UNIVERSITY OF TORONTO
FACULTY OF APPLIED SCIENCES AND ENGINEERING
Mid-term examination
BME 205S: Engineering Biology
Examiner: M.V.Sefton
February 21, 2005
Answer all questions
All questions are of equal value
Answer each question in a different book and label with your name and the
question
1. (a) The cytoskeleton is made up of elaborate arrays of protein fibers,
which form networks throughout the cytosol.
i.
What general function does the cytoskeleton perform?
ii. What are the three major types of protein filaments that make up
the cytoskeleton and what are their primary functions?
(b) The F-class proton pump is one of a family of ATP powered pumps
involved in the transport of ions and various small molecules against their
concentration gradients.
i.
How is the typical operation of the F-class proton pump different
from the other “ATP powered” pumps?
ii. In a mammalian cell, which membrane are you most likely to find
this pump on?
iii. This pump, as indicated by its name, is involved in proton transport.
Name two strategies the cell can use to operate this pump without
generating an electrical potential across the relevant membrane.
2. A novel glucose sensor is being created with an optical readout based on a
change in colour of a strip of immobilized enzyme that also contains a
reagent that changes colour when the pH changes (Figure 1). The concept is
to produce a linear gradient of immobilized enzyme (along the x direction);
i.e., more enzyme at one end than at the other. Adding the substrate (a
solution containing glucose) uniformly over the strip for a known time results
in no colour where the strip has too low a glucose concentration to effect
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significant colour change and a strong colour if the glucose concentration is
high enough. This results in a discontinuity in colour at a particular value of x.
A calibration curve linking the position of the colour discontinuity to glucose
concentration results in a user determining the glucose concentration in a test
sample, through visual determination of the position of the discontinuity for
that sample. In order to detect colour using the intended reagents requires at
least 15 µmol/cm2 of glucose to react; assume the discontinuity is very sharp.
The minimum glucose concentration to be detected is 1 mM and the
maximum is 25 mM. At one end, there is 0.5 mg/cm2 of enzyme immobilized
(enough to detect the low concentration limit). The enzyme specific activity
(kr) is 172 µmol/minmg of enzyme and the Michaelis-Menten constant, Km is
1.5 mM.
(a) What is the required minimum exposure time to see the needed colour
change.
(b) What is the required gradient in enzyme concentration if the strip is to be
3 cm long.
(c) Sketch the scale that could be marked on the strip to enable a direct
readout of glucose concentration from the colour discontinuity. Comment
on the problem implicit in this scale.
Immobilized enzyme – uniform in y direction;
linear gradient in x direction (i.e., there is more
enzyme at one end than at another)
Figure 1
Coloured zone
y
x
Position of coloured front denotes concentration of
glucose in test sample (the discontinuity)
3. In the European Union there is strong pressure to eliminate the use of
animals in research, and thus there is a major focus on developing tissues
that can serve as materials in toxicology tests. Wagner et al.1 are developing
1
Wager, H., et al., Interrelation of permeation and penetration parameters obtained from in
vitro experiments with human skin and skin equivalents. J. Control. Release, 75:283-295(2001).
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a new tissue-engineered skin called Epiderm for use in testing dermal
penetration and toxicity. This skin is made from living human donor cells,
grown into a dermal equivalent (the soft, underlying layer of skin) and an
epidermal equivalent (the surface layer) in culture.
One important
assessment is how it compares to normal skin (Figure 2) in terms of
permeability.
outside
epidermis
Stratum
corneum
dermis
Figure 2: normal skin
An experiment is conducted in which the test “skins” are placed in a two
chamber diffusion apparatus (Figure 3). The surface area of the skin between
the two chambers is 2 cm2. The amount of a model compound (a “penetrant”,
flufenamic acid, a lipophilic molecule) that diffuses across the skin is
determined in the sink (downstream or receiving) chamber as a function of
time and used to calculate the amount permeating as a function of time. In
these experiments, the concentration of the molecule in the source chamber
is much greater than that in the sink chamber, so the concentration profile in
the skin can be assumed to be steady state after a very short initial transient.
The data, collected in Table 1, were all taken in the steady state regime.
Initially the sink is filled with fluid that contains no penetrant and the small
increases in concentration, although negligible compared to the source
concentration, are used to calculate the cumulative amount of penetrant that
has permeated through the “skin”.
It is determined in a separate experiment that the partition coefficient
(“solubility”) for the penetrant in epidermis is 1.5, in stratum corneum (a thin
layer between the epidermis and dermis) is 5.6, in the full skin is 0.8 and in
Epiderm is 1.3. The partition coefficient, H, is defined here as H = C skin layer /
Csource. The concentration in the source chamber is kept constant at 4.5
mg/cm3 (Csource). On the basis of the data in table 1:
a) Calculate the steady-state flux in each case;
b) Calculate the diffusion coefficient in each case;
c) Explain how close is Epiderm to Full skin and why might it be different?
Include discussion of thicknesses, diffusivities and fluxes.
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Test Skins
Epidermis,
Skin
Normal
Stratum
Corneum,
Normal Skin
Full skin, Normal Skin
Epiderm
Thickness = 2910 m
Thickness = 67.3 m
Time
(hr)
Amount
sink (g)
Time
(hr)
Amount
sink (g)
Thickness = 75.2 m
Thickness = 12.2 m
Time
(hr)
Amount
sink (g)
Time
(hr)
Amount
sink (g)
5
9.5
5
10
5
3.5
5
115
10
19
10
20
10
7
10
230
20
38
20
40
20
14
20
460
in
Source
in
in
in
Sink
Csource
X=0
Csink
X=l
Test skins
Figure 3
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