NN HAREN. The Netherlands
Protein translocation in Escherichia. coli is mediated by the translocase which comprises
a protein conducting pore SecYEG, a motor protein, SecA, and a molecular chaperone,
SecB. SecB targets secretory proteins (preproteins) to the SecYEG-bound SecA. The
SecYEG complex forms a narrow channel in the membrane that allows passage of
preproteins in an unfolded state only, and a major unresolved question is whether
preproteins are actively unfolded prior translocation. Indeed, the SecA (and ATP)
requirement for translocation has been proposed to depend on the folding stability of the
mature preprotein domain suggesting that preproteins retain substantial tertiary structure
while bound to SecB. Using optical tweezers and all-atom molecular dynamics
simulations, we determined the effect of SecB on the folding and unfolding pathways of
maltose binding protein (MBP). Optical tweezers allow studies of protein folding at the
single molecule level. In the absence of SecB, the MBP polypeptide first collapses into a
molten globule-like compacted state and then folds into a stable core structure, onto
which several α-helices are finally wrapped. Interactions with SecB completely prevent
stable tertiary contacts in the core structure, but have no detectable effect on the folding
of the external α-helices. It appears that SecB only binds to the extended or molten
globule-like structure and retains MBP in this latter state. Biochemical studies further
demonstrate that the effect of folding stabilizing and destabilizing mutations in the
mature MBP are drastically overruled by the signal sequence and SecB, Consequently,
the kinetics and energetics, and the SecA-dependence of the translocation of the folding
mutants are indistinguishable from those of wild-type preMBP. Thus during SecBdependent translocation, energy is required only to remove SecB and not to disrupt
tertiary interactions in the preprotein.
Suugested further reading:
Bechtluft, P., van Leeuwen, R., Tyreman, M., Tomkiewicz, D., Nouwen, N., Tepper,
H.L., Driessen, A.J.M., Tans, S.J. (2007) Direct Observation of Chaperone-induced
Changes in a Protein Folding Pathway. Science 318: 1458-1461.
Tomkiewicz, D., Nouwen, N., Driessen, A.J.M. (2008) Kinetics and Energetics of the
Translocation of Maltose Binding Protein Folding Mutants. J. Mol. Biol. 373: 83-90.
Text that focusses on the methods to study protein folding and interaction with the
molecular chaperone SecB:
Driessen, A.J.M., de Wit, J.G., Nouwen, N. (2004) Chapter 27. SecB In: Protein Folding
Handbook, Vol. 5. Eds. J. Buchner, T. Kiefhaber. Wiley-VCH Verlag, Weinheim,
Germany, pp. 919-937.
Structural Basis of N2,N2-Guanosine dimethyltransferase Trm1,
Essential for the L-shape Formation of tRNA
Ihsanawati1, Rie Shibata1, Madoka Nishimoto1, Mikako Shirouzu1,
Yoshitaka Bessho1,2, Shigeyuki Yokoyama1,2,3
1
Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro, Tsurumi,
Yokohama 230-0045, 2RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 6795148, 3Department of Biophysics and Biochemistry, Graduate School of Science, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; email: (ihsanawa@gsc.riken.jp)
Transfer RNA (tRNA) is a crucial adapter molecule in protein biosynthesis. For being the adapter
molecule, tRNAs should have proper length and fold, by extensive cellular processes such as removal
of the 5’ and 3’ additional sequences by RNaseP, endo and exonucleases and nucleotide modifications
by corresponding modification enzymes.1; 2 Modified nucleosides in tRNAs are commonly found in
the core (including the D- and T stems interactions) and anticodon regions. In the core region,
modified nucleosides such as m1A58, Gm at positions 18 and 19, m2G, and m22G at positions 10 and
26 are linked to the correct folding and stability of the three-dimensional structure of tRNAs.
Modifications in the anticodon stem and anticodon region (position 34-36) have functions in repairing
the decoding process and avoiding codon-anticodon mismatch.3 Thus in general, modified
nucleosides in tRNAs are important for the fidelity and efficiency of the protein biosynthesis although
as yet mostly unknown. One of the tRNA modification enzymes, Trm1, catalyses mono- (m2G) and
dimethylation (m22G) of guanosine at position 26, a junction between the D- and anticodon stems,
employing S-adenosyl-L-Methionine (AdoMet) as a methyl donor.4 Both m2G26 and m22G26
modifications are found in most archaeal, eukaryotic, and Aquifex aeolicus tRNAs.4; 5
To explore the Trm1 methyl transfer mechanism, we determined the crystal structures of Trm1
from Pyrococcus horikoshii liganded with AdoMet or S-adenosyl-L-homocysteine. The protein
comprises N-terminal and C-terminal domains with class I methyltransferase and novel folds,
respectively. The methyl moiety of AdoMet points toward the invariant Phe27 and Phe140 within a
narrow pocket, where the target G26 might flip in. Mutagenesis of Phe27 or Phe140 to alanine
abolished the enzyme activity, indicating their role in methylating G26. Structural analyses revealed
that the movements of Phe140 and the loop preceding Phe27 may be involved in dissociation of the
monomethylated tRNA•Trm1 complex prior to the second methylation. Moreover, the catalytic
residues Asp138, Pro139, and Phe140 are in a different motif from that in DNA 6-methyladenosine
methyltransferases, suggesting a different methyl transfer mechanism in the Trm1 family.
References
1. Hopper, A. K. & Phizicky, E. M. (2003). tRNA transfers to the limelight. Genes Dev 17, 162-80.
2. Nakanishi, K. & Nureki, O. (2005). Recent progress of structural biology of tRNA processing and
modification. Mol Cells 19, 157-66.
3. Grosjean, H. (2005). Fine-Tuning of RNA Functions by Modification and Editing, NY Springer Verlag,
Berlin-Heidelberg.
4. Constantinesco, F., Motorin, Y. & Grosjean, H. (1999). Characterisation and enzymatic properties of
tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus. J Mol Biol
291, 375-92.
5. Awai T., Takehara T., Takeda H. & Hori H. (2005). Aquifex aeolicus Trm1[tRNA (m22G26)
methyltransferase] has a novel recognition mechanism of the substrate RNA. Nucleic Acids Symp Ser
(Oxf) 49, 303-304.
ABSTRACT
Mutation of a functional gene may lead to an incomplete, deleted, aborted, or complete
translations but eventually paralyse its protein functions.
Researchers have developed techniques in analyzing mutations at gene level to predict geneassociated disease for example, in quickest and easiest ways. One of the technique widely used is
electrophoresis on poly-acrylamide gels (PAG) following PCR amplification of gene of interest.
Such PAG approaches are DGGE (denaturing gel gradient electrophoresis), TGGE (temperature
gradient gel electrophoresis), SSCP (single stranded conformational polymorphism), takes longer
preparation and practical steps to get results.
Recently, real time amplification of a gene can be used to analyze mutations in post-run fashions.
Real time PCR is a new technique to analyze gene amplifications and mutations in real time mode
by incorporating fluorescent moieties into the reactions and examining the signals arise from
modulated signals in conjunction with DNA amplifications. Several fluorescents have been
chosen as reporters during the amplifications and these can be measured accordingly.
Critical parameters underlying the ability to analyze mutations are temperature and illumination
variations. At level of +0.01 Centigrade variations and extremely even illuminations the DNA
fragments can be analyze on its single base difference by so called high resolution melting
(HRM) analysis.
ACTN3 encodes actinin-3 protein will be used as a model in this respect to distinguish the
appearance of wild type, mutant and homozygote species by post-run real time PCR using
RotorGene 6000 rotary analyzer.
The beauty of RotorGene rotary analyzer design will also be discussed in depth.
Djonady Sugiaman
Short Background:
Born in 1956
1976
Graduated from Sekolah Analis ITB (4-5 years training)
1977-1979
Worked as First Class Chemical Analyst at PT INCO, a Canadian mining
company, Soroako, South Sulawesi
1980-1986
Drs in chemistry, Padjadjaran University GPA 3.12 (BSc 1983, Elective Thesis
1985 in nickel deposition and brightening at National Metallurgy Agency, Major
Thesis 1986 in amino acids bioavailability in vivo studies of lamtoro by using
high voltage electrophoresis
1987
Appointed as Assistance Factory Manager at Sandoz Cimanggis Factory
1988-1989
MSc in molecular physiology, Dept of Animal Physiology, Faculty of Biology
and Earth Sciences, Stockholm University, studies on c-myc interplay in brown
fat cells recruitment in physiologically induced mice, Sweden, Prof. Barbara
Cannon
1990-1993
Guest Research Scientist, Dept of Tumor Biology (now Microbiology and Tumor
Biology Center, MTC), Karolinska Nobel Institute, studies on Rb suppressor
gene reconstitution in Rb-truncated osteosarcoma cells, Sweden, Prof. George
Klein
1993-1995
Research Scientist at PAU Bioteknologi ITB, Bandung
1995-1998
Appointed as Product Application Manager at PT Hilab Sciencetama, Jakarta
1998-now
Appointed as Director at PT Sentra Biosains Dinamika (Sentra BD), Jakarta
Djonady has collected sixteen certificates as lecturer and trainers in many
institutions on PCR applications.
PT Sentra BD has been appointed as representative of Corbett Research in Indonesia,
manufacturer of genuine RotorGene and PalmCycler, Australia
Human Mitochondrial DNA and Protein Mutations in Disease
Achmad Saifuddin Noer
Division of Biochemistry, Institut Teknologi Bandung
Almost all human cells consist of twenty-three pairs of chromosome and one
extrachromosomal mitochondrial DNA. The publication of 16,569 base pairs of the
human mitochondrial DNA complete sequence was a milestone in the study of DNA and
protein mutations in normal and disease individual. The mitochondrially synthesized
subunits of respiratory enzyme complexes are the ND1 to ND6 subunits of complex I,
apocytochrome b of complex III, the COI, II and III subunits of complex IV, and ATP8
and 9 subunits of the ATP synthase. Recently there are more than two-thousand and fivehundred normal and disease variants of mitochondrial DNA have been documented.
Among them, around three-hundred and sixty DNA mutations have been demonstrated
either causal or only associated mutation in human disease. The features of these
mitochondrial DNA and protein mutations related to human mitochondrial diseases will
be discussed in this paper.
Masafumi YOHDA
Department of Biotechnology and Life Sciences,Tokyo University of Agriculture & Technology,
2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Proteins are synthesized as linear polypeptides by ribosomes based on nucleotide
sequences of mRNAs. They acquire specific functions after folding into the 3D conformations
specified by their amino acid sequences. Although most proteins can fold spontaneously in their
optimal conditions, many proteins require the assist of other proteins, molecular chaperones, for
correct folding in the cell. Molecular chaperones are proteins that selectively recognize and bind
to the exposed hydrophobic surfaces of non-native proteins, subsequently preventing protein
aggregation and facilitating correct folding of non-native proteins in vivo. Molecular chaperones
are also involved in many important aspects of protein homeostasis, degradation and subcellular
trafficking.
Aberrant protein folding does not only result in the malfunction of the protein itself but
also cause other effects by forming aggregation in the cell. Protein folding diseases are diseases
induced by the malfunction of cells, which are caused by the aggregation or abnormal
conformation of proteins. Alzheimer’s disease (AD) neuropathology is the most famous protein
folding disease. AD is characterized by loss of synapses and neurons in the brain and the
accumulation of senile plaques and neurofibrillary tangles of the 39–43 amino-acid Aβ peptides
which are cleaved from the parental amyloid precursor protein (APP). These insoluble fibrillar
forms were thought to cause neurotoxicity through oxidative stress. Aberrant folding and fibrillar
aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in
Huntington's disease and other neurodegenerative disorders. Prion diseases are also well known
protein folding diseases. Because of the nature of the proteins responsible for the such protein folding
diseases, molecular chaperones take important roles in protecting cells from the cells from the effects
of such proteins in the abnormal conformation and also progression of the abnormal conformations.
Hsp104 is required for the progression of prion in Saccharomyces cerevisiae. Small heat shock
proteins exhibit protective effects on the effects of polyglutamines.
In this lecture, I will review the recent progresses of the researches on molecular chaperones
and protein folding diseases. If time allows, I will talk on our recent researches on the functional
mechanism of some molecular chaperones, prefoldin, group II chaperonin and also small heat shock
proteins.
My abstract for the 30 min talk in the seminar is as follows.
Disulphide bond formation during protein folding in the cell: Most proteins processed through the secretory
pathway in eukaryotic cells contain disulphide bonds which are essential to their stability and function.
This includes many physiologically and economically significant groups of proteins including digestive
enzymes, antibodies, growth factors, blood clotting enzymes and storage proteins, such as the major
proteins in eggs, milk and plant seeds. Formation of the correct disulphide bonds is a key step in the initial
folding of these proteins in vivo. The seminar will review our knowledge of how these proteins fold in the
cell and of the cellular machinery involved; it will also consider how this knowledge can be applied to
produce high-value recombinant proteins.
My abstract for the 60 min lecture in the workshop is as follows.
Protein folding in vivo: the role of helper proteins Before 1990, most textbooks of Biochemistry and Cell
Biology did not discuss the question of how proteins fold following biosynthesis and it was often assumed
that this was a spontaneous process. However, a number of different lines of work during the 1980s
challenged this view and established a new paradigm, namely that protein folding in the cell is facilitated
by a large number of helper proteins or 'molecular chaperones'. There are a number of structural and
functional classes of molecular chaperones including the chaperonin family and the hsp70 family. The
cellular roles of many molecular chaperones are now known but we have relatively little information about
the exact mechanism by which these chaperones assist the folding of their substrate proteins. The lecture
will discuss the limited mechanistic information available for the chaperonin family and the protein
disulphide-isomerase family
Robert Freedman