National Science Foundation

Research Experiences for Undergraduates

Structure and Function of Proteins
Summer 2011


Research Reports








University of South Alabama

This program was supported by a grant from the
National Science Foundation (# 0751684)




1

Participants

Undergraduate students who participated in the NSF-REU site on Structure
and Function of Proteins at the University of South Alabama































Back Row: Matthew Hurton, Mitchell Ingram and Gregory Schalla
Front Row: Lauren Cichon, Liriany Pimentel, Kanika Topiwala,
Katherine Bauer and Allison Roth


2

TABLE OF CONTENTS


Characterizing the Cytotoxic Properties of a Natural Diterpenoid from Rabdosia
rubescens
By Katherine Bauer, University of Alabama Page 3
Research Mentor: Dr. Richard Honkanen, Dept. of Biochemistry and Molecular
Biology

Hyperspectral Imaging Microscopy And Linear Unmixing Of Fret Signals
By Lauren Cichon, Aquinas College Page 13
Research Mentors: Dr. Diego Alvarez, Dr. Silas Leavesley and Dr. Thomas Rich, Dept. of
Medicine,
Chemical Engineering and Pharmacology

Qualitative Evaluation of Autofluorescence in Drosophila Brain and Hindgut
By Matthew Hurton, Providence College Page 27
Research Mentor: Dr. Robin Mockett, Dept. of Biomedical Sciences

Proteomic Analysis of the Response of Escherichia coli and Staphylococcus aureus to the
Photodynamic Activity of Phloxine B
By Mitchell Ingram, Jacksonville State University Page 35
Research Mentor: Dr. Lewis Pannell, Mitchell Cancer Institute

Correlation of Mitochondrial Membrane Potential and Velocity in Endothelial Cells
By Liriany Pimentel, Bryn Mawr College Page 51
Research Mentor: Dr. Abu Bakr Al Mehdi, Dept. of Pharmacology

Enzyme-Loaded Titania Nanoparticles for Organic Synthesis
By Allison Roth, Truman State University Page 65
Research Mentor: Dr. Scott Miller, Dept. of Chemistry

The Role of the NarU Nitrite/Nitrate Transporter in Nitrate
Reductase Z-Dependent Resistance to Environmental Stresses
By Gregory Schalla, Beloit College Page 77
Research Mentor: Dr. Michael Spector, Dept. of Biomedical Sciences

Glucose Consumption and Lactate Production in Nih-3t3 Fibroblasts Carrying
Mutations in Mitochondrial DNA
By Kanika Topiwala, University of Arkansas at Little Rock Page 85
Research Mentor: Dr. Mikhail Alexeyev, Dept. of Cell Biology and Neurosciences






3




Characterizing the Cytotoxic Properties of a Natural
Diterpenoid from Rabdosia rubescens












Katherine Bauer
University of Alabama

















Research Mentor: Dr. Richard Honkanen
Department of Biochemistry and Pharmacology





4

Abstract

Oridonin is a biologically active compound contained in an herbal extract that has
been used as a Traditional Chinese Medicine to treat cancer and other ailments. In recent
studies, oridonin has been shown to induce cell cycle arrest after DNA replication (G
2
/M-
phase growth arrest) and to kill cells via apoptosis. However, the molecular mechanisms
leading to the onset of growth arrest or apoptosis are not clear. In this study, time-lapse
microscopy of live HeLa cells expressing a histone 2B-green fluorescent protein fusion
protein, allowing high-quality chromosome imaging without affecting chromosome structure,
was used to document the progression of cells through mitosis. In cells treated with oridonin,
the chromosomes fail to align properly at the metaphase plate and arrest. Several affects were
observed: 1) metaphase arrest and apoptosis before the onset of anaphase; 2) apoptosis after
anaphase; 3) successful anaphase and cytokinesis, or 4) successful anaphase followed by
failure to complete cytokinesis.

Introduction

Traditional Chinese medicine is a system of medicine based on the teachings of
Taoism. It originated in China more than 2000 years ago, and remains in wide use today.
However, scientific evidence still needs to be gathered to document the efficacy of many of
the treatments used. Two of the most commonly used treatments are acupuncture and herbal
medicines.
One herbal extract used in Traditional Chinese medicine is produced from Rabdosia
rubescens, and is known as dong ling cao in Chinese and blushred rubescens in English.
This herbal concoction has been used to treat bacterial infections, inflammation, and cancer
(1,2,3,4,5,6). Rabdosia rubescens is found in the Yellow River valley in China (1), and is a
member of the Isodon family, two other members of which have been used as similar
remedies in Japan and Korea (2,3). Oridonin was isolated from these members of the Isodon
family, and is believed to represent the main biologically active component of Rabdosia
rubescens (2,3).
Oridonin has been shown to cause apoptosis in several lines of cultured cells,
including HepG2 (1), L929 (4), MT-1, MT-2, U266, RPMI 8226, murine macrophage
RAW264.7, Jurkat (5), Kasumi-1, and U937 (2). Apoptosis is programmed cell death, and it
is characterized by the appearance of fragmented DNA, dissolution of the nuclear envelope,
condensation of the cytoplasm, cell membrane blebbing, and phagocytosis by neighboring
cells (4). Apoptosis is brought about by the activation of procaspases, which become
caspases. The activation of caspases can be induced from outside the cell by the binding of
the Fas ligand to the Fas protein on the cell surface and from inside the cell by the activation
of Apaf-1. Some proteins in the Bcl-2 family, such as Bcl-2 and Bcl-x
L
, inhibit apoptosis,
while others, such as Bax, induce apoptosis. Several pathways, such as the PTK survival
pathway, inhibit apoptosis (4). Other pathways, such as some of the p53 pathways, induce
apoptosis, [i.e. p53 can induce the increased expression of Bax(4) ].
At low concentrations oridonin has been shown to cause G
2
/M arrest prior to the
onset of apoptosis (1,4,5). The cell cycle is divided into four major stages (4): 1) G
1
phase,
the gap before S phase, 2) S phase, the phase during which DNA is replicated, 3) G
2
phase,
the second gap occurring before M phase and 4) M phase, the mitotic phase during which the


5

chromosomes are condensed and aligned in preparation for anaphase and cell division (4).
The cell cycle is regulated by checkpoint control mechanisms, such as the G
2
checkpoint,
which checks to make sure all of the DNA is replicated and undamaged before allowing
cell cycle progression into M phase, and the metaphase checkpoint, which checks that all
chromosomes are attached to the mitotic spindle before allowing progression into anaphase.
Cyclin-dependent kinases, or Cdks, and cyclins, act as key component of these cell cycle
checkpoint controls. Interference with expression of Cdks or cyclins can stop the progression
of the cell cycle (4). The activation of p53, for example, increases the expression of p21,
which binds to CDK-cyclin complexes to inhibit their activity and induce growth arrest (4).
The molecular mechanisms associated with oridonins cytotoxic effects are unknown,
though oridonin has been shown to inhibit the PTK survival pathway (4), p38 MAPK (which
activates p53), telomerase (1), and NF-B (which increases levels of Bcl-x
L
, allowing cells to
live longer and increase drug resistance) (5), and activate effector caspases through the
cleavage of AML1-ETO protein (2) and p53 (4). To date, research efforts to characterize the
cellular actions of oridonin have not fully addressed the effects of oridonin on the cell cycle
progression. The observation and classification of these effects could provide more
information on the G
2
/M arrest caused by oridonin.
To document the effects or oridonin on cell cycle progression, a HeLa cell line stably
expressing histone 2B fused to green fluorescent protein (HeLa-H2b-GFP cells) was used in
combination with time-lapse fluorescent microscopy. This cell line allows for excellent
chromosome imaging without affecting chromosome structure for behavior, and is a useful
model system to study cell cycle progression through mitosis (7).

Materials and Methods

Cell culture
HeLa-H2B-GFP cells were a kind gift from Dr. Kevin Sullivan (7), were cultured in
35 mm dishes with 2 or 3 mL of media: 89% DMEM, or Dulbeccos Modified Eagles
Medium, 9% of 10% Fetal Bovine Serum, 1% Pen/Strep and 1% L-Glutamine, or in 60mm
dishes with 6 mL of media. They were cultured at 37C in 5% CO
2
.

Oridonin
Oridonin was provided by Dr. Richard Honkanen. It was reported 97% pure. To treat
cells, oridonin was dissolved in dimethylformamide, purchased from Sigma-Aldrich.
Working stocks (10 or 100 mM solutions) were stored at at 20C.

Live cell imaging and Time-lapse Fluorescent Video Microscopy
The HeLa-H2B-GFP cells were incubated at 37C in 5% CO
2
in a Neuve-live cell
chamber on an Eclipse Nikon TE 2000-U microscope from Nikon Instruments Inc. in
Melville, NY. Images were taken using a COOLSNAP ES monochrome camera and
processed using NIS-Elements AR 3.0 software by Nikon.

Cell passage
HeLa-H2B-GFP cells were cultured in 25 and 75 cm
2
cell culture flasks with 8 and 15
mL of media above, respectively. When the flask was approximately 90% confluent, the cells
were passed. The media was drained and the cells were rinsed with approximately 5 mL of


6

PBS. 2-3 mL of trypsin/ EDTA were added to each flask, and the flask was replaced in the
incubator. The cells were observed under a microscope until almost all cells were rounded.
The flask was then tapped against a hard surface to free the cells. 10 mL of media were
added and the mixture was subjected to centrifugation for 1 minute. The supernatant was
poured off and the pellet of cells was resuspended in 10 mL of media. The cells were
resuspended in the culture media and then aliquots (0 .5 to 1 mL, respectively) were added to
either the 25 or 75 cm
2
flask containing growth media (DMEM, supplemented with 10%
FBS).

Results

Time-lapse video microscopy was used to characterize the effects of oridonin on
HeLa-H2B-GFP cells. In control cells (Figure 1), chromosomes condense at the beginning of
prophase and align at the metaphase plate after approximately 20 minutes. Anaphase occurs
half an hour to two hours after chromosome alignment. Metaphase arrest is observed in less
than 3% of control cells (7).
In cells treated with low (<30 M) concentrations of oridonin, some cells do not
achieve metaphase alignment after prophase. In the cells that do not achieve normal
metaphase alignment, chromosomes that do not align at the metaphase plate, hereafter
referred to as lagging chromosomes, are often observed (Figure 2a). The chromosomes of
these cells appear to move in a random manner (Figure 2b). Cells that fail to achieve
metaphase alignment also arrest prior to the onset of anaphase, spending more than the
typical 2 hours in an aberrant form of metaphase (Figure 3). Some cells that arrest proceed to
anaphase; others simply remain in an aberrant metaphase, while others become apoptotic. Of
those that proceed to anaphase, many successfully complete cytokinesis. However, one or
both of the daughter cells often die, often after considerable delay. Still others complete
anaphase and then fail to complete cytokinesis, forming one cell with two 2N nuclei or one
cell with one nuclei that appears to contain 4N DNA. On occasion, a cell formed a Y-shaped
metaphase alignment and then divided into three daughter cells. These three daughter cells
may either survive or become apoptotic. The percentage of mitotic cells increases in a
concentration-dependent manner until the concentration of oridonin reaches 25 M.
Concentrations greater than 25 M still contain more mitotic cells per total number of cells
than the control group, but the number is far lower than it is at lower doses (Figure 4).
However, the number of cells affected by the extract increases, and at higher concentrations
more dead cells are observed (Figure 5). At lower concentrations, <25M, oridonin does not
appear to have an effect on cells that do not enter prophase.
The percentage of total cells that becomes apoptotic increases in a dose-dependent
manner. Oridonin has negligible effects on cells treated with concentrations less than 10 M.
Treatments of 10 and 15 M oridonin affect relatively few cells, and only rarely induced
apoptosis. Treatment with 20 M oridonin caused apoptosis, generally after inducing a
mitotic arrest. The number of cell deaths increases sharply in cells treated with 30 M
oridonin. In cells treated with high (>45 M) concentrations of oridonin, the cells do not
arrest in metaphase, but become apoptotic, generally within 19 hours. Cell death in cultures
treated with >45 M oridonin was not dependent on cell cycle progression, as death was
observed in prophase cells. The apparent IC
50
, or concentration that kills 50% of cells, of
oridonin is approximately 35 M (Figure 6).


7

During experimentation, preliminary data indicate that changing the media directly
before treatment with oridonin may enhance the killing effects of the extract (Figure 7). The
number of aberrant cells in dishes treated immediately after a media change was
approximately 4 times that observed in dishes treated a day or more after a media change.
The reason for this is currently unknown.

Discussion

The results of this study show that low concentrations of oridonin induce mitotic
arrest and prevent cells from achieving normal metaphase alignment. Arrested cells remain in
the resulting aberrant metaphase, proceed to anaphase, or become apoptotic. The percentage
of apoptotic cells increases in a dose-dependent manner. High concentrations of oridonin
induce apoptosis in all cells, not just cells that are arrested during progression into mitosis.
Conclusions: The molecular mechanisms affected by oridonin leading to mitotic
arrest are not yet clear. The failure of some chromosomes to align at the metaphase plate
could be due to interference with the mitotic spindles. Mitotic spindles attach to the sister
chromatids at the centromeres, bring the sister chromatids to and hold them at the metaphase
plate, and pull them apart upon the cleaving of cohesin. If any of the sister chromatids were
not connected to the spindle, the metaphase checkpoint would prevent the cell from
proceeding to anaphase. Likewise, interference with Cdc20 or the anaphase-promoting
complex (APC), which activates separase to cleave cohesin, would inhibit cell progression to
anaphase. Since the sister chromatids are aligned at the metaphase plate by the tension from
opposing spindle fibers, premature cleavage of cohesin would allow the sister chromatids to
be pulled to opposite ends of the cell prior to metaphase alignment. Premature loss of
cohesion could also produce the observed effects. Therefore, future studies of oridonin
should consider these as possible targets of oridonin.

References

1. Wang, H., et al. 2010. Proteomic identification of proteins involved in the anticancer
activities of oridonin in HepG2 cells. Phytomedicine doi: 10.1016/j.phymed.2010.06.011.

2. Zhou, G.B., Kang, H., Wang, L., Gao, L., Liu, P., Xie, J., Zhang, F.X., Weng, X.Q.,
Shen, Z.X., Chen, J., Gu, L.J., Yan, M., Zhang, D.E., Chen, S.J., Wang, Z.Y. and
Chen, Z. 2007. Oridonin, a diterpenoid extracted from medicinal herbs, targets AML1-
ETO fusion protein and shows potent antitumor activity with low adverse effects on
t(8;21) leukemia in vitro and in vivo. Blood 109:3441-3450.

3. Zhou, G.B., Chen, S.J., Wang, Z.Y. and Chen, Z. 2007. Back to the future of oridonin:
again, compound from medicinal herb shows potent antileukemia efficacies in vitro and
in vivo. Cell Research 17:274-276.

4. Cheng, Y., Qiu, F., Ye, Y.C., Tashiro, S.I., Onodera, S. and Ikejima, T. 2009.
Oridonin induces G2/M arrest and apoptosis via activating ERK-p53 apoptotic pathway
and inhibiting PTK-Ras-Raf-JNK survival pathway in murine fibrosarcoma L929 cells.
Archives of Biochemistry and Biophysics 490:70-75.


8

5. Ikezoe, T., Yang, Y., Bandobashi, K., Tsuyako, S., Takemoto, S., Machida, H.,
Togitani, K., Koeffler, H.P. and Taguchi, H. 2005. Oridonin, a diterpenoid purified
from Rabdosia rubescens, inhibits the proliferation of cells from lymphoid malignancies
in association with blockade of the NF-B signal pathways. Mol Cancer Ther 4:578-586.

6. Zhang, M., Zhang, Y.M., Lu, W. and Nan, F.J. 2011. Synthesis and revision of
stereochemistry of rubescensin S. Org Biomol Chem 9:4436-4439.

7. Bonness, K., Aragon, I.V., Urban, G., Huang, X.Z., Dean, N.M. and Honkanen, R.E.
Studies into the antitumor activity of fostriecin reveal a critical role for ser/thr protein
phosphatase 4 (PP4) in the regulation of mitotic progression into anaphase.

Figures











A
B
Figure 1. Localization of H2B-GFP protein in control HeLa cells. Representative fluorescent and corresponding
differential interference contrast (DIC) microscopic images of live control HeLa cells expressing H2B-GFP in various
phases of the cell cycle. Prophase (2 min), pro-metaphase (20 min), metaphase (70 min), anaphase (80-86 min), and
telophase (132 min) are illustrated. Elapsed time from the onset of chromosome condensation is shown above image
pairs. The images are representative of approximately 97% of cells studied.
Figure 2. Localization of H2B-GFP protein in oridonin-treated HeLa cells. Representative fluorescent microscopic
images of live oridonin-treated HeLa cells expressing H2B-GFP. Cells treated with 10-30 M oridonin showed no
difference from control cells during prophase (0 min) and pro-metaphse (35 min). However, some cells then failed to
achieve metaphase alignment, with several chromosomes, called "lagging chromosomes," failing to align at the
metaphase plate (6.5 hrs, indicated by "A"). The chromosomes of these cells moved in an apparently random fashion
(8.5 hrs, indicated by "B"), and arrested in an aberrant form of metaphase. Many arrested cells become apoptotic
(9.25 hrs), while others remain arrested in metaphase and others proceed to anaphase.


9










0
10
20
30
40
50
60
70
80
90
0 500 1000 1500
C
e
l
l
Time(min)
Timeinmetaphase
Control
10uM
20uM
0
2
4
6
8
10
12
14
16
Control 10uM 15uM 20uM 25uM 30uM
P
e
r
c
e
n
t

m
i
t
o
t
i
c
Dose(uM)
Dosev.percentmitoticat24hours
Figure 3. Metaphase arrest. Graph comparing the tme spent in metaphase by control cells, cells
treated with 10M oridonin, and cells treated with 20M oridonin. Each data point is the time one
cell, randomly assigned a number, spent in metaphase as observed using time-lapse microscopy, as
described in methods. All but one control cell spent less than 100 min in metaphase. Cells treated
with 10 and 20M oridonin spend anywhere from 0 to >1500 minutes in metaphase before either
proceeding to anaphase or becoming apoptotic.
Figure 4. Percent of total cells in mitosis. Graph comparing the percentage of cells in mitosis at 24 hours after treatment
with different concentrations of oridonin. The percentage of cells in mitosis was determined using images of the cells
taken 24 hours after treatment.


10









0
10
20
30
40
50
60
70
Control 10uM 15uM 20uM 25uM 30uM
P
e
r
c
e
n
t

o
f

t
o
t
a
l

c
e
l
l
s
Dose(uM)
Percentmitoticandpercentdeadat24hours
%dead
%mitotic
0
20
40
60
80
100
120
0 20 40 60
P
e
r
c
e
n
t

d
e
a
d
Concentration
Percentdead
%dead
Figure 5. Percent of total cells dead or in mitosis. Graph showing the percentage of total cells in mitosis at
24 hours and the percentage of total cells that are dead at 24 hours, stacked to depict the percentage of total
cells affected by oridonin. As described in Figure 4, the percentage of total cells in mitosis at 24 hours
increases in a dose-dependent manner until ~25M, after which the percentage of mitotic cells is still higher
than in control cells. The percentage of dead cells at 24 hours increases with increasing concentrations of
oridonin.
Figure 6. Percent of total cells dead at 24 hours. Graph showing the percentage of total cells dead at 24 hours
induced by different concentrations of oridonin. The percentage of dead cells increases with increasing
concentration of oridonin. The apparent IC
50
is 35 M.


11











0
5
10
15
20
25
30
35
40
45
Fed Notfed
P
e
r
c
e
n
t

d
a
e
d

o
r

a
b
e
r
a
n
t
Timeofmediachangev.percent
dead/aberrantcells
Series1
Figure 7. Effect of time after media change on observed effects of oridonin. Graph of the percentage of
total cells affected by oridonin, as observed in cells whose media was changed directly before treatment
with oridonin and cells whose media was changed a day or more before treatment. Approximately 4 times
more affected cells (dead or aberrant) were observed in cells whose media was changed directly before
treatment than in cells whose media was changed a day or more before treatment. This data, however, is
preliminary, and more trials need to be conducted to confirm these observations.



12









13





Hyperspectral Imaging Microscopy And Linear
Unmixing Of Fret Signals











Lauren Cichon
Aquinas College








Research Mentor: Dr. Diego Alvarez
Center for Lung Biology - Department of Internal
Medicine

Dr. Silas Leavesley
Department of Chemical and Biomedical Engineering

Dr. Thomas Rich
Department of Pharmacology





14

Abstract

Measuring FRET is becoming increasingly more important for tracking protein-
protein interactions within a cell, with different approaches used to measure it. To fulfill the
need for higher specificity and sensitivity for FRET measurements, hyperspectral analysis
techniques including linear unmixing have been applied to fluorescence microscopy. In this
study, we have used widefield and confocal microscopy with linear unmixing to measure
FRET signals from time series fluorescence images. A cAMP-sensitive FRET probe was
used. Forskolin and rolipram were used to increase intracellular cAMP levels, and the
subsequent FRET change was measured. Spectral fluorescence microscopy images were
analyzed using linear unmixing and the FRET signal was estimated. Hyperspectral imaging
and linear unmixing proved to have many advantages over traditional analysis methods.
However, it cannot be assumed that this is the best method for quantifying FRET without
further experimentation.

Introduction

Frster Resonance Energy Transfera mechanism of non-radiative energy transfer
known as FREThas become increasingly important for tracking protein-protein
interactions within a cell, by providing unprecedented information with high specificity and
sensitivity
1
. This spectroscopic ruler occurs between a pair of two different fluorophores
that are attached to the protein molecule(s) of interest. The efficiency of energy transfer is
dependent on the distance separating the fluorophores. When proteins bound to the
fluorophores interact, or when a protein containing both fluorophores interacts with its
corresponding substrate, there is a change in the distance between the fluorophores that
affects the FRET efficiency. When FRET occurs, there is a transfer of energy between the
FRET pair, from the donor fluorophore to the acceptor fluorophore. There are several ways
to measure FRET, including fluorescence microscopy using two filter sets, three filter sets, or
spectral filtering. Two filter set imaging uses an algorithm that corrects for crosstalk and
acceptor photobleaching. Three filter set imaging uses three different excitation ranges to
excite fluorophores at different wavelengths. The most common approach to measure FRET
is to use three-filter sets and calculate FRET with respect to the native concentration
(FRETN).
2
Nonetheless, the use of current fluorophores constitutes a major problem as
evidenced by the amount of false positives, even after using three filter sets. Improvements
in this area are in need.
Choosing fluorophores can be difficult. For instance, if the spectra between
fluorophores overlaps too much there can be cross-talk (or bleed-through) between the two
fluorophores, which means that the transfer of energy from the donor to the acceptor
becomes non-specific and type I errors develop. On the other hand, if the spectra between
the donor and acceptor are too far apart the overlap integral, J(), is reduced which decreases
the detectability of the FRET signal, which means that the transfer of energy is not efficient
and type II errors develop
3
. Correct calibration of FRET ratios have also been challenging.
The CFP-YFP pair of fluorophores has been particularly successful for FRET. However, the
extenuated tail at the right end of the CFP emission spectrum causes bleeding of the CFP
emission into the YFP emission range and increases non-specific FRET detection
4
. Another


15

complication in data acquisition is calculating accurate levels of molecules, such as cAMP,
within a cell.
cAMP is a second messenger responsible for numerous intracellular functions and
physiological processes. Traditional biochemical approaches for measuring cAMP do not
allow insight into the second messenger dynamics; therefore, FRET biosensors have been
developed for the study of real-time dynamics of cAMP signaling in living cells.
5
One of
these FRET biosensors involves CFP and YFP that are genetically fused to exchange protein
directly activated by cAMP (EPAC). FRET biosensors have become essential tools for
detecting cAMP dynamics in various subcellular microdomains.
5
When cAMP binds to the
sensor (EPAC) there is a conformational change in the enzyme. This change is accompanied
by an increase in distance between the fluorophores, which results in a decrease in FRET
efficiency. As FRET occurs, the donor emission is decreased and the acceptor emission is
increased, resulting in a decrease of the donor-acceptor ratio, an index of FRET.
2,6
CFP-
EPAC-YFP displays significant energy transfer, which rapidly diminishes following a rise in
intracellular cAMP levels and increases again as cAMP levels decrease.
6
cAMP
concentrations can be regulated with the use of forskolin, an alkaloid that stimulates
synthesis of cAMP, and rolipram, a phosphodiesterase inhibitor that precludes the
metabolism of cAMP. Treatment of cells with forskolin and rolipram results in elevation of
cAMP levels, and thus, decreases in FRET. Accurate calculation of cAMP concentration
from FRET ratios has been challenging for several reasons, one of which is that the basal
cAMP concentrations are difficult to estimate.
5

The many challenges encountered when trying to calculate FRET has led to new
techniques such as hyperspectral imaging and linear unmixing to increase the sensitivity and
specificity of the measurements. Hyperspectral imaging consists of collecting both the donor
and acceptor spectra, which enables the determination of the amount of donor fluorescence
and acceptor fluorescence. In hyperspectral imaging, pixels of interest are frequently a
combination of numerous disparate components, which has introduced a need to extract
increasingly detailed information by performing spectral unmixing.
7
Linear spectral
unmixing uses a collected spectral library and an algorithm to quantitatively decompose
mixtures within a pixel. The algorithm requires the fluorescence emission spectra of each
fluorophore to be measured and saved prior to analysis. The measured spectra of a mixed
pixel is decomposed into a collection of constituent spectra, or endmembers, and a set of
corresponding fractions, or abundances, that indicate the proportion of each endmember
present in the pixel.
7

Hyperspectral analysis techniques have advanced the quality and applications of
fluorescence imaging. Hyperspectral imaging allows for complex-color images to be
separated into a set of single channel images, which is particularly useful if several stains are
used. It also provides information on the co-localization of structure, proteins, and other
entities within the cell. Use of linear unmixing algorithms allows detection of FRET signals
with increased sensitivity and can compensate for significant overlap of donor and acceptor
fluorophores. The aim of the current study is, therefore, to determine the best method for
quantifying the FRET response of cAMP sensors using widefield and confocal spectral
microscope systems and hyperspectral analysis techniques including linear unmixing.





16

Materials and Methods

Cell and sample preparation
HEK293 cells were used. Cells were grown either on coverslips or in 60 mm round
cell culture dishes. Cells on coverslips were imaged while cells from cell culture dishes were
suspended and used for parallel spectrofluorimeter measurments. Cells were transfected with
CFP, YFP, or the CFP-EPAC-YFP reporter using fugene-6 reagent. Tests were also done
with cells containing adenovirus.

Preparation of cells in suspension
Cells were suspended in PBS EDTA after removal of media. The cell suspension was
centrifuged in a test tube, the supernatant was removed, and the cells were re-suspended in a
buffer without EDTA (1X Tyrodes buffer). The cells and buffer were mixed with a vortex
mixer. Either 1 mL of suspended cells were allowed to settle on a coverslip or cells grown
on a coverslip were covered with 1 mL of 1X Tyrodes buffer.

Instrument configuration and image acquisition
Calibration
Calibration was performed using a NIST-traceable lamp (LS-1-CAL-INT, Ocean
Optics, Inc.).

Spectral microscope
Fluorescence microscopy was performed using an inverted fluorescence microscope
(TE2000-U, Nikon Instruments, Melville, NY) and a 40X-oil immersion objective. A liquid
light guide-coupled Xe arc lamp (Lambda DG-4, Sutter Instrument Company, Novato, CA)
was used for fluorescence excitation. A variable-bandwidth acousto-optic tunable filter
(AOTF) that was specifically configured for microscopic use (Hsi-300 Hyperspectral
Imaging System, ChromoDynamics, Orlando, FL) was used for spectral filtering of the
fluorescence emission. A 430 nm excitation was used with a 450 nm long-pass dichroic
beamsplitter. Images were collected using a cooled, back-illuminated, charge-coupled device
(CCD) camera (Cascade 512B, Photometrics, San Diego, CA). The tunable filter and CCD
camera were both controlled using Manager software (Vale Lab, UCSF).

Image acquisition
Spectral images of fluorescence were taken from 450-650 nm at 5 nm increments
using a bandwidth of 8.5 nm. An exposure time of 100 ms was used. The CCD gain was set
to 3 and the electron multiplier gain was set to 3500.

Single image
For each sample, a bright field image and a fluorescence image were taken at two
different fields of view (FOV) (Figure 1 A, B). These images were used for initial
verification of FRET and to build the spectral library.



17


Figure 1: Suspended HEK 293 cells that have settled onto a coverslip, as observed with a widefield microscope. (A)
Bright field image. (B) Fluorescence image of the expressed CFP-EPAC-YFP FRET probe.

Time series images
Time series experiments were performed in which images were acquired every three
minutes for 25 minutes and 10 L of forskolin 10 M concentration and/or 10 L of rolipram
10M concentration were added at two minutes and at ten minutes, respectively.

Confocal microscope
Confocal microscopy was performed using an A1 confocal microscope with a
spectral detector (A1 Confocal, Nikon Instruments, Melville, NY) and a 40X-oil immersion
lens.

Image acquisition
Images were acquired using an excitation wavelength of 404.5 nm. The scan size was
kept at 512 and images were averaged either 4 or 16 times. A confocal pinhole of 3.5 Airy
disk units was used. The laser power was varied to ensure an adequate dynamic range.

Single image
For each sample, a fluorescence image was taken at two different FOV (Figure 2 B).
A z-stack image with 20 steps was taken for some samples to visualize the cell in the x, y,
and z planes.

A B


18


Figure 2: Confluent HEK 293 cells grown on a coverslip, as observed with an A1 confocal microscope. (A) Bright
field image. (B) Fluorescence image of the expressed CFP-EPAC-YFP FRET probe.

Time series images
Time series experiments were performed in which images were acquired every two
minutes for 30 minutes. 10 L of forskolin 10 M concentration and/or 10 L of rolipram 10
M concentration were added after the scan at two minutes and the scan at ten minutes,
respectively.

Analysis
Four regions of interest (ROI) were marked on each fluorescence image (Figure 3A).
The spectra peak intensity data of the unmixed image was exported to Excel (Microsoft
Corporation, Redmond, WA) for manipulation, normalization, and plotting. The comparison
of different ROIs at one time as well as comparison of one ROI throughout the duration of
the scanning was plotted. Graphs were made using both Excel and MATLAB (The Math
Works, Inc., Natick, MA).

Spectral analysis
Hyperspectral images were analyzed using ENVI software for linear unmixing (ITT
Visual Information Solutions, Boulder, CO).
CFP and YFP expressing cells were used as the pure CFP and YFP signal in the
spectral library for both the widefield and confocal microscopes. For each FOV, cellular and
background ROIs were defined through thresholding. Thresholding caused pixels at or
below the background threshold to be categorized as background, and pixels at or above the
cellular threshold were categorized as the cell spectrum. The pixel-averaged background
spectrum was subtracted from the pixel-averaged cell spectrum, resulting in a background-
corrected fluorescent protein spectrum. The averaged spectra were saved in the spectral
library.

A B


19


Figure 3: Cytosolic EPAC as observed with an A1 confocal microscope. (A) Fluorescence image. (B) FRET image
after unmixing. Image was obtained by applying the algorithm YFP/(CFP+YFP) using the unmixed YFP and
unmixed CFP images from the hyperspectral analysis.

The spectral library was used with a linear least squares approach to calculate the
abundance of each component in each pixel of an image. The FRET efficiency was
estimated using a band math equation of YFP/(YFP+CFP) applied to the unmixed images
(Figure 3 B).

Results and Discussion

In this study, we have used different techniques to measure and calculate FRET.
Widefield microscopy data were compared to confocal data, and unmixed data was compared
to data before unmixing. Time series experiments were performed with the widefield
microscope on HEK 293 cells expressing the CFP-EPAC-YFP FRET probe.

Signal Intensity
To select the optimum EPAC expression level, fluorescence images were taken of
several EPAC samples including some that used a fugene-6 transfection reagent to encode
DNA as well as cells with adenovirus. EPAC samples with fugene had significantly stronger
signal at 524 nm than the other samples (Figure 4) and were therefore primarily used for
further experimentation.

A B


20

0
5
10
15
20
25
30
35
40
45
Cells
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
EPAC2cyt_(Adenovirus)_1
EPAC2cyt_(Adenovirus)_2
EPAC2_Cyt_virus_1
cAMP_EPAC2_fugene(1-
1)_1
cAMP_EPAC2_fugene(1-
2)_2
0
5
10
15
20
25
30
35
40
45
Cells
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
EPAC2cyt_(Adenovirus)_1
EPAC2cyt_(Adenovirus)_2
EPAC2_Cyt_virus_1
cAMP_EPAC2_fugene(1-
1)_1
cAMP_EPAC2_fugene(1-
2)_2

Figure 4: Peak Intensities at 524 nm in EPAC cells.

Widefield
Time series imaging was performed with treatments of 10 M forskolin or 10 M
rolipram to observe the effects on FRET intensity of changes in intracellular cAMP
concentration.
In the trial with forskolin (Figure 5), FRET intensity decreased rapidly after 2
minutes, and slightly increased after 7 minutes, decreased again after 9 minutes, and then
increased after approximately 13 minutes. As expected, the forskolin stimulates synthesis of
cAMP, which binds to EPAC and causes a conformational change in the enzyme. This
conformational change is accompanied by an increase in distance between fluorophores and
leads to a decrease in FRET. cAMP synthesis became slower after a few minutes and FRET
intensity rose until forskolin was added for a second time and the same pattern repeated.



21

50
55
60
65
70
75
80
85
0 5 10 15 20
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
50
55
60
65
70
75
80
85
0 5 10 15 20
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
70
75
80
85
90
95
100
105
110
115
0 5 10 15 20
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
70
75
80
85
90
95
100
105
110
115
0 5 10 15 20
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4


Figure 5: EPAC cells were treated with forskolin (For) at 2 minutes and 9 minutes and observed for 25 minutes
with a widefield microscope. Hyperspectral images were unmixed and used to calculate FRET over time.



Figure 6: EPAC cells were treated with rolipram (Rol) at 2 minutes and 9 minutes and observed for 25 minutes
with a widefield microscope. Hyperspectral images were unmixed and used to calculate FRET over time.

For
For
Rol
Rol


22

0
0.2
0.4
0.6
0.8
1
1.2
450 470 490 510 530 550 570 590 610 630 650
Wavelength (nm)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
1 min
5 min
8 min
11 min
14 min
17 min
20 min
25 min
0
0.2
0.4
0.6
0.8
1
1.2
450 470 490 510 530 550 570 590 610 630 650
Wavelength (nm)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
1 min
5 min
8 min
11 min
14 min
17 min
20 min
25 min
In the trial with rolipram (Figure 6) FRET intensity decreased slightly with time, but
was fairly constant because rolipram prevents the breakdown of cAMP and the basal cAMP
synthesis levels in HEK 293 cells is relatively low. Therefore, cAMP neither increased nor
decreased significantly and consequently, FRET intensity differed only slightly over time. In
both experiments, there was an initial steep increase in FRET from zero to three or four
minutes, which was due to thermal drift. These two experiments were combined into a time
series experiment with forskolin being added between 1 and 2 minutes and rolipram being
added between 9 and 12 minutes (Figure 7, Figure 8).



Figure 7: EPAC cells measured over a period of 25 minutes with 10 M forskolin and 10 M rolipram added at
1 minute and 9 minutes, respectively. Intensities were normalized to the YFP peak and CFP peak fluctuation
was observed as a result of FRET. Note that these relatively small changes can represent a large shift in the
amount of FRET.

FRET in the cytosolic EPAC cells (Figure 8 B) decreased quickly when forskolin was
added at 2 minutes and remained at a constant intensity when rolipram was added at 10
minutes. By contrast, FRET in the plasma membrane EPAC cells (Figure 8 D) decreased
slowly when forskolin was added at 2 minutes and decreased very gradually throughout the
experiment after rolipram was added at 10 minutes. The cytosolic EPAC cells experienced a
significantly larger decrease in FRET than the plasma membrane EPAC cells. This could
mean that more cAMP is present in the cytosol than at the plasma membrane, or that
forskolin-induced cAMP synthesis is primarily cytosolic in nature, or that the near-membrane
cAMP levels are somehow buffered.


23


Figure 8: FRET response of CFP-EPAC-YFP expressing HEK 293 cells on coverslip as measured with the widefield
microscope. (A) Cytosolic EPAC control (untreated). (B) Cytosolic EPAC treated with 10 M forskolin (For) at 2
minutes and 10 M rolipram (Rol) at 12 minutes. (C) Plasma membrane EPAC control (untreated). (D) Plasma
membrane EPAC treated with 10 M forskolin at 2 minutes and 10 M rolipram at 12 minutes.
Confocal
The same time series experiment with forskolin and rolipram was performed on the
confocal microscope, which yielded similar results (Figure 9). The FRET curves from the
confocal microscope are not as consistent as the FRET curves from the spectral microscope
with the exception of the control cells (Figure 9 A, C). However, the spectral images had
much more background signal (DC offset) than the confocal microscope. The background
spectra for the spectral microscope could have overcompensated for the amount of noise in
the measured spectra, giving similar FRET curves for each region of interest. In addition,
out-of-focus fluorescence may also contribute to background levels on the widefield
microscope. The detectors on the confocal microscope produce less DC offset than the
widefield microscope. In addition, the confocal pinhole reduces out-of-focus fluorescence.
However, the confocal detector produces a relatively high noise level. Combining these
characteristics, we believe the confocal microscope produces a more specific FRET
measurement. The cytosolic and plasma membrane EPAC cells decreased in FRET by
approximately the same amount and both tests had an additional decrease in FRET at 12
minutes for one ROI. Overall the results give the same information as the spectral
microscope, but the confocal microscope is more specific and subtracts out-of-focus light
more accurately.
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y
ROI 2
ROI 3
ROI 4
ROI 1
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Ti me (mi n)
I
n
t
e
n
s
i
t
y
ROI 2
ROI 3
ROI 4
ROI 5
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y
ROI 1
ROI 2
ROI 3
ROI 4
0
0.2
0.4
0.6
0.8
1
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Ti me (mi n)
I
n
t
e
n
s
i
t
y
ROI 4
ROI 2
ROI 1
ROI 3
A B
C D
Rol
For
Rol
For


24

0
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4
0
20
40
60
80
100
120
140
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (min)
I
n
t
e
n
s
i
t
y

(
A
.
U
.
)
ROI 1
ROI 2
ROI 3
ROI 4



Figure 9: FRET response of EPAC-CFP-YFP expressing HEK 293 cells on coverslip as measured with the A1
spectral confocal microscope. (A) Cytosolic EPAC control (untreated). (B) Cytosolic EPAC treated with 10 M
forskolin at 2 minutes and 10 M rolipram at 12 minutes. (C) Plasma membrane EPAC control (untreated). (D)
Plasma membrane EPAC treated with 10 M forskolin at 2 minutes and 10 M rolipram at 12 minutes.

FRET calculations without unmixing of confocal images
FRET calculations were also done without unmixing for the time series images
acquired with the confocal microscope (Figure 10). The algorithm YFP/(CFP+YFP) was
applied to the peak CFP and YFP emission wavelengths from the confocal microscope. The
FRET curves from before and after unmixing are similar with the exception of the control,
but unmixing allows for more specificity. Unmixing allows for more specific classification
of components within pixels as well as correction for background signal. In addition,
unmixing the fluorescence image yields separate images for CFP, YFP, and RMS signals,
which aid in visualization and quantification of cell components.

A B
C D Rol
For
Rol
For


25

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25 30 35
ROI 1
ROI 2
ROI 3
ROI 4
N
o
r
m
a
l
i
z
e
d

F
R
E
T
Time (min)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25 30 35
ROI 1
ROI 2
ROI 3
ROI 4
N
o
r
m
a
l
i
z
e
d

F
R
E
T
Time (min)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25 30 35
ROI 1
ROI 2
ROI 3
ROI 4
N
o
r
m
a
l
i
z
e
d

F
R
E
T
Time (min)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20 25 30 35
ROI 1
ROI 2
ROI 3
ROI 4
N
o
r
m
a
l
i
z
e
d

F
R
E
T
Time (min)
0.56
0.57
0.58
0.59
0.6
0.61
0.62
0 5 10 15 20 25 30 35
ROI 1
ROI 2
ROI 3
ROI 4
N
o
r
m
a
l
i
z
e
d

F
R
E
T
Time (min)
0.56
0.57
0.58
0.59
0.6
0.61
0.62
0 5 10 15 20 25 30 35
ROI 1
ROI 2
ROI 3
ROI 4
N
o
r
m
a
l
i
z
e
d

F
R
E
T
Time (min)



Figure 10: FRET response of CFP-EPAC-YFP expressing HEK 293 cells on coverslip as measured with the A1
spectral confocal microscope. FRET was measured by using the band math equation YFP/(YFP+CFP) on single
wavelength fluorescence time series images. (A) Plasma membrane EPAC treated with 10 M forskolin at 2
minutes and 10 M rolipram at 12 minutes. (B) Cytosolic EPAC treated with 10 M forskolin at 2 minutes and 10
M rolipram at 12 minutes. (C) Cytosolic EPAC control (untreated).

Future Work

Further research includes developing experiments to determine whether unmixing
yields better FRET measurements. One way this could be done is by quantitatively
comparing spectral unmixing and single wavelength results. Another informative experiment
would be to develop a dual excitation method that corrects for changes in YFP
photobleaching.

Acknowledgements

Funding for this study was provided by NSF and NIH. I would like to thank my
mentors, Dr. Alvarez, Dr. Rich, and Dr. Leavesley, for all of their guidance and support, and
Dr. Turrens, for organizing the NSF REU program. I would also like to thank Andrea Britain
for preparing all of the cells and Thomas Baker, Gulnara Fayzulina, and Tiffany Stedman for
their input and collaboration.
A B
C
Rol
Rol


26

References

1. Jares-Erijman, E.A. and Jovin, T.M. 2006. Imaging molecular interactions in living
cells by FRET microscopy. Current Opinion in Chemical Biology 10:409-416.

2. Gordon, G.W., Berry, G., Liang, X.H., Levine, B. and Herman, B. 1998. Quantitative
fluorescence resonance energy transfer measurements using fluorescence microscopy.
Biophys J 74:2702-2713.

3. Piston, D.W. and Kremers, G.J. 2007. Fluorescent protein FRET: the good, the bad and
the ugly. Trends in Biochemical Sciences 32:407-414.

4. Pollok, B.A. and Heim, R. 1999. Using GFP in FRET-based applications. Trends in Cell
Biology 9:57-60.

5. Borner, S. et al. 2011. FRET measurements of intracellular cAMP concentrations and
cAMP analog permeability in intact cells. Nat Protocols 6:427-438.

6. Ponsioen, B. et al. 2004. Detecting cAMP-induced Epac activation by fluorescence
resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep 5:1176-1180.

7. Keshava, N. and Mustard, J.F. 2002. Spectral unmixing. IEEE Signal Processing
Magazine 19:44-57.




27






Qualitative Evaluation of Autofluorescence in
Drosophila Brain and Hindgut







Matt Hurton
Providence College














Research Mentor: Dr. Robin Mockett
Department of Biomedical Sciences









28

Abstract

Studies on lipofuscin, a granular accumulation connected with oxidative stress and
aging, have been a valuable part of recent research on senescence. Lipofuscin is believed to
play a part in the senescent death of certain tissues, and studies in mammals varying from
mice to humans have suggested this to be the case. In this study, Drosophila melanogaster
specimens were studied in order to determine whether or not lipofuscin is present in
Drosophila central nervous system and GI tract, and if there is a substantial difference in
young and old fly tissue. Also to be determined was whether confocal fluorescence
microscopy would be useful for quantifying lipofuscin accumulation in both a two- and
three-dimensional analysis, and whether use of Alexa Fluor 633, plasma membrane stain,
would aid in outlining the region of interest in the tissue. Preliminary results indicate
confocal microscopy may be somewhat useful for quantifying lipofuscin, Alexa Fluor 633
aids in outlining the tissue, and there is presence of lipofuscin in Drosophila GI tract.
However, there is some inconsistency with results that should be further explored.

Introduction

Among various mechanisms and molecules potentially involved in the aging process,
lipofuscin is conspicuously present across tissue types and organisms. In post-mitotic cells,
lipofuscin, a brown-yellow material that autofluoresces at certain wavelengths
1
, is shown to
accumulate. It is shown to autofluoresce when excited at tissue-dependent wavelengths in the
300-to-400 nm range, and progressively increases in concentration over time in these post-
mitotic cells
2
. Various explanations have been given for this positive correlation between
aging and lipofuscin accumulation, and for the possible role of lipofuscin in aging.
Current research suggests that lipofuscin accumulation is a result of oxidative stress
and accumulation of oxidized, nonfunctional molecules
3,4
. Oxidative stress, occurring
naturally in all cell types, can lead to the oxidation of proteins and other molecules within the
cytoplasm of the cell, leading to termination or limitation of their original function and
aggregation with other oxidized materials. These aggregated materials are then targeted for
breakdown and recycling by lysosomal degradation
3,4
. In relation to this, an additional
possible effect this oxidative stress is believed to have on cellular function is the decreased
functionality of lysosomes and mitochondria, leading to progressively more oxidative
damage as the cell becomes more incapable of dealing with these aggregates
1,2,3,4
. This
aggregation is far more noticeable in post-mitotic cells, as cellular division otherwise dilutes
the lipofuscin concentration
1,3,4
. After a certain point this defect might become severe enough
to limit the life of the cell, leading to organismal senescence.
Accumulation of this autofluorescent material is known to occur in various post-
mitotic cell and tissue types in rats
5,6
, humans
7,8,9
, and other organisms, even, in some
instances, in fruit flies
10
. Previous techniques for quantification of lipofuscin accumulation in
tissue samples involved various lipid extraction techniques, while visualization mainly
involved electron microcscopy.With the advent of fluorescent confocal microscopy, it might
now be possible to use microscopy to directly quantify and compare lipofuscin accumulation
between young and aged samples. The goal of this experiment was to determine whether
using these new techniques, more accurate three-dimensional quantification of lipofuscin can
be achieved. Furthermore, this experiment was conducted as an attempt to determine


29

whether, as in other organisms, lipofuscin progressively accumulates with age in both brain
and gut tissue in Drosophila melanogaster.

Materials and Methods

Food Preparation
Standard Drosophila food was prepared and stored in vials and bottles. Preparation
consisted of using 950 mL of distilled, deionized water and adding 69.3 g of cornmeal (equal
amounts white and yellow), 19.5 g of Torula yeast, 6.55 g agar, and 16.3 g glucose. After this
mixture was heated on a hot plate and boiled for two minutes, it was then allowed to cool to
60C before adding 1.875 g methylparaben in 15 mL of ethanol ( 99.5%) as well as 6 mL of
an acid mix. The acid mix consisted of 3.243 mL water, 2.508 mL propionic acid, and 249
L of phosphoric acid per 6 mL of mixture.

Fly stocks
Stocks of y w
2
flies were reared in vials and bottles of standard fly food, being
maintained and transferred to attain flies at certain ages. Individuals were reared in large
stock bottles initially, collected in groups of twenty-five young adult males or virgin females,
and then transferred to new vials every one to two days to provide fresh food.

Dissection
Flies selected for dissection were anesthetized with carbon dioxide gas. Anesthetized
flies were then soaked for a few seconds in concentrated ethanol ( 99.5%) to kill them, then
transferred to a PBS solution to keep tissue from drying out. Dissection was carried out with
tweezers as well as 0.5 mm insect pins. Dissection took place with an Olympus SZ61
microscope. Standard protocol was followed, with legs and wings removed first in obtaining
gut and brain tissue. After removal of legs and wings, hindgut tissue was obtained by
carefully cutting through and removing the cuticle of the thorax, then likewise removing the
abdominal cuticle. Hindgut tissue was spooled out carefully so as to avoid damaging or
splitting the Malpighian tubules. In retrieving brain tissue, after removal of legs and wings
the proboscis was removed to create an opening in the skullcap. Skull cap and retinal tissue
remains were then carefully pulled away so as to reveal the brain. The cuticle of the thorax
was then carefully opened so as to access the ventral nerve cord along with the brain.

Plating of Dissected tissue
Dissected fly tissue was either transferred to or directly dissected on glass
microscope slides. Slides were coated around their edges with a number of layers of nail
polish so as both to create wells for tissue as well as to prevent damaging of the tissue upon
placement of cover slips over the tissue. PBS was applied to the wells in the slides in order to
prevent drying out of the tissue and to immerse tissues for microscopy.

Alexafluor Staining
Certain tissue samples were stained with Alexa Fluor prior to confocal micrsocopy to
aid visualization by demarcating boundary between tissue and external environment.
Samples were submerged in a solution of light-sensitive Alexafluor and PBS and shielded
from light for one hour. Liquid was then drawn away using a micropipette and the tissue was


30

covered in a mixture of PBS and glycerol to prevent it from drying out and aid in
microscopy.

Confocal microscopy
Dissected fly tissues were either viewed directly or stained with Alexafluor to
visualize with confocal fluorescence microscopy using a Nikon MODEL. Samples were
viewed under spectral detection settings, with excitation at 404 nm and emission at 570 nm,
with appropriate settings for gain and intensity. Z-stacks were used for three-dimensional
quantification.

Image Analysis and Quantification
NIS elements software was used to analyze samples for relative intensity and to draw
conclusions concerning relative fluorescence across wavelengths.

Results

Dissected thick tissue sample preparation can be used for confocal fluorescence
microscopy
Samples were taken of two distinct tissue types; the hindgut and the Malpighian
tubules; and the brain, along with the ventral nerve cord. Careful dissection of said tissue
provided suitable samples for use with confocal microscopy. Use of nail polish or any other
such layering agent before applying the cover slip also proved useful in preventing tissue
damage. Application after sealing also prevented the PBS/glycerol in which the tissue was
covered from leaking out and potentially causing damage or otherwise causing problems with
visualization.

Thick tissue sample autofluorescence is visible using confocal fluorescence microscopy
Prepared samples were used along with a Nikon confocal fluorescence microscope
for imaging of autofluorescence, as would be expected of tissue containing samples of
lipofuscin. Spectral detection was observed with excitation wavelengths of 404.5, 488, 562,
and 640 nm. Fluorescence with these settings was visible both young and old tissue
particularly with hindgut tissue showing at least minimal fluorescence at some emission
wavelengths, although others showed minimal to no autofluorescence (Fig. 2)

Alexa Fluor 633 aids in confocal visualization of autofluorescent tissue
While autofluorescence was visible in the given tissue types, visualization was
difficult either due to the faintness of the fluorescence or the inability to determine the
boundary of the tissue and therefore the area of interest for study. Alexafluor dye is a plasma
membrane staining fluorophor that has proven useful in visualization of tissue. Knowing this,
we compared tissue samples stained with Alexa Fluor 633 and left untreated, and determined
that Alexa Fluor 633 was useful in visualization of the Drosophila tissue at the 640 nm
wavelength (Fig. 3).

Emission at 640 nm is best for visualizing stained tissue with confocal microscopy
Visualization of the stained tissue, while moderately successful in some samples at
the lower emission wavelengths, was best accomplished at 640 nm (Fig 4). This fits with the


31

Alexa Fluor used, designed to emit at 633 nm. Unstained tissue samples, however, showed
minimal autofluorescence and showed more so at the lower emission wavelengths.
Therefore, although the use of this Alexa Fluor is useful in demarcating tissue, it may not be
as helpful with autofluorescent tissue as expected.

Difference between old and young tissue
While the protocol has been developed for visualization of lipofuscin in this tissue,
there has been no statistically significant quantification of the difference in accumulation
between old and young tissue. Future work with NIS Elements software will be done in order
to determine whether or not aged tissue has a more significant build-up of lipofuscin than
that of young flies. Some of the tissue samples did show punctuate spots of fluorescence at
certain wavelengths (data not shown) with subjective evidence that there is more
fluorescence in older samples. However, the data is inconsistent from trial to trial and there
are not enough images for quantification, and so further study is necessary for a definitive
conclusion.

Discussion

Use of Drosophila as a model organism has taken place through the history of
modern genetics. Its similar features to humans as an animal and genetic model, and the
intricate and detailed knowledge we currently have of its genetic make-up, make it suitable
for various forms of study, including the aging experiments carried out in this study.
Lipofuscin accumulation is a well-known phenomenon in mammalian models. As
stated before, its accumulation has been documented. Mechanisms and effects of that
accumulation have been hypothesized for mouse and human tissue. These results, gathered
by use of various methods of lipid extraction and quantification, have proven useful in
identifying the problems associated with lipofuscin; and although they also have issues with
representation, that does not alter the original composition in vivo. This study utilized a new
approach, determining whether confocal fluorescence microscopy can be used to visualize
and quantify lipofuscin via the natural autofluorescence of tissue containing lipofuscin.
The results obtained indicate that this is a possibility. With proper methods used for
dissection, preparation, and staining of Drosophila brain and hindgut tissue, detailed
visualization was possible. Furthermore, using spectral detection settings with wavelengths
similar to those used in previous lipofuscin studies in mammals, some tissue
autofluorescence was visible, and with the aid of analysis software, we hope to make it
quantifiable in a meaningful way.
However, there are some issues with this approach. While the use of tissue-staining
dyes like Alexa Fluor aids in visualization of the tissue itself, it may make it more difficult to
visualize autofluorescence. Additionally, further study will have to be conducted to
determine whether there is a significant, quantifiable difference between autofluorescence in
young and old tissue, as well as to determine that this autofluorescence is in fact due to
lipofuscin. With further study, we hope to show, with use of NIS Elements software, that
autofluorescence due to lipofuscin occurs in young and old tissue at statistically different
intensities.
Lipofuscin is held to be a hallmark of aging, but its quantification is not yet routine.
The studies described here should be continued to establish whether autofluorescence in


32

Drosophila is indeed age-dependent at the associated excitation and emission wavelengths
with lipofuscin in other species.

Acknowledgments

Special thanks to all members of the Mockett Laboratory for their help and
instruction in carrying out the various techniques, protocols, and experiments discussed.
Special thanks also to Trudy Cornwell for instruction in use of the Nikon A1R Confocal
Microscope.

References

1. Armstrong, D. Chapter 13. Advanced Protocols in Oxidative Stress II. [S.l.]: Humana,
2010. Print

2. Terman, A. and Brunk, U.T. 1998. Lipofuscin: Mechanisms of formation and increase
with age. Apmis 106.1-6:265-76.

3. Porta, E.A. 2002. Pigments in aging: An overview. Ann NY Acad Sci 959:57-65.

4. Brunk, U.T. and Terman, A. 2002. Lipofuscin: Mechanisms of age-related
accumulation and influence on cell function. Free Radical Biology and Medicine
33(5):611-619.

5. Amenta, F. et al. 1989. Reduced lipofuscin accumulation in senescent rat brain by long-
term acetyl-L-carnitine treatment. Arch Gerontol Geriatr 9:147-153.

6. Bodner, L., Dayan, D. Chimovitz and N., Hammel, I. 1991. Lipid pigment (lipofuscin)
accumulation in rat tongue muscle with aging. Experimental Gerontology 26:89-95.

7. Goyal, V.K. 1982. Lipofuscin pigment accumulation in human brain during aging. Exper
Geront 17:481-487.

8. Dayan, D., David, R. and Buchner, A. 1979. Lipofuscin in human tongue muscle.
Journal of Oral Pathology 8:121-125.

9. Dayan, D., Abrahami, I., Buchner, A., Gorsky, M. and Chimovitz, N. 1988. Lipid
pigment (lipofuscin) in human perioral muscles with aging. Exper Geront 23:97-102.

10. Miquel, J., et al. 1974. Fluorescent products and lysosomal components in aging
Drosophila melanogaster. Journal of Gerontology 29(6):622-637.


33

Figures









































Figure 1 A general model of the hypothesized role of lipofuscin in aging and oxidative
stress.

C D
A B
Figure 2 Autofluorescence is visible via certain excitation and emission wavelengths. Various brain and
hindgut samples from young and old adult Drosophila exposed to excitation at 404 nm and emission at various
visible light wavelengths showed appreciable autofluorescence. Particular wavelength and color was dependent
on tissue type. A) Young hindgut tissue (10 days),
ex
= 404.5, 488, 561.8, 640 nm. B. Old hindgut (72 days),
ex
=
404.5, 488, 561.8, 640 nm. C. young brain,
ex
= 404.5 nm. D.) old brain,
ex
= 404.5 nm.
Figure 1 retrieved from: Armstrong, Donald. Chapter 13. Advanced Protocols in Oxidative
Stress II. [S.l.]: Humana, 2010.


34















































C D
Figure 3 Alexa Fluor 633 aids in visualization of autofluorescent tissue and distinguishes tissue from background. In A and
B, gut samples from ten day old flies were either left untreated or treated with Alexa Fluor 633 and viewed via confocal
fluorescence microscopy, respectively; In C and D, brain samples from 72 day old flies were similarly compared in regards to
application of Alexa Fluor 633. Excitation was at 632 nm and emission at 647 nm.
A B
C D
Figure 4 Stained Tissue Fluorescence is most visible at 640 nm. Tissue samples were excited at 404 nm and then emission
spectrum were collected over three different emission wavelengths. Here each wavelength is tested in a separate image, with
fluorescence at
ex
= 488 (A), 562 (B), and 640 nm (C) shown. Note that there is also substantial fluorescence at the 404 nm
excitation wavelength (D).
A
B
C D


35




Proteomic Analysis of the Response of Escherichia
coli and Staphylococcus aureus to the Photodynamic
Activity of Phloxine B











Mitchell D. Ingram
Jacksonville State University, Jacksonville, AL











Research Mentor: Dr. Lewis Pannell
USA Cancer Research Institute and
Department of Biochemistry and Molecular Biology





36

Abstract

The photodynamic inhibition of bacteria incorporates a photosensitizer that, when
exposed to light of the proper wavelength in the presence of oxygen, releases reactive oxygen
species in solution. This photodynamic effect causes photodegradation of cellular
components and consequently, cell damage or death. One such photosensitizer, phloxine B,
is used commercially as a pesticide and a color additive in many foods, beverages, drugs, and
cosmetics. The inhibitory effects of phloxine B were evaluated using two model bacteria,
Escherichia coli and Staphylococcus aureus. Quadropole-Time of Flight and Ion Trap mass
spectrometry were used to analyze any proteomic changes resulting from exposure to
phloxine B and light. E. coli is a gram-negative bacterium and is far less sensitive to
photodynamic action, showing no growth inhibition and no obvious proteomic changes upon
incubation with phloxine B. Growth of gram-positive S. aureus, however, was profoundly
retarded within 45 minutes of exposure to light, and exhibited changes in the regulation of
proteins controlling DNA synthesis and redox balance.

Introduction

Photodynamic therapy involves the use of a photoactive dye, or photosensitizer,
which is activated by exposure to light of a proper wavelength. In the presence of oxygen,
the transfer of energy from the activated compound leads to the formation of reactive oxygen
species (ROS): singlet oxygen, hydroxyl radical, superoxide anion, hydrogen peroxide,
hydroperoxides. This is referred to as photodynamic effect or photodynamic action. These
ROS are responsible for localized cell damage and cell death by photodegradation of lipids,
proteins, nucleic acids, and other cellular components (Konopka & Goslinski, 2007). The
principles of photodynamic action are currently being applied to the treatment of cancers,
bacterial and fungal infections, rheumatoid arthritis, age-related macular degeneration, and
diagnosis of malignant oral lesions (Konopka et al., 2007).
The range of photosensitizers available is quite extensive and thousands of
synthesized and natural compounds have photodynamic capabilities (Konopka et al., 2007).
Many photosensitizers are aromatic and grouped according to structure (Wainwright, 1998).
The structure of the photosensitizer often indicates the site and mode of action of the
compound. More familiar photoactive dyes would be crystal violet and methylene blue, used
commonly as microbiological stains. Most notably, the ideal photosensitizers possess (i) low
toxicity and fast elimination from skin and epithelium, (ii) high quantum yield of singlet
oxygen in vivo, (iii) high solubility, and (iv) commercial availability and cost effectiveness
(Konopka et al., 2007).
Upon illumination in the presence of oxygen, photoactive compounds transition from
a low energy ground-state to a high energy singlet state. Compounds may revert back to the
ground state and fluoresce, or they may transition into the high energy triplet state. The high
energy triplet state may react with endogenous oxygen to form ROS which cause oxidative
damage to target tissue. There are two mechanisms by which photosensitizers form ROS.
Type I photodynamic reactions entail the removal of electrons/hydrogen from the
photosensitizer to form ions, or electron/ hydrogen removal from target molecules to form
radicals. Type II reactions involve the formation of highly reactive and unstable singlet
oxygen, all of which damages subcellular components (Konopka et al., 2007).


37

Photodynamic antimicrobial chemotherapy (PACT) incorporates the premise of
photodynamic action in the treatment of bacterial, viral, and fungal infection. The dramatic
increase of antibiotic resistance in bacteria has spurred the research effort to uncover novel
antimicrobial therapies. PACT is showing increasing promise in the treatment of infection,
especially since the decline of discovery and synthesis of new -lactam antibiotics. It is
postulated the bacteria would not easily acquire resistance to photodynamic action (Embleton
et al., 2005).
While some photosensitizers are more effective than others, gram-negative species of
bacteria remain to be much less susceptible to photodynamic inhibition than gram-positive
bacteria. Neutral or anionic photosensitizers are generally efficient at binding to and
inactivating gram-positive bacteria, but not gram-negative bacteria. Gram-positive bacteria
have a porous peptidoglycan layer and lipoteichoic acid surrounding the cell membrane to
which photosensitizers may infiltrate to sensitive areas. Gram-negative bacteria, however,
have a thick outer membrane that prevents the diffusion of photoactive compounds (Konopka
et al., 2007). Photosensitizers may be linked to more cationic molecules by membrane active
agents or conjugated to monoclonal antibodies (Konopka et al., 2007). Even bacteriophages
have been used to deliver photosensitizers to target cells (Embleton et al., 2005).
Phloxine B is a photoactive, halogenated xanthene dye. It is a common additive in
foods, cosmetics, drugs and is approved for consumption by the FDA at 1.25 mg/kg of body
weight. Phloxine B has been shown to be effective at inactivating 99.99% of Methicillin-
Resistant Staphylococcus aureus at 100 g/ml concentration with a minimum inhibitory
concentration of approximately 25 g/ml (Rosooly & Weisz, 2002). While phloxine B has
been widely used in agriculture as a pesticide, its potential as a photosensitizer is somewhat
unresearched.
The objective of this study is observe the changes, if any, in the protein expression of
two model gram-positive and gram-negative bacteria, Staphylococcus aureus and
Escherichia coli, respectively, that have been exposed to phloxine B at a concentration of
100 g/ml. Using mass spectrometry, in-house software, and online databases, I aim to shed
more light on the responses of bacteria to photodynamic events. It is expected that gram-
negative Escherichia coli shall be far less sensitive to photodynamic inhibition than
Staphylococcus aureus; and I hypothesize that expression of proteins that function in the
control of ROS may increase in either organism. Additionally, it is hypothesized that random
oxidative damage to the intracellular proteins brought on by the photosensitizer may be
observed.

Discussion of Objectives and Protocols
The aim of this study is to better understand the effect of photodynamic action on
bacteria and evaluate photosensitizing capabilities of phloxine B. The starting point of this
study, however, was to use mass spectrometry to verify the differences in the gut bacterial
profile of individuals with and without colorectal cancer (CRC) and in the various stages of
the disease.
CRC is predominantly an illness affecting Western culture, associated most with
affluent nations. It is the third most common cancer and the fourth leading cause of cancer
death worldwide. It is currently understood that a relationship does exist between colonic
bacteria and colorectal carcinogenesis. The exact mechanisms by which gut microbiota


38

contribute to CRC is not fully understood nor has a causal relationship been established
between suspect bacteria and CRC.
It is known, however, that certain bacteria are associated with CRC and other chronic
illnesses, such as inflammatory bowel disease. Helicobacter pylori, Enterococcus faecalis,
various Clostridium spp. and Bacteroides spp., Streptococcus bovis/gallolyticus, and
sulphate-reducing bacteria have all been implicated in the pathology of CRC. Still evidence
shows that other bacteria, such as, Bifidobacterium longum, enterotoxigenic Escherichia coli,
and lactic-acid producing bacteria are involved in the suppression of mutagens and lower
incidence of CRC.
This aim proved to be challenging for various reasons. Our initial method for
preparing the bacterial samples for analysis by the mass spectrometers was inadequate and
needed adjustment. There was also some difficulty identifying the bacteria cultured from the
human gut.
Method Optimization
The methods and conditions of cell lysis were the largest components in the development of
our protocol. The bacterial cells must be lysed in order to extract the intracellular proteins
for mass spectrometry analysis. Sonication, freeze/thaw cycles, and bead beat
homogenization were performed and evaluated for their ability to return satisfactory data.
Data was judged by the spectra and Mascot search results of the bacterial proteins. An online
search engine, Mascot utilizes mass spectrometry data and primary sequence databases to
identify proteins. The program compares experimental masses of peptides or fragment ions
to calculated mass values, incorporating tryptic cleavage. Algorithms sort peptides and
match them to proteins, giving each peptide and protein a score: the greater the score, the
greater the homology of the peptide to the protein, and the greater the confidence in ones
results
Sonication of samples was performed at maximum amplitude with or without glass
beads. The principle behind integrating glass beads in sonication is that the beads would
work in concert with sonication to aid in breaking apart cells. Sonication without glass beads
failed, whereas sonication with glass beads returned reasonable data. However, when
sonicating with glass beads the solution changed to a deep black color. This same occurrence
was observed upon sonication of simply water and glass beads, and it was inferred that the
beads presumably were becoming scorched by the heat of sonication. This result is probably
inconsequential to the outcome of the sample; however, I cannot definitively state it had no
detrimental effect. Casting doubt on the integrity of the lysate, this method was pursued no
further.
Freeze/thaw cycles incorporate immediate freezing to trigger the swift rupture of
cells. Using -80 C freezers, sample pellets were frozen 20 minutes then thawed in a 65 C
water bath; this was repeated 4-5 times. This method did not return acceptable data.
Bead beat homogenization is a means of cell lysis that incorporates minute glass or
silica beads to rupture cells. The premise is that, under violent agitation, the beads act as
projectiles, penetrating the cells and mechanically lysing them.
Bead beat homogenization was the most practical, efficient method of cell lysis for
our purposes. The initial protocol employed bead beat homogenization in 1.5 ml bead beat
tubes. Just enough 0.1 mm beads were used to cover the bottom of the tube, but this quickly
proved to be highly inadequate to achieve proper lysis. I determined, however, that when
filled to about 1/3 of their volume these tubes can provide satisfactory lysis. Aimee Tucker,


39

a graduate student at the University of South Alabama, kindly provided 500 l bead beat
tubes. These tubes operate under a much smaller volume, allowing for a more concentrated
lysate. The amount of beads needed to give superior lysis using this format was found to be
enough to fill about half of this much smaller tube. While either type of tube may be
considered comparable in their effects, the smaller volume tubes were recommended by
Tucker and seemed to yield more consistent results.
Several variables besides those mentioned were tested in effort to improve our bead
beating protocol. These shall be discussed very briefly.
It was speculated that prior freezing would aid in the rupture of the cell and augment
bead beat homogenization. I observed no real benefit to freezing bacterial pellets overnight
and then lysing them.
Bead beating in urea achieved best results compared to bead beating in water or
trifluoroethanol, a solvent used in cell lysis for other mass spectrometry applications.
The effect that the optical density of the culture had on the outcome of the sample is
inconclusive. However, it stands to reason that a greater optical density would mean a larger
pellet and, consequently, a more concentrated lysate.
After homogenization, it is best that the lysate mixture does not cool excessively. For
example, lysates should not be allowed to sit on ice after or during bead beating to prevent
proteolysis, nor centrifuged below room temperature. As the sample cools, proteins will
stick to the glass beads, potentially affecting the outcome of the sample.
Figure 1 gives a general overview of the refinement of cell lysis protocol. Figure 2
depicts the spectra and Mascot search results for two bacterial samples: one that was lysed
according to our optimized method, another which was lysed in trifluoroethanol.
Bacterial protein samples were analyzed using two unique mass spectrometers.
Samples were first evaluated utilizing a Quadropole-Time of Flight mass spectrometer
(QTOF) with electrospray ionization. The QTOF scans and collects mass spectrometry data,
and, when a peptide is detected, rescans and collects sequence data of the peptide.
Upon initial readings using the QTOF mass spectrometer, phloxine B-study samples
were scrutinized using a LTQ-Orbitrap mass spectrometer (Orbitrap). The Orbitrap, a much
more sensitive and faster machine, scans and collects mass spectrometry data and sequences
data simultaneously. These parallel scans are a result of the machines high sensitivity and
resolution. Much more data is collected as compared to the QTOF; and typically proteins
can be detected at 95% confidence from a bacterial prep.
The spectra of each sample were converted to a chromatogram for analysis, and
peptides were sequenced and cataloged using Mascot. Data was searched using the
bacteria species.
Bacterial Identification
It became difficult to make a conclusion about the sub-species and, in some cases, the
species of the individual bacteria. This was caused by the homology of many bacterial
proteins and, given that I was using very heterogeneous cultures, made profiling the bacteria
problematic. With many results, Mascot can assign alternative proteins, and, consequently,
alternative taxa, to which the peptide masses may also correspond. In some cases there can
be twenty or more alternatives. Coupled with the complexity of judging the individual scores
of the fragment ions, results of this undertaking were inconclusive.
After an optimized protocol was developed, I focused my attention on phloxine B studies.



41

Bead beat
homogenization
500 l tube
Minimal bead
amount
Intermediate
bead amount
Beat in 8M
urea
Beat 4 min
Spun 4 C
Spun 25 C Best results
Beat in three
30 sec
intervals
Beat in eight
30 sec
intervals Ice, spun 4 C
No ice, spun
25 C
Beat in H
2
O
Beat in TFE*
Maximum
bead amount
1.5 ml tube
Sonication
Glass beads
Scorched
beads
Compromised
Without glass
beads
Poor results
Freeze/Thaw -80 C Poor results
Bacterial Cell Lysis
Figure 1: General development of cell lysis procedures. An overview of our refinement of a
lysis protocol, this flow chart illustrates the method of cell lysis, as well as, the manipulation of
variables involved. Bead beat homogenization using smaller volumes and 8M urea, while
prohibiting lysates from cooling, yielded consistently better results. While sonication in glass
beads also yielded reasonable data, the beads were scorched, turning the solution black and
compromising the samples integrity. *Trifluoroethanol


42





Figure 2: Comparison of methods. The topmost figures depict the chromatogram and protein search results of a bacterial sample prepared according to our refined
method, while the lower figures those of one that was not. Our optimized method consistently yielded better chromatograms and more proteins with higher scores.
Note the clean peaks and greater number of proteins observed in the sample prepared using our optimized protocol. Compare to the noise found in the other
chromatogram and the single protein observed.
Noise

43


Materials and Methods

Phloxine B solutions
Phloxine B solutions were made by dissolving 0.5 g of phloxine B in 100 ml of 0.7%
aqueous NaCl. For testing, dilutions of this solution were made giving a final concentration
of 100 g/ml. These solutions were syringe filtered through 0.2 m filters into sterile bottles,
wrapped in foil, and stored at room temperature.

Bacterial preparation
Escherichia coli colonies were grown on nutrient agar, an intermediately rich, non-
selective media used in the general culture of microorganisms. Staphylococcus aureus
colonies were grown on mannitol salt agar which is selective for gram-positive Staphylococci
species. Plates were incubated 24 h at 37 C.
Super Optimal Broth (SOB) was used to culture both bacteria. Isolated colonies from
either plate were used to inoculate 20 ml of sterile SOB. Broth cultures were incubated
shaking for 19-24 h at 37 C and 240 RPM (OD, ~1.5). Dilutions were prepared by
inoculating an additional 18 ml of sterile SOB with 2 ml of culture (OD, 0.2-0.3).
Throughout this study, optical densities were measured at 595 nm using a BioTek Synergy 4
microplate reader.

Growth inhibition assay
In testing the photodynamic inhibition of growth, 7 ml of sterile SOB was added to
two dilute cultures. To one culture was added 3 ml of 100 g/ml phloxine B and to the other
3 ml of 0.7% NaCl syringe filtered in 0.2 m filters, which served as a control. To facilitate
uptake of the dye by the bacteria, cultures were incubated covered for 1 h at room
temperature, aerating vigorously at 240 RPM. Cultures were then exposed to four hours of
light under the same conditions using a LumaPro 15 watt indoor fluorescent lamp. Optical
density measurements were taken after incubation in the absence of light and every 45 min
for over 4 h upon exposure to light. This procedure was performed for both bacterial species.

Mass spectrometry preparation
Five milliliter aliquots of bacteria/photosensitizer were taken after incubation covered
and twice every two hours during illumination. These were centrifuged at 5000 RPM.
Supernatants were discarded and pellets were washed three times in 1x phosphate-buffered
saline (PBS). Depending on pellet size, pellets were re-suspended in either 250 l or 500 l
8M urea (urea, 0.5 M ethylenediaminetetraacetic acid [EDTA], 200 mM ammonium
bicarbonate [ABC], 0.5 M tris(2-carboxyethyl)phosphine [TCEP]) and lysed using bead beat
homogenization.
Suspensions were transferred to 500 l bead-beat tubes containing 0.1 m glass beads
and were homogenized continuously for four minutes. Lysates were then centrifuged at
13,200 RPM and 25 C for 15 min to separate intracellular protein from membrane bits and
glass beads. Approximately 100-250 l of supernatant was filtered in 0.2 m cellulose
acetate spin filter tubes at 13,200 RPM for 1 minute to remove any remaining cellular debris


44

that could potentially damage the mass spectrometers. Twenty microliters of filtrate was
added to 60 l of 50 mM ABC/10 mM TCEP and 3 l trypsin in a sterile 1.5 ml eppindorf
tube.
Sample digests were incubated shaking overnight at 37 C and 600 RPM. Following
digestion, samples were centrifuged at 13,200 RPM for 15 min and transferred to mass
spectrometry vials for analysis.
Each of the components of sample preparation play a specific role. Urea and heat
during cell lysis work in tandem to unfold and denature proteins from the globular state and
allow for greater ease of trypsin to access cleavage sites in the sample. Ammonium
bicarbonate serves to buffer the trypsin enzyme at its optimal working pH of 7.8. TCEP is a
powerful reducing agent and is used to break disulfide bonds. Finally, trypsin is a serine
protease produced by the pancreas and secreted into the digestive tract of many vertebrates.
Its function is to cleave proteins at the carboxyl terminus of lysine and arginine residues. In
proteomics, trypsin serves to hydrolyze protein samples into individual polypeptide chains to
be analyzed by mass spectrometry.

Mass spectrometry
MS analysis was performed on our LTQ Orbitrap instrument using triplicate
injections onto an Agilent SB300 C18 column running at 1L/min flow rate. The A solvent
is 0.2% formic acid in water containing 3% AcN and and B is 0.2% formic acid in AcN
containing 3% water. The gradient runs from 5% B (95% A) to 40% B over 80 minutes and
then to 90% B over the next 20 minutes. The total run time is 120 minutes and an injector
wash and 2 blanks are run between samples to monitor and ensure there is no carryover. The
total per sample cycle time is 8 hours.
The MS system acquires MS (peptide m/z data) in the Orbitrap at 60,000 resolution
while acquiring up to 5 MS/MS (peptide sequence m/z data) in the LTQ. An exclusion list is
maintained to ensure that the maximum number of peptides is sampled by MS/MS. The
cycle time for each scan group is approximately 1.2 seconds and data is acquired by the
Thermo Xcalibur software. At the end of each acquisition, data is automatically archived to
a second site.
Data from the acquisitions was turned into a search file for use with the Mascot
search engine (http://matrixscience.com). Each set of three runs was condensed into a single
search file that was searched against the NCBI RefSeq database. In searching E. coli, the
sub-species used was Escherichia coli 0157:H7 EDL933. Conversely, when searching S.
aureus, the species used was Staphylococcus aureus subspecies aureus Mu50.

Results

Growth Inhibition Assay
Growth inhibition studies showed that E. coli was much less susceptible to
photodynamic inhibition with phloxine B than was S. aureus (Figure 3). This agreed with
my hypothesis, in that, the gram-negative E. coli showed no inhibited growth when compared
with a non-treated control culture. Treated and control E. coli growth was practically
identical.


45

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300
A
b
s
o
r
b
a
n
c
e
,

5
9
5

n
m
Minutes
Escherichia coli growth
Control
Treated
Staphylococcus aureus, on the other hand, demonstrated an extreme sensitivity to
photodynamic inhibition (Figure 4). Growth was inhibited within 45 minutes upon exposure
to light, and optical density remained consistently at ~0.4 throughout the
remainder of the experiment. These observations fit appropriately with what is known about
gram-positive bacteria being more vulnerable to photodynamic challenges.























Figure 3: Escherichia coli growth over 4 h. Optical density measured every 45 minutes after
incubation 1h covered. There is no difference in the rate of growth of the control culture and the
culture treated with 100 g/ml phloxine B. E. coli is highly resistant to photodynamic inhibition
using photosensitizer alone, due to the thick outer membrane of gram-negative bacteria.
Figure 4: Staphylococcus aureus growth over 4 h. Optical density measured every 45 minutes after
incubation 1h covered. Treatment with phloxine B dramatically retards the growth of S. aureus
within 45 min of exposure to light. Growth is inhibited and optical density halts at ~0.4.
Staphylococcus aureus, a gram-positive bacterium, is highly susceptible to photodynamic inhibition
of growth using phloxine B.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 50 100 150 200 250 300
A
b
s
o
r
b
a
n
c
e
,

5
9
5

n
m
Minutes
Staphylococcus aureus growth
Control
Treated


46

Proteomic Analysis
In comparing the proteomes of the two species incubated covered 1 h and exposed to
light 2 h and 4 h, the amount of data collected was quite tremendous. Hundreds of proteins
were detected for either species and these were compared for protein position (rank in the
catalog of detected proteins) and protein score. While data analysis is still in progress, trends
in protein expression were observed.
In E. coli, no obvious changes in protein expression between the control and treated
cultures were observed. Many proteins seemed to follow the same basic trend shown in
Figure 5, in which protein expression remained quite comparable. It appears that E. coli does
not undergo very conspicuous proteomic changes under photodynamic treatment with
phloxine B.
Protein expression in Staphylococcus aureus, on the other hand, appeared to be highly
influenced by photodynamic treatment in some cases. Figures 6 and 7 illustrate these
observations. Some proteins were observed to be up-regulated in response to photodynamic
stress. Still others seemed down-regulated, depending presumably on the function of the
protein. Other proteins did not reflect these types of trends, however, and protein expression
in the treated culture seemed to remain comparable to the control culture, as was observed in
E. coli.
Additionally, initial searches using oxidative modifications yielded many oxidized
residues in S. aureus, particularly, methionine residues. This may be a result of an
interaction between the ROS produced by phloxine B and the bacterial proteins. It is unclear
why it appears to be methionine being oxidized.







0
50
100
150
200
250
300
350
400
450
0 2 4
P
r
o
t
e
i
n

S
c
o
r
e
Elapsed Time, hours
Escherichia coli: Serine Hydroxymethyl
Transferase expression
control
treated
Figure 5: Protein expression in Escherichia coli. This graph illustrates a trend observed in many of the
proteins detected in E. coli, not just serine hydroxymethyl transferase. There were no obvious changes in
protein expression levels, and many proteins presented comparable score between the treated and control
cultures of E. coli.


47

0
10
20
30
40
50
60
70
80
0 2 4
P
r
o
t
e
i
n

S
c
o
r
e
Elapsed Time, hours
Staphylococcus aureus: DNA Pol III
expression
Control
Treated












Figure 6: Down-regulation of protein in treated Staphylococcus aureus. Obvious changes in protein
expression were observed in S. aureus. In the case of DNA polymerase III subunit expression, this protein
was increasingly down-regulated over time upon exposure to light. Presumably because, treated S. aureus
cells are actively responding to oxidative stress and do not have the energy to devote to reproduction.
Figure 7: Up-regulation of protein in treated Staphylococcus aureus. Cysteine synthase appeared to be up-
regulated over time upon exposure to light and, conversely, down-regulated in the control. Cysteine
synthase has been implicated in the oxidative stress response and thiol redox balance of Staphylococcus
aureus.
0
50
100
150
200
250
0 2 4
P
r
o
t
e
i
n

S
c
o
r
e
Elapsed Time, hours
Staphylococcus aureus: Cysteine Synthase
expression
Control
Treated


48

Conclusion

In accordance with my hypothesis, Escherichia coli cultures were indeed very
resistant to photodynamic inhibition with phloxine B. No obvious changes in protein
expression were observed, with many treated-culture proteins exhibiting the same behavior
as control-culture proteins. Staphylococcus aureus is very sensitive to photodynamic
inhibition with phloxine B, and protein expression can be affected upon exposure to
fluorescent light in the presence of the photosensitizer. A massive amount of data was
collected for either bacterial species. Data analysis is still in progress, and further
examination of protein expression and modification between control and treated cultures for
each species should be conducted.

Acknowledgements

I would like to thank Lewis Pannell and the other members of the Pannell lab for this
wonderful opportunity and for their profound guidance and patience in helping me to
accomplish this project. I would also like to thank Dr. Julio Turrens for his leadership of the
NSF/REU program and for teaching me many valuable things. A special thanks to Dustin
Ingram for providing materials and assistance essential to the phloxine B studies. And
finally, I would like to thank the National Science Foundation and the University of South
Alabama for making this experience possible to undergraduates like myself.

References

1. Abdulamir, A.S., Hafidh, R.R. and Bakar, F.A. 2011. The association of
Streptococcus bovis/gallolyticus with colorectal tumors: The nature and the underlying
mechanisms of its etiological role. J Experimental Clinical Cancer Research 30:11.

2. Demidova, T.N. and Hamblin, M.R. 2005. Photodynamic inactivation of Bacillus
spores, mediated by phenothiazinium dyes. Appl and Environ Microbiol 71:6918-6925.

3. Embleton, M.L., Nair, S.P., Heywood, W., Menon, D.C., Cookson, B.D. and Wilson,
M. 2005. Development of a novel targeting system for lethal photosensitization of
antibiotic-resistant strains of Staphylococcus aureus. Antimicrob Agents Chemother
49:3690-3696.

4. Gad, F., Zahra, T., Hasan, T. and Hamblin, M.R. 2004. Effects of growth phase and
extracellular slime on photodynamic inactivation of gram-positive pathogenic bacteria.
Antimicrob Agents Chemother 48:2173-2178.

5. Huycke, M.M. and Gaskins, H.R. 2004. Commensal bacteria, redox stress, and
colorectal cancer: mechanisms and models. Exp Biol Med 229:586-597.

6. Konopka, K. and Goslinski, T. 2007. Photodynamic therapy in dentistry. J Dent Res
86:694-707.



49

7. Rasooly, A. and Weisz, A. 2002. In vitro activities of phloxine B and other halogenated
fluoresceins against Methicillin-Resistant Staphylococcus aureus. Antimicrob Agents
Chemother 46:3650-3653.

8. Scanlan, P.D., Shanahan, F., Clune, Y., Collins, J.K., OSullivan, G.C., ORiordan,
E.H., Wang, Y. and Marchesi, J.R. 2008. Culture-independent analysis of the gut
microbiota in colorectal cancer and polyposis. Environmental Microbiology 10:789-798.

9. Sobhani, I., Tap, J., Roudot-Thoraval, F., Roperch, J.P., Letulle, S., Langella, P.,
Corthier, G., Nhieu, J.T.V. and Furet, J.P. 2011. Microbial dysbiosis in colorectal
cancer (CRC) patients. PLoS ONE 6:e16393.doi:10.1371/journal.pone.0016393.

10. Wainwright, M. 1998. Photodynamic antimicrobial chemotherapy. J Antimicrob
Chemo 42:13-28.

11. Zhang, M., Cheng, J., Xia, L., Lu, Y. and Wu, X. 2006. Monitoring intestinal
microbiota profile: A promising method for the ultra early detection of colorectal cancer.
Medical Hypotheses 76:670-672.




50




51






Correlation of Mitochondrial Membrane Potential
and Velocity in Endothelial Cells








Liriany Y. Pimentel
Bryn Mawr College









Research Mentor: Dr. Abu-Bakr Al-Mehdi
Department of Pharmacology


52

Abstract

Previous studies have shown that mitochondrial movement is not random. When
mitochondria move, they are able to create signaling microdomains in the cell. Membrane
potential is not the same in every mitochondrion. It is known that mitochondria are
hyperpolarized when there is an abundance of protons in the intermembrane space that
facilitates ATP generation. Depolarized mitochondria are considered to be metabolically
inactive. Considering the presence of significant variation in mitochondrial velocity and
their membrane potential, we asked the following question: Is there a relationship between
mitochondrial membrane potential and velocity? Cultured rat pulmonary microvascular cells
were stained with JC-1, a mitochondrial potential-sensitive probe, and a fluorescence time-
lapse imaging of mitochondria was performed. Velocities of hyperpolarized and
depolarized mitochondria were calculated. Our results show that depolarized mitochondria
had faster average velocity compared with hyperpolarized mitochondria (0.0540.002 m/s
vs. 0.0470.002 m/s; P<0.05; n=65). This suggests an important role of mitochondrial
membrane potential in the regulation of the mitochondrial transport process.

Introduction

Eukaryotic cells are known to contain an organelle considered as the power house
of the cell, also known as mitochondria. In contrast to other organelles, mitochondria
maintain their own DNA and demonstrate significant motility. Previous studies have shown
that mitochondria do not move randomly. They depend on certain conditions and stimuli to
move to specific locations within the cell. For example, perinuclear clustering of
mitochondria was seen in cells after transfection with herpes simplex virus (Murata 2000).
In pancreatic acinar cells, mitochondria were localized in perigranular, subplasmalemmal,
and perinuclear areas (Johnson 2003). Mitochondria translocate in the oocyte during
fertilization to cluster around the pronuclei and can remain in a perinuclear pattern during
embryo development (Bavister 2006; Katayama 2006; Tarazona 2006). In order to
understand phenomena like these, it is essential that the mechanism of their movement be
studied. Considering the heterogeneity of membrane potential of mitochondria, we asked
the following question: Is mitochondrial velocity related to their membrane potential?
The cytoskeletal element microtubule serves as the track that mitochondria use to
move around with the help of motor proteins kinesin and dynein. There are approximately
two microtubule-organizing centers (MTOC) in each cell. These centers control the number
of microtubules formed, their location, and orientation throughout the cell (Alberts 2010, p.
579). These microtubules grow out of the y-tubulin ring complex within the MTOC, also
known as the minus end, and grow through the plus end outward throughout the periphery of
the cell (Alberts 2010, p. 582). They then serve as the tracks for mitochondria to move
around with the help of motor proteins.
Mitochondria move with the help of two motor proteins - kinesin and dynein (Alberts
2010, p. 583). These motor proteins use the energy produced during ATP hydrolysis to
move in a single direction. Kinesins move toward the plus end, and dyneins move toward the
minus end (Alberts 2010, p. 583). They both possess globular heads that act as enzymes in
order to walk along the microtubules, and tails that connect to a cargo, such as a
mitochondrion (Alberts 2010, p. 584).


53

When a mitochondrion moves, it is able to create signaling microdomains. These
microdomains can be enriched with ATP, calcium, or reactive oxygen species (ROS). ATP
is the energy currency of the cell produced most efficiently as a result of the activity of the
electron transport chain. This energy is essential for a cell to perform all of its functions.
Mitochondria are able to buffer calcium in the cell. Mitochondrial electron transport chain
can potentially leak single electrons to oxygen and convert it into superoxide anion, a
progenitor ROS (Andreyev 2005). Too much ROS may cause cellular damage and death.
These mitochondrial functions are in part regulated by its membrane potential.
All mitochondria differ in membrane electrical potential, where its matrix is negative
and intermembrane space is positive. This charge difference results primarily from the
activity of the electron transport chain, which pumps out protons from the matrix into the
intermembrane space. In the electron transport chain, the energy released by the electrons,
donated by NADH and succinate, is used to pump out H
+
, from the matrix into the
intermembrane space. Strong electrical gradient in hyperpolarized mitochondria drives the
movement of H
+
through the ATPase back into the matrix to help the production of ATP.
Therefore, hyperpolarized mitochondria have more potential to generate ATP than
depolarized mitochondria. In any given cell, a wide distribution of mitochondrial membrane
potential can be observed. In this research we have distinguished two categories of
membrane potentials. Hyperpolarized mitochondria are ones to exhibit a high active electron
transport chain, therefore they are able to pump out more protons and generate an abundance
of ATP. Depolarized mitochondria are considered to possess a less active electron transport
chain, not pump out as many protons, and not produce sufficient ATP.
Mitochondrial membrane potential plays an important role in cellular functions such
as import and export of adenine nucleotides, regulation of apoptosis (physiological cell
death), and generation of reactive oxygen species. Due to the mitochondrial membrane
potentials ability to regulate these cellular and mitochondrial properties, it could be a
possibility that it could also regulate aspects of mitochondrial movement.
Hyperpolarized mitochondria are theoretically predisposed to generating more ATP.
If there are some mitochondria that are capable of having a more negative membrane
potential, they are also capable of producing higher amounts of ATP by pumping out more
protons. If the mitochondrion were producing more ATP, it would only be likely that it will
move faster. The faster movement will be favorable because motor proteins use the energy
produced by mitochondria to move along the microtubule tracks. Thus, we hypothesized that
mitochondria with more negative membrane potential possess greater movement in terms of
velocity.

Materials

Microvascular Endothelial Cells (Rat)
These microvascular cells were procured from the Cell Culture Core three to four
days before the expected experiment. The cells are prepared in the Core lab, where
specialized lab technicians scrape cells out of the rats lung lining, and through many
experiments segregate the endothelial cells. Once retrieved, the lab technicians prepare them
for experimental use in other labs.




54

JC-1
JC-1 is a cationic carbocyanine membrane-permeant dye that is taken up my
mitochondria and emits green fluorescence (FITC channel). In hyperpolarized mitochondria,
the dye is taken up at higher concentrations (>1 M) that causes its aggregation (de Proost,
2008, sec. 2.4). At concentrations that are higher than 1 M, the dye emits a red
fluorescence (TRITC channel).

Krebs Buffer
Krebs Buffer is a salt solution that is used as experimental medium for endothelial
cells. 10 mM d-glucose is added as a nutrient and 5% dextran is added as an osmotic agent.
Krebs Buffer is made by putting 4 L of double-distilled H
2
O in a beaker or flask with
a stir bar inside. The beaker or flask is placed on the stir plate with no heating. 0.18 g of D-
glucose and 5.00 g of Dextran for every 100 mL is added to the 4 L of DD H
2
O. Once
added, the ingredients are left stirring on the stirrer plate to dissolve, and then filtered and
refrigerated. The pH is adjusted to 7.4 prior to each use.

Nikon TE2000 Inverted Fluorescence Microscope
The Nikon TE2000 Inverted Fluorescence Microscope is an inverted microscope used
to view live cell imaging. It is equipped with two light sources and three glass filters that
specialize in allowing particular wavelengths to excite the fluorescent probe JC-1 within the
mitochondria. The excitation and emission wavelengths depend on the filter channel being
used. Once the molecules are excited, the fluorescence microscope then takes a picture of its
emitted light. Special cover-glass bottomed petri dishes were used to take pictures with a
60x oil-immersion objective.
The microscope is equipped with hardware and Metamorph software for image
acquisition, processing, and analysis. In this experiment these three functions were utilized
under two specific channels: TRITC and FITC. TRITC channel is able to detect
hyperpolarized mitochondria. These two channels are distinguishable under the fluorescence
microscope by using separate filter cubes allowing the TRITC channel to appear as red, and
FITC channel as green.

Incubator and Hot Water Bath
The incubator is heated at a constant 37 C with 5% of CO
2.
This incubator is used to
culture cells.
The hot water bath is also heated at a constant 37 C. The hot water bath is used to
warm up some materials being used in experiments, such as Krebs Buffer, and DMEM
media.

Methods

Sterilization Method
In order to maintain a sterilized environment for all of the materials used, especially
the cells, gloves were worn at all times. The gloves, as well as every area that was used for
the experiments, were sprayed with ethanol. The bottom of the cell dishes and every material
that was transferred from one place to another, was cleansed with ethanol to maintain
sterility. The hood, where all of the experiments were conducted, was cleansed before and


55

after each use. In general, everything had to be cleansed with ethanol before and after each
usage.

Rat Microvascular Endothelial Cells Preparation
The Microvascular Endothelial Cells also known as MV cells, were ordered three to
four days before usage. Once ready, the MV cells were looked at under a compound light
microscope for confluence. If they appeared to be sub-confluent, then they were available
for an experiment that same day. If not, the MV cells were put back into the incubator for
one more day. To expedite their growth, the media was changed.
For a media change, the DMEM media was taken out of the refrigerator and warmed
in a hot water bath at 37C for approximately 15 minutes. After it reached 37C, it was taken
into the hood along with the dish that needed a media change. Both, media and cell dish,
were cleansed with ethanol before being placed inside the hood.
The cell dishs top was taken off and placed facing up. The original media the cells
had was aspirated, and then 2-3 mL of DMEM media was transferred into the dish. Once the
new media was transferred in, the cell dish was closed with its top, cleansed with ethanol,
and placed back in the incubator until the next day.

Mitochondrial Staining Procedure with JC-1
Once the MV cells looked ready for the experiment they were placed back in the
incubator. The Krebs buffer, in a beaker covered with parafilm, was taken out of the
refrigerator and warmed in the hot water bath for approximately 15 minutes. After the Krebs
buffer was warm, it was taken to measure its pH.
When measuring the pH of the Krebs buffer, the beaker containing the Krebs buffer
was placed in a stir plate with a stirrer inside the beaker, at a level four. While stirring, the
pH stick was washed with H
2
O and then placed inside the beaker with the least amount of
space opened to air. Depending what the pH measured, the appropriate acid or base was
used; HCl was used when above 7.4, and NaOH was used when below 7.4. The acid or base
was added in increments starting at 1.0 L and then lowered as the pH slowly reached 7.4.
Once at 7.4, the pH stick was taken out of the beaker, washed with H
2
O, and placed back into
its original holder. The stir plate was stopped, and the beaker containing Krebs buffer was
taken back to the lab, cleansed with ethanol, and placed inside the hood.
The rest of the materials needed for the experiment were then collected and placed
inside the hood as well. JC-1 dye was taken out of the freezer, and covered quickly with
aluminum foil due to its light sensitivity. Before placing the JC-1 dye in the hood it was
normally shaken by a shaker to make sure the particles didnt stay at the bottom of the tube.
A 15 mL Falcon tube, a 0.5 100 L pipette, and the dish of MV cells were all collected,
cleansed with ethanol, and placed inside the hood.
3 mL of Krebs buffer and 0.60 L of JC-1 were added to the 15 mL Falcon tube,
closed with cap, and shook. The media in the MV cell dish was aspirated and the mixture in
the 15 mL Falcon tube of Krebs buffer and JC-1 was transferred into the MV cell dish. The
dish was closed with its lid, cleansed with ethanol, and placed in the incubator for 30
minutes.
After 30 minutes, the MV cells dish was taken out of the incubator, cleansed with
ethanol, and placed inside the hood. The mixture of Krebs buffer and JC-1 it contained was
aspirated. 1 mL of Krebs buffer was transferred into the dish as a wash and aspirated after. 3


56

mL of Krebs buffer was transferred into the dish and left inside the dish. The dish was then
closed with its lid, cleansed with ethanol, and taken to the Fluorescence Microscope.
The rest of the Krebs cycle was placed back into the refrigerator, the JC-1 was placed
back in the freezer, and the used pipette tips and tubes were placed in a Biohazard container.

Fluorescence Time-lapse Imaging of Mitochondria
Before being able to actually see the cells through the fluorescence microscope, there
were many important steps that needed to be done in order to get a proper view. Before
placing anything on the fluorescence microscope, it was vital that the lens be cleansed with
residual oil remover. This cleaner was also used to clean the bottom of the dish before
placing it in the chamber. After cleaning the lens, a drop of lens oil was dropped on the eye
of the lens. Once this was done, the chamber was placed on the fluorescence microscope but
only after it was well equipped.
The chamber is where the cell dish is placed in order to properly move the dish and
be able to see all of the cells the dish contains through the fluorescence microscope. The
chamber has an indented circular area in the center that is ideal for a 35mm dish placement.
The dish, after having its bottom cleansed, was placed there and locked by the tightening of
the two silver metals on each end. Water was then added to the rim of the chamber, a proper
indented outline was visible for this purpose. Once the water and dish are added, the
chamber was then closed, and placed on the fluorescence microscope. Once there, the CO
2

tube was connected to the chamber.
After the chamber was properly located, the lens was maneuvered and the focus was
adjusted for proper view. Once a good view was found, finding a good group of cells was
the next step. Once the cells were found, pictures were taken under the two channels used to
see the JC-1 dye fluorescence: TRITC and FITC.
Metamorphs Multidimensional time-lapse was used to take the pictures. This system
specifically facilitates the use of taking pictures under multiple channels at the same time
within a desired time length. The time used for both channels was every 5 seconds for 20
minutes, providing a total of 241 pictures for each channel. During the time-lapse, it was
crucial to check when the fluorescence microscope lost focus because it was important to
make sure that the pictures were as clear as possible.
Once the pictures were taken, they were saved for further analysis. The chamber was
taken off of the fluorescence microscope. The CO
2
was detached, and the dish was taken off
of the chamber. The fluorescence microscope lens were cleansed once more and turned off.
The Krebs buffer on the dish was discarded and the dish was thrown away in the proper
waste bin.

Analyzing Data
Once the time-lapse was done, the pictures for TRITC and FITC were separated with
the use of Multidimensional Data in the Metamorph Software. This self-automated system
separated the best-focused pictures one channel at a time. It then created a movie, which
were back-to-back pictures of the same channel within a time-lapse.
After retrieving independent movies for each channel of each time-lapse for a dish,
the movies were transferred to another computer. At this computer Metamorph, Excel, and
PowerPoint were used for further analysis. The Track Points fucntion of Metamorph was
used to track five mitochondrion in each channel for each movie. In this case, each


57

mitochondrion was tracked within a range of 100 pictures that appeared the most focused,
like shown in Figure 1. For consistency purposes, the majority of these pictures were the
first 100 of each movie. After tracking, Metamorph automatically calculated the
mitochondrial velocities for each of the 100 pictures that were tracked. Metamorph also
provided a graph to show the velocities of the mitochondrion during the 100 pictures, like
shown in Figure 2. The graph and a picture of the tracking done in Metamorph were saved in
PowerPoint. Therefore, each mitochondrion had its own velocities, and everything was
saved for future use.



Figure 1 represents how a mitochondrion's movement looks after being tracked through 100 pictures.




Figure 2 represents how the velocity calculated for one mitochondrion looks like when graphed.
After tracking and saving velocities, averaged velocities for each mitochondrion were
calculated using Excel. Once the average for each mitochondrion were known, an overall
average was calculated for each channel. Both channels, which represented both types of
membrane potentials, were then compared with graphs.
The fluorescence microscope was able to also help detect interesting facts about
depolarized and hyperpolarized mitochondria. One of the first things detected was that not
all mitochondria were hyperpolarized. Therefore, as seen in Figure 3, some cells that were
visible under the FITC channel werent in the TRITC channel.


58




Figure 3 represents how some cells that are visible under the FITC channel, depolarization detector, are not visible
in the TRITC channel, hyperpolarization detector.
It was also identified that the majority of depolarized mitochondria tend to retain the
fluorescence dye for longer periods when compared to hyperpolarized mitochondria, as
shown in Figures 4A, 4B, 5A, and 5B.


4A 4B
Figures 4A and 4B Figure 4A is the first picture under the FITC channel of the first 100 pictures analyzed. Figure
4B is the 100th picture.

5A 5B
Figure 5A and 5B Figure 5A is the first picture under the TRITC channel of the first 100 pictures analyzed. Figure
5B is the 100th picture.


59

After taking additional pictures of these fascinating facts, both channels, which
represented both types of membrane potentials, were then compared with graphs using the
calculations previously found.

Statistical Analysis
Bar graphs for each, hyperpolarized and depolarized, were created through Sigma
Plot Software. A total of 8 bins were used to view if there was any relationship to the
Gaussian or normal distribution, but also most importantly to detect which membrane
potential contained a faster velocity.


6A 6B
Figures 6A and 6B Figure 6A on the left represents the average velocities of depolarized mitochondria. Figure 6B
on the right represents the velocities for hyperpolarized mitochondria.
8-bin bar graphs were also done for the intensities of each, hyperpolarized and
depolarized.


7A 7B
Figure 7A and 7B Figure 7A on the left represents the intensities for depolarized mitochondria. Figure 7B to the
right represents the intensities for hyperpolarized mitochondria.
Depolarized
Average Velocity, microm/s
0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
N
u
m
b
e
r

o
f

M
i
t
o
c
h
o
n
d
r
i
a
0
5
10
15
20
Depolarized
Intensity, AFU
20 40 60 80 100 120
N
u
m
b
e
r

o
f

M
i
t
o
c
h
o
n
d
r
i
a
0
5
10
15
20
Count
Hyperpolarized
Intensity, AFU
40 60 80 100 120 140 160 180 200 220
N
u
m
b
e
r

o
f

M
i
t
o
c
h
o
n
d
r
i
a
0
5
10
15
20
25
30
Count
Hyperpolarized
Average Velocity, microm/s
0.02 0.04 0.06 0.08 0.10
N
u
m
b
e
r

o
f

M
i
t
o
c
h
o
n
d
r
i
a
0
2
4
6
8
10
12
14
16
18


60

After analyzing them independently, another bar graph was done using both
hyperpolarized and depolarized data in order to compare and detect any correlation amongst
both.
Results

Depolarized mitochondria move faster than hyperpolarized mitochondria.
Average velocities were calculated for hyperpolarized and depolarized mitochondria
separately. Our results show that depolarized mitochondria had faster average velocity
compared with hyperpolarized mitochondria (0.0540.002 m/s vs. 0.0470.002 m/s;
P<0.05; n=65; Fig. 6.).



Figure 8 represents the Velocity vs. Membrane Potential graph that shows how depolarized mitochondria have a
faster velocity.

Membrane potential and velocity show no relationship within each group of
depolarized and hyperpolarized mitochondria.
With the help of the Sigma Plot Software, the depolarized mitochondria intensities
and velocities were compared to one another. The same was done for the hyperpolarized
mitochondria, as shown in these graphs:

A
v
e
r
a
g
e

v
e
l
o
c
i
t
y
,

m
/
s
0.00
0.01
0.02
0.03
0.04
0.05
0.06


61


9A 9B
Figure 9A and 9B Figure 9A represents the Velocity vs. Intensity of depolarized mitochondria with the best-fit
regression line. Figure 9B represents the same information but for the hyperpolarized mitochondria.
Based on these graphs, it was concluded that although the depolarized mitochondria
moved faster than hyperpolarized mitochondria as a group, there were no direct correlation
between intensity and velocity within each group. Intensity is directly related to membrane
potential, thus this information also states that membrane potential and velocity do not
depend on each other within each group. This contrasts with the finding in Figures 9A and
9B where there is an inverse relationship between groups of depolarized and hyperpolarized
mitochondria.

Depolarized and hyperpolarized mitochondria share similar fluorescence intensity
distribution.
After analyzing the depolarized and hyperpolarized intensity bar graphs (Figures 7A
and 7B), it was concluded that both demonstrate how the majority of the mitochondria tend
to have less intensity within each group. Consequently, this skews both graphs toward the
left, also showing how there are few mitochondria that exhibit high intensities or high
membrane potentials.

Discussion

The mitochondrial characteristics of membrane potential and movement in terms of
velocity in this experiment proved that depolarized mitochondria have an overall faster
velocity when compared to hyperpolarized mitochondria. Depolarized mitochondria being
faster than hyperpolarized mitochondria could be due to many things, but one possible reason
could be their differential attachment to motor proteins. It is known that dynein tends to
move faster when compared to kinesin. It could then be that depolarized mitochondria, being
less electronegative when compared to hyperpolarized ones, is attracted to dynein and
therefore moves faster than hyperpolarized mitochondria.
Depolarized mitochondria obtaining an average faster velocity could also be due to
their inactive use within the cells. If it is thought that hyperpolarized mitochondria are
defined as the ones working and depolarized mitochondria as the ones not in use, then what
could depolarized mitochondria possibly be doing? It was visibly obvious that depolarized
mitochondria outnumbered hyperpolarized mitochondria within the cells. When there is a
big population of, in this case depolarized mitochondria, it is important that they each move a
Depolarized Velocity vs Intensity
Intensity, AFU
0 20 40 60 80 100 120 140
A
v
e
r
a
g
e

V
e
l
o
c
i
t
y
,

m
i
c
r
o
m
/
s
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
Intensity (AFU) vs V(m/sec)
x column 1 vs y column 1
Hyperpolarized Velocity vs Intensity
Intensity, AFU
20 40 60 80 100 120 140 160 180 200 220 240
A
v
e
r
a
g
e

V
e
l
o
c
i
t
y
,

m
i
c
r
o
m
/
s
0.00
0.02
0.04
0.06
0.08
0.10
0.12
hyper. intensity vs Col 4
x column 2 vs y column 2


62

specific way in order to keep the cell functioning properly by not intervening with the other
active organelles. Thus, making sure that these hyperpolarized mitochondria get to their
energy-needed sites is crucial. But, in order to make sure that these active mitochondria
complete their duties, it is important that they do not get disturbed while working. Having an
excessive amount of depolarized mitochondria could increase the chances of disturbance if
their movements arent controlled. A way they could be controlled is by having specific
velocities that do not intrude with the varying velocities of those working hyperpolarized
mitochondria. Additionally, it only makes sense for these depolarized mitochondria to retain
a velocity that is similar amongst all, where no matter the velocity of the hyperpolarized
mitochondrion, they are able to not interfere with its path. This point could also further
explain why in their individual velocity graphs (Figures 6A and 6B), depolarized
mitochondria exhibited a closer relationship to the Gaussian or normal distribution and a
slightly tighter range when compared to the hyperpolarized velocity graph.
Size and shape of mitochondria could also be a factor that contributes to depolarized
mitochondria being faster. In research done on organelle transport velocity measurements, it
was found that mitochondria with more oxidized thiol redox status have lower membrane
potentials and are smaller in size (Gerencser and Nicholls 2008). This could not only
introduce that fact that depolarized mitochondria might be smaller in size but also that they
produce more oxidants than hyperpolarized mitochondria. Depolarized mitochondria
obtaining small sizes can directly correlate with speed because the less mass they have, the
smaller they are, the more options they have to move, and the more likely they are of having
faster velocities. In contrast to hyperpolarized mitochondria, where if they were to highly
vary in size then they are limited to the places they can move, which could also explain why
their velocities ranged more. This thought directly connects to the previous idea of
depolarized mitochondria obtaining similar velocities due their excess in amount within the
cell. If they are able to have more space, a tighter velocity range will be necessary in order to
control interference with other active organelles. Being smaller in size and having a more
controlled velocity range, could be beneficial for active mitochondria, whose size range may
be bigger or smaller than inactive mitochondria; and therefore, space would be crucial to
have for the variety of pathways they move through.
Depolarized mitochondria being able to produce more oxidants could then explain
why they are not in use. Energy is something that cells highly depend on to function in all
sorts of ways. In most differentiated animal cells polarization is a reflection of the polarized
systems of microtubules in its interior, which help position organelles within the cell and
guide streams of traffic between one part of the cell to another (Alberts 2010, pg. 582). In
order for this to traffic of streams to occur in mammalian cells, energy must be needed.
Therefore, the active mitochondria could be used in mammalian cells for that traffic and
energy transportation, while depolarized mitochondria might not be needed in any site. If
they arent needed in any site, then besides being able to move wherever they desire, they
could be generating more of something besides energy; that could be oxidants. Since they
arent producing as much ATP, their electron transport chain could be producing more
oxidants

instead. Depolarized mitochondria not being in use could be the reason for this
inversely correlated result amongst membrane potential and velocities.
Unlike depolarized mitochondria, hyperpolarized mitochondria are active. As
previously stated, hyperpolarized mitochondria are not only used for energy production, but
they also offer other services to places where needed. This point could be the reason why


63

their membrane potentials are inversely correlated with their velocities as well. If
hyperpolarized mitochondria depend on these sites, then this could further explain why active
mitochondria have a more varying range of velocities when compared to depolarized
mitochondria. Their slower velocities may facilitate longer residence times that in turn
would provide opportunity for sustained delivery of ATP, calcium, and ROS.
In conclusion, the fact that depolarized mitochondria move faster than hyperpolarized
mitochondria opens up the door for future research that can hopefully help understand the
mechanism of mitochondrial movement.

Acknowledgements

I want to thank Dr. Abu-Bakr Al-Mehdi for giving me the awesome opportunity of
becoming involved in this program, especially his lab. Thank you so much for guiding,
supporting, and instructing me throughout this program. I dont think my progress and
knowledge would have excelled if it werent for your dedication and time.
I want to thank Dr. Julio Turrens for making this program possible. Every aspect of it
was full of enriched and essential information that will come in handy later on in life. I want
to thank you for your warm welcome, and for making sure that everything was working well
and that we were doing well throughout these ten weeks.
I want to thank Mita Patel for the time you took to train me in Dr. Al-Mehdis lab. I
dont know what I wouldve done without you and your amazing help throughout this
project. I really appreciate you teaching me and being there, always, whenever I needed
help.
This also goes to Jamie Hill. Thank you so much for making sure I was on the right
track when it came to my PowerPoint and data analysis. I really appreciate the time you took
to help me out and make sure I was doing OK. Thank you for allowing me to see how life
would be as a graduate student, and for sharing your insights on what to do when my time
comes.
I want to thank Dr. Mark N. Gillespie for supporting and guiding me as well. I really
appreciate the care and time you took to show, explain, and converse with me my project and
future plans. It was really helpful having you around whenever I needed help, and taking
part in your famous Friday meetings.
Lastly, I want to thank Dr. Gillespies lab for being supportive of my work. I really
appreciate the time they took to see and judge my presentations.




64

References

1. Alberts, B.; Bray, D.; et al. 2010. Essential Cell Biology Third Edition. Garland
Science, Taylor and Francis Group: New York and United Kingdom 579-584.

2. Andreyev, A. Yu., et al. 2005. Mitochondrial metabolism on reactive oxygen species.
Biochemistry (Mosc) 70(2):200-214, Original Russian Text Copyright. Retrieved July 23,
2011, from http://protein.bio.msu.su/biokhimiya/contents/v70/pdf/bcm_0200.pdf

3. Bavister, B.D. 2006. The mitochondrial contribution to stem cell biology. Reprod Fertil
Dev 18(8):829-838.

4. Dash, P. (n.d.), Role of Mitochondria in Apoptosis. In Reproductive Cardiovascular
Research Group. Retrieved, July 23, 2011, from
http://www.sgul.ac.uk/depts/immunology/~dash/apoptosis/mito.htm

5. De Proost, I. et al. (n.d.), Studying Mitochondrial Health. In JC-1 Details. Retrieved
July 20, 2011, from http://www.invitrogen.com

6. Gerencser, A.A. and Nicholls, D.G. 2008. Measurement of instantaneous velocity
vectors of organelle transport: mitochondrial transport and bioenergetics in hippocampal
neurons. Biophys J 95(6):3079-3099.

7. Johnson, P.R., Dolman, N.J., Pope, M., Vaillant, C., Petersen, O.H., Tepikin, A.V.
and Erdemli, G. 2003. Non-uniform distribution of mitochondria in pancreatic acinar
cells. Cell Tissue Res 313(1):37-45.

8. Katayama, M., Zhong, Z., Lai, L., Sutovsky, P., Prather, R.S. and Schatten, H. 2006.
Mitochondrial distribution and microtubule organization in fertilized and cloned porcine
embryos: implications for developmental potential. Dev Biol 299(1):206-220.

9. Murata, T., Goshima, F., Daikoku, T., Inagaki-Ohara, K., Takakuwa, H. Kato, K.
and Nishiyama, Y. 2000. Mitochondrial distribution and function in herpes simplex
virus-infected cells. J Gen Virol 81(Pt 2):401-406.

10. Safiulina, D., Veksler, V., et al. 2006. Loss of mitochondrial membrane potential is
associated with increase in mitochondrial volume: physiological role in neurones. J Cell
Physiol 206(2):347-353.

11. Tarazona, A.M., Rodriquez, J.I., Restrepo, L.F. and Olivera-Angel, M. 2006.
Mitochondrial activity, distribution and segregation in bovine oocytes and in embryos
produced in vitro. Reprod Domest Anim 41(1):5-11.




65






Enzyme-Loaded Titania Nanoparticles
for Organic Synthesis











Allison Roth
Truman State University











Research Advisor: Dr. Scott Miller
Department of Chemistry



66

Abstract

Enzymes are catalysts with properties desirable for synthetic chemistry, including
high specificity and mild pressure and temperature requirements. However, their high cost,
difficulty of separation from reaction mixtures, and low activity retention in storage have
limited their usefulness in this discipline. Enzyme encapsulation is a process which has the
potential to enable more extensive use of enzymes in industrial and pharmaceutical
applications by lowering these barriers. A biomimetic process is used to synthesize enzyme-
containing mesoporous silica and titania nanoparticles. Several enzymes are encapsulated
within these particles, and their activities are examined by UV spectroscopy, HLPC, and LC-
ESI-MS. Alcohol dehydrogenase is encapsulated in titania with an efficiency of 57.4% and is
shown to catalyze the conversion of propionaldehyde to isopropyl alcohol. Evidence is
presented for the successful conversion of glutamine to glutamic acid by titania-encapsulated
glutaminase, and an attempt to reduce a-ketoglutarate to glutamic acid using titania-
encapsulated glutamate dehydrogenase is discussed.

Introduction

The function of enzymes is to catalyze chemical reactions in biological systems by
creating an environment in which the activation energy of that reaction is lowered.
1
As
interest in the manipulation of biological molecules has grown, interest in the use of enzymes
as catalysts to perform those manipulations has grown as well.
1
In addition to applications
taking advantage of their bioactivity, enzymes are used as indicators and synthetic reagents.
2

They are highly selective and are marked by high enantiomeric specificity. Enzymes can
replace often hazardous reagents used in organic synthesis reactions, and their re-usability
reduces waste.
1
They function in an aqueous environment, further reducing hazardous waste,
and demonstrate high activities.
3

Despite their advantages, several characteristics of these biomolecules limit their
applicability, and recent research has sought to reduce those barriers. The high specificity of
enzymes can inhibit their use in reactions that vary from those they perform in nature, but
custom design now enables the production of biocatalysts for non-natural applications.
1,3

Though cost has inhibited widespread use of enzymes, the demands of industry have
produced new biochemical techniques which have reduced the cost of enzyme products and
broadened opportunities for their application as biocatalytic materials on a large scale.
1

Several remaining challenges, including lack of stability over time, difficulty of separation
from reaction mixtures, and activity loss in non-aqueous environments, are being approached
using the technique of enzyme immobilization.
2,3

Previous studies in this laboratory have focused on a type of immobilization known
as encapsulation, in which the enzyme is trapped within a material that contains the enzyme
while still allowing it to function.
4,3
One method of encapsulation traps the enzyme within a
silica sphere, which is then coated in layers of a nanocomposite material; this has been shown
to protect the enzyme from proteolysis.
5
Another encapsulation method, that which is studied
by this lab, traps the enzyme within the pores throughout a mesoporous silica nanoparticle in
a manner inspired by the production of silica structures in nature.
3

The intricate shells of diatoms, a type of unicellular algae, are formed by the
biomineralization of monosalicic acid (Si(OH)
4
) in their environment into silica (SiO
2
).
6
This


67

condensation mechanism was found to involve templating proteins known as silaffins,
characterized by their heavily post-translationally modified lysine side chains.
7
Further study
showed that it is the high local concentration of amines provided by the silaffins that enables
the condensation of silica from monosalicic acid.
8
Dendrimers - generational branched
polymers - with amine termini have since been used as biomimetic templates around which
silica can form. Polyamidoamino dendrimer (PAMAM) has successfully been used to form
silica nanoparticles from monosalicic acid in aqueous solution, and enzymes have been
encapsulated within these particles while retaining their activity.
2
A similar method has been
shown to condense titania (TiO
2
) from a conjugate of titanium in aqueous solution.
9

This project explored enzyme encapsulation within mesoporous nanoparticles of both
silica and titania. The silica is condensed from hydrolyzed TMOS and the titania from
Ti(BALDH). The precipitation reaction occurs at near-neutral pH and ambient temperature
and pressure and takes minutes to go to completion.
10,2
The amine-terminated dendrimer
forms a template around which the mineral precipitates, and enzymes in the solution are
captured inside the nanoparticle as it forms.
2
A buffered solution is used in order to enhance
the encapsulation efficiency of the precipitation.
2

In order to advance the study of the encapsulation of enzymes within mesoporous
nanoparticles, it was determined to confirm encapsulation of three new enzymes in titania
and investigate three characteristics of encapsulated enzymes: activity retention or loss over
time, the particles internal environment, and their pore size. The conditions within these
particles, specifically the mobility and orientation of enzymes and the extent of their
interactions with the metal and oxygen atoms, are largely unknown. These conditions, along
with the size of the pores in the particles, may affect the activity of encapsulated enzymes by
preventing or enabling interaction with potential substrate molecules. Clearer understanding
of the morphological features and internal environment of these particles will enable
improvements in their manipulation and more effective application of this immobilization
technique, and expanding the list of successfully encapsulated enzymes will enable wider
study of the techniques applications.
One study makes use of the autolytic properties of the enzyme trypsin. Since trypsin
cleaves itself, a sample of it will tend to lose activity over time when stored in solution.
11

Similar activity loss in encapsulated trypsin may indicate that autolysis is occurring under
those conditions as well, which would reveal that the internal environment of the silica
particles may accommodate multiple enzymes which are capable of orienting themselves
appropriately for cleavage. We studied the changes in activity of encapsulated and free
trypsin stored in solution in order to explore this possibility.
The supernatant of a digest of bovine serum albumin (BSA) performed by
encapsulated trypsin was analyzed by HPLC in order to reveal any differences between its
peptide contents and those of a digest performed by free trypsin. These differences were
anticipated to illustrate qualities of the silica particles internal environment and the size of
their pores. The presence and types of peptide fragments in the encapsulated trypsin digest
would indicate three things: that multiple proteins, at least one enzyme and one substrate
molecule, were being encapsulated within single particles; the location of proteolysis on the
substrate molecule; and the upper size limit on peptide fragments capable of escaping the
particles and being released into solution.
Three enzymes were selected in order to explore encapsulation in titania.
Glutaminase (GLN, EC 3.5.1.2) is an enzyme that converts the amino acid glutamine into


68

glutamic acid (glutamate). Performing the conversion of the amide functionality on
glutamine into an acid functionality with typical organic synthesis methods could require a
multi-step process, which a enzyme-catalyzed reaction would bypass. Glutamate
dehydrogenase (l-glutamic dehydrogenase, GDH, EC 1.4.1.2) participates in the citric acid
cycle, interconverting a-ketoglutaric acid (a-ketoglutarate) and glutamic acid. Encapsulating
GDH would enable both of these reactions to be accomplished using only GDH-loaded
nanoparticles and the necessary cofactors, instead of the two different processes required to
convert the two molecules with traditional organic synthesis methods. Successful
encapsulation of both GLN and GDH would enable studies in which the two enzymes work
together to form a-ketoglutarate from glutamine. A similar system, consisting of tyrosinase
and alcohol dehydrogenase each encapsulated in silica, was shown in a previous study to
produce 4-hydroxybenzyl alcohol from phenol.
12
A study of this kind in titania would be
useful in order to investigate the effects of encapsulating multiple enzymes within individual
titania particles, enabling the single-batch synthesis of biocatalytic reagents capable of
performing consecutive synthetically useful reactions. Alcohol dehydrogenase (ADH, EC
1.1.1.1) was also selected for encapsulation in titania. Successful encapsulation of ADH in
titania would enable side-by-side studies comparing the activity of ADH when encapsulated
in either silica or titania, furthering understanding of the attributes of the nanoparticles
formed by this biomimetic process.

Experimental

Assay for Trypsin Activity
A solution of trypsin in Tris/HCl buffer (0.1 M, pH 8) was prepared. A 600 L
aliquot was combined with 400 L of BAEE. After 5 minutes, A
340nm
was measured. A
standard curve was prepared using 1-3 g/mL trypsin in the final solution.

Tryptic digest of Bovine Serum Albumin
Bovine Serum Albumin (Fraction V, from Fisher) in aqueous solution was digested
by trypsin (E.C. 3.4.21.4, 1:250 virus-free from MP Biomedical) by a protocol adapted from
that used by the Proteomics Facility at La Jolla Neuroscience.
13
In a culture tube, an aliquot
of aqueous BSA (250 L) was combined with 30 L of 0.1 M dithiothreitol (DTT, 99% from
Acros) and incubated 30 minutes in a water bath at 60 C. At room temperature, 50 L 0.1 M
iodoacetamide (98%, from Acros Organics) was added to the reaction mixture, and it was
incubated 15 minutes in the dark. 50 L 0.1 M cysteine (l-cysteine, 98% from Sigma) was
added to the mixture and after 15 minutes, 250 L distilled water was added. 2 L of an
aqueous trypsin suspension (at a concentration to give 1:1 enzyme:substrate ratio in the final
mixture) was then added, and the mixture was incubated with agitation at 37 C for 4 to 24
hours.

Chromatographic separation and HPLC analysis of BSA digests
A column was prepared using G-50 Sephadex and equilibrated with Tris/HCl buffer
(pH 8.2). Using the same buffer as the mobile phase, each digest was separated into 0.5-mL
fractions. A 100 L aliquot of each fraction was diluted 1:10 in distilled water and tested for
absorbance at 220 and 280 nm for protein presence. The fractions with signals at those
wavelengths were combined, and the mixture was subjected to HPLC analysis using a C12


69

column from Phenomenex and a 47.5-minute solvent gradient from 95% water/5%
acetonitrile (with 0.1% TFA) to 100% acetonitrile (with 0.1% TFA).

Trypsin encapsulation
The encapsulation of trypsin (E.C. 3.4.21.4) from MP Biomedical (1:250 virus-free
from porcine pancreas) was achieved by the formation of silica particles in the presence of
the enzyme. Phosphate buffer (0.1 M, pH 7.0) was diluted 1000-fold and used to prepare a
stock solution of trypsin (2 mg/mL). Generation 4 PAMAM dendrimer from Sigma (10%
w/w in MeOH) was used as the template for the precipitation of tetramethylorthosilicate
(TMOS). A 200 L aliquot of stock trypsin was combined with 12 L dendrimer and 20 L
of HCl-hydrolyzed 1M TMOS in a 1.5 mL bullet tube. The mixture was agitated to
encourage particle formation, and the reaction was allowed to run to completion overnight at
room temperature. The resulting particles were centrifuged (10,000 RPM) and washed three-
fold with Tris/HCl buffer (pH 8). Particles were stored in Tris/HCl buffer at 4 C. When this
protocol was doubled or tripled, each multiple was referred to as one batch.

GLN Encapsulation
Glutaminase from Sigma Life Sciences (lyophilized; stored in frozen 1 mL aliquots of
1 un/mL) was encapsulated in titania by following the procedure described for silica
encapsulation of trypsin, with the substitution of an enzyme stock solution of 1 un/mL GLN
in 1:1000 diluted phosphate buffer and 1M Ti(BALDH) in place of TMOS. Particles were
centrifuged and washed three-fold with phosphate/Tris buffer (pH 8.6) and stored in the same
at room temperature.

ADH Encapsulation
Alcohol dehydrogenase (lyophilized, from MP Biomedical) was encapsulated in
titania in the same manner as GLN, using a stock solution of 2 mg/mL ADH in 1:1000
diluted phosphate buffer. Particles were washed with undiluted phosphate buffer (pH 7.5)
and stored in phosphate the same at room temperature.

GDH Encapsulation
Glutamate dehydrogenase from MP Biomedical (suspension, buffer) was
encapsulated in titania in the same manner as GLN, using a stock solution of 2 mg/mL GDH
in 1:1000 diluted phosphate buffer. Particles were washed with undiluted phosphate buffer
(pH 7.5) and stored in triethanolamine (TEA) buffer (pH 7.3) at room temperature.

ADH Photometric Assay
Each sample was dissolved to 2 mg/mL in distilled, deionized water, diluted 1:2 in
0.1% bovine serum albumin, and further diluted 1:50 in 0.1M phosphate buffer (pH 7.5). An
aliquot of the dilute ADH solution was further diluted to 500 L in phosphate buffer in a
quartz cuvette. 300 L NAD+ (25mM) and 200 L ethanol (2M) were combined in a 5 mL
culture tube then added to the cuvette. Two minutes after mixing, A
340nm
was recorded. A
standard curve was created using final concentrations of ADH from 0.2 to 0.6 un/mL.





70

GLN Photometric Assay
GLN was detected using a Bradford assay. 200 L Bradford solution (Coomassie
blue in phosphate buffer from OmniPur) was added to 800 L of glutaminase in water. The
mixture was transferred to a quartz cuvette and allowed to react for 5 minutes, then A
595nm

and A
450nm
were measured and A
595nm
/A
450nm
calculated. A standard curve was created using
final concentrations of BSA from 10 to 200 g/mL.

GDH Kinetic Rate Assay
A mixture of 3.0 mL TEA buffer (pH 7.3, 100 mM), 1.3 mL alpha-ketoglutarate
(200mM, pH 6.5-7.5), 330 L ammonium acetate (3.2 M), 200 L NADH (10 mM) and 200
L EDTA (25 mM) was prepared and the pH adjusted to 7 with 1M NaOH or HCl as needed.
A 500 L aliquot of this mixture was transferred to a quartz cuvette, then 500 L GDH in
cold pH 7.3 TEA buffer was added to the cuvette. The decrease in A
340nm
was recorded for 5
minutes and the rate from 200-300 seconds was calculated. For the creation of a standard
curve, final concentrations of GDH ranged from 0.004 to 0.02 un/mL.

Alcohol Dehydrogenase Reaction Mixture


Scheme 1. Reduction of propionaldehyde by alcohol dehydrogenase.

Encapsulated ADH was suspended in 200 L phosphate buffer (pH 6.9) per batch of
particles. 100 L of this suspension was combined with 400 L phosphate buffer (pH 6.9),
1.5 mL propionaldehyde (Acros), and 0.0206 g NADH. (Scheme 1) Immediately after
mixing, and at points in time afterward, a 250 L aliquot of the reaction mixture was
removed, diluted 1:2 in phosphate buffer (pH 6.9), and centrifuged. The supernatant was then
removed for HPLC and LC-ESI-MS analysis.

Glutaminase Reaction Mixture

OH
O
NH
2
N H
2
O
OH
O
NH
2
O
-
O
NH
3 +
glutaminase
H
2
O

Scheme 2. Oxidation of glutamine to glutamic acid by glutaminase.

Encapsulated GLN was combined with 49 L phosphate/Tris buffer (pH 8.6) per
batch of particles. 125 L of this mixture was combined with 1.875 mL phosphate/Tris buffer
(pH 8.6) and 0.0384 g glutamine. (Scheme 2) At two points in time after initial mixing, a 250
L aliquot of the reaction mixture was removed, diluted 1:2 in phosphate/Tris buffer (pH
8.6), and centrifuged. The supernatant was then removed for HPLC and LC-ESI-MS
analysis.



alcohol dehydrogenase
H
+
, NADH
C H
3
O
C H
3
CH
3
O
H
+
NAD
+


71

Glutamate Dehydrogenase Reaction Mixture


Scheme 3. Reduction of a-ketoglutarate to glutamic acid by glutamate dehydrogenase

Encapsulated GDH was suspended in 100 L triethanolamine buffer (pH 7.3) per
batch of particles. 10 L of this suspension was combined with 0.30 g a-ketoglutarate and
0.02 g NADH in 2 mL triethanolamine buffer (pH 7.3). (Scheme 3) At three points in time
after initial mixing, a 250 L aliquot of the reaction mixture was removed, diluted 1:2 in
triethanolamine buffer (pH 7.3), and centrifuged. The supernatant was then removed for
HPLC and LC-ESI-MS analysis.

Reaction Monitoring by HPLC
Each reaction sample was analyzed using a C18 column (Luna, from Phenomenex)
and an isocratic separation. Each run consisted of a 10-minute equilibration with 0.2%
acetonitrile in water (with 0.1% TFA) at 1 mL/min, followed by a sample injection of 10 L,
20 minutes of data collection using the same solvent and flow rate, and a 1 minute flush with
a solvent mixture of 50.1% acetonitrile (with 0.1% TFA) and 49.9% water (with 0.1% TFA),
and was monitored for Abs
220nm
.

Reaction Monitoring by LC-ESI-MS
For each reaction supernatant analyzed, a 30 L aliquot of the sample prepared for
HPLC analysis was added to 70 L acetonitrile (with 0.1% formic acid). Each run consisted
of a 10-minute equilibration with 2% acetonitrile and 98% water (with 0.1% formic acid) at 1
mL/min, followed by a sample injection of 10 L, 20 minutes of data collection using the
same solvent and flow rate, and a 1 minute flush with a solvent mixture of 50% acetonitrile
and 50 % water (with 0.1% formic acid). Each run was monitored for Abs
220nm
and for
masses between 100 and 700 mass units.

Results and Discussion

Trypsin Stability
Trypsin encapsulated by the above method was assayed for activity four times over
two weeks. These measurements were compared to tests of both non-enzyme-containing
particles and unencapsulated trypsin stored under the same conditions. Free trypsin showed
steady loss of activity, and empty particles showed no significant change in activity.
Encapsulated trypsin demonstrated an overall increase in activity, but with high uncertainty
due to large deviations within the measurements on each day. The experiment was repeated,
and this second trial also produced indeterminate results from both photometric and kinetic
UV-visual spectrophotometric methods. The settling of the particles may have interfered with
absorbance readings, causing a decrease in absorbance over time which competed with the
increase that indicated trypsin activity. Attempts to control and account for this effect did not
succeed in producing consistent measurements.
OH
O
NH
2
O
-
O
OH
O
O
O
-
O
+
NADH
+
NH
3 +
H
+
glutamate dehydrogenase
NAD
+


72

Tryptic Digest
The HPLC analysis method described above did not detect any peptide fragments
produced by the digest of BSA with unencapsulated trypsin. Therefore analysis of a digest
using encapsulated trypsin was not performed.

Original enzyme
concentration (g/mL)
Activity of supernatant
(g/mL)
Encapsulation efficiency (% of
initial concentration)
ADH 0.800 0.459 57.4

Table 1. Summary of encapsulation data for alcohol dehydrogenase.

Encapsulation and Activity of ADH
Using the encapsulation method, assay, and standard curve described above, the
encapsulation efficiency and effective activity of alcohol dehydrogenase were determined.
(Table 1)



Figure 1. Chromatograms for initial ADH reaction sample (darkest blue), 17 hours (medium blue), and 20 hours
(lightest blue).

The supernatant of the reaction mixture was analyzed by HPLC soon after initial
mixing and at two points in time afterward, illustrating the appearance of isopropyl alcohol.
(Figure 1) Confirmation of peak identity was accomplished by HPLC analysis of the 20-hour
reaction sample with additional isopropyl alcohol doped in and LC-ESI-MS analysis of the
17-hr reaction sample.

Encapsulation and Activity of GLN
Using the encapsulation method, assay, and standard curve described above, the
encapsulation efficiency and effective activity of titania-encapsulated glutaminase were
isopropyl alcohol (by
doping)
enzyme
(by LC-ESI-MS)
NAD+
(by LC-ESI-MS)


73

tested. However, the data produced by tests of the unknown samples did not fall within the
range included in the standard curve, and could not be converted into meaningful
measurements of encapsulation rate. A reaction mixture for the study of the activity of GLN
was prepared according to the method described above. The supernatant of the reaction
mixture was separated by HPLC at two points in time (Figure 2), and the two samples were
each doped with either glutamine or glutamic acid. (Figure 3)



Figure 2. Chromatograms of GLN reaction samples at two points in the progress of the reaction; the sample at Time
1 was not analyzed by LC-ESI-MS



Figure 3. Chromatogram of GLN reaction sample at time 1 after sample was doped with glutamine, and
chromatogram of GLN reaction sample at time 2 after sample was doped with glutamic acid
Time 1
Time 2
Time 2,
doped
Time 1,
doped
glutamic acid
(doped in)
glutamine
(doped in)
glutamic acid
(by LC-ESI-MS)
glutamine
(by LC-
ESI-MS)


74

In order to confirm that glutamine was being converted into glutamic acid over time,
the reaction sample at Time 2 was analyzed by LC-ESI-MS to determine the identity of the
main peak. Both glutamine and glutamic acid were identified within the sample; glutamine
with a retention time of 2.64 minutes and glutamic acid at 2.77 minutes. The appearance of a
second peak in the HPLC chromatogram after the Time 2 sample had been doped with
glutamic acid indicates that the main peak in the original sample is not glutamic acid, but
glutamine.
Addition of glutamine to the Time 1 sample appears to increase the intensity of the
larger, longer-retained peak of the two with retention times near 2.5 minutes. This indicates
that the HPLC method used causes glutamine to elute after glutamic acid. However, LC-ESI-
MS analysis of the Time 2 sample indicated the elution of glutamine before glutamic acid.
These contradictory results prevented quantitative analysis of the reaction mixture.

Encapsulation and Activity of GDH
Using the encapsulation method, assay, and standard curve described above, the
encapsulation efficiency of glutamate dehydrogenase was tested. However, the data
produced, by tests of the unknown samples, did not fall within the range included in the
standard curve and could not be converted into meaningful measurements of encapsulation
rate. A reaction mixture for the examination of the activity of encapsulated GDH was
prepared by the method above. The supernatant of the reaction mixture was separated by
HPLC soon after initial mixing and at two points in time afterward. In each chromatogram,
a large, broad signal was centered between 3 and 4 minutes, along with smaller, sharper
peaks that resemble those of glutamine and glutamic acid. The lack of baseline resolution at
those retention times inhibited quantification of the changes in peak areas, but a relative
increase and then decrease in the height of the peak centered around 3.3 minutes was
apparent. In order to determine if any of the changing peaks and shoulders represented the
anticipated product, the reaction sample from Time 3 was doped with glutamic acid. A new
peak was visible in the doped sample with a retention time of 3.2 minutes, which may
indicate that the peaks at 3.3 minutes in the original samples represented glutamic acid. The
shape of the broad peak between 5 and 6 minutes was greatly altered in the doped sample
compared to the original sample. The cause of this change is unclear. The altered peak shape
in the doped sample and the interference of the broad peak in all samples prevent confident
identification of the peaks at 3.3 minutes as glutamic acid.

Conclusions

The study of the stability of encapsulated trypsin was inconclusive due to
inconsistencies in activity measurements among multiple tests of the same samples. This
variation prevented significant comparison of the free and encapsulated enzyme and the
unloaded silica particles. The study of the digest products of encapsulated trypsin acting on
BSA did not take place due to the lack of polypeptide fragments detected by HPLC from a
digest using unencapsulated enzyme; this could indicate either a lack of trypsin activity, a
fault in the digestion procedure, or an inappropriate HPLC method.
Encapsulated alcohol dehydrogenase was used to catalyze the conversion of
propionaldehyde to isopropanol, the appearance of which was monitored by HPLC.
Encapsulated glutaminase was used to produce glutamic acid from glutamine, evidenced by


75

the presence of both glutamine and glutamic acid in the first reaction sample. The
observation that the second sample contained glutamic acid only in levels detectable by LC-
ESI-MS, but not HPLC, is not explained by the analyses performed. The discrepancy in
elution order between LC-ESI-MS and HPLC, along with the lack of baseline resolution in
the HPLC chromatograms, prevents quantification of shifts in the composition of the reaction
mixture.
Continuing study of glutaminase should both repeat the method and reaction used in
this study and, if these results are replicated, alter the method, taking into consideration the
possibility of a reverse reaction. Method development may also require alternative strategies
for separating glutamine and glutamic acid. Use of an ion-exchange column may enable
greater separation of the amide and acid and improve the baseline resolution of the
separation.
Encapsulated glutamate dehydrogenase was indicated by HPLC analysis to have
converted a-ketoglutarate to glutamic acid. However, further analysis of the unidentified
peaks in the chromatograms, particularly the broad peak which coeluted with other analytes,
should occur in order to confirm this conclusion. Once all reaction components are identified,
other separation methods may be employed to more effectively analyze GDH reaction
mixtures and definitively determine the effectiveness of encapsulated GDH. Future GDH
reaction mixtures should be altered to include NH
3
, which may help drive the conversion
from a-ketoglutarate to glutamic acid.
Further investigation of the studies composing this project may include: investigation
and improvement of the protocols used for the tryptic digest and its analysis; optimization of
the assay for the activity of encapsulated trypsin for the study of its activity retention over
time, optimization of a specific activity assay for glutaminase; precise determination of the
specific activity of ADH, GDH, and GLN when encapsulated; and the combination of titania-
encapsulated enzymes to form a system of sequential reactions.

Acknowledgements

Dr. Sandra Stenson, Dr. Jason Coym, Dr. Scott Miller, Dr. Julio Turrens, University
of South Alabama Department of Chemistry, National Science Foundation

References

1. Wong, C. and Whitesides, G.M. 1994. Enzymes in Synthetic Organic Chemistry.
Elsevier Science Ltd.: Tarrytown, New York, xiii-xiv.

2. Miller, S., Hong, E. and Wright, D. 2006. Rapid and efficient enzyme encapsulation in
a dendrimer silica nanocomposite. Macromolecular Bioscience 6:839-835.

3. Sheldon, R. 2007 Enzyme immobilization: The quest for optimum performance. Adv
Synth Catal 349:12891307. http://largens.com/biochemlab/peroxidase2.pdf (accessed
Jun 17, 2011).

4. Swartz, J., Miller, S., and Wright, D. 2009. Rapid production of nitrilase containing
silica nanoparticles offers an effective and reusable biocatalyst for synthetic nitrile
hydrolysis. Org Proc Research and Development [Online].


76

5. Wang, Y. and Caruso, F. 2005. Mesoporous silica spheres as supports for enzyme
immobilization and encapsulation. Chemistry of Materials [Online] 17(5):953-961
http://pubs.acs.org/doi/full/10.1021/cm0483137 (accessed Jun 17, 2011).

6. Kroger, N., Deutzmann, R. and Sumper, M. 1999. Biomimetic formation of micro and
nanostructured titanium and protein encapsulation. Science 286:1129-1132.

7. Sumper, M., Lorenz, S. and Brunner. E. 2003. Biomimetic control of size in the
polyamine-directed formation of silica nanospheres. Angewandte Chemie
International 42(42):5192-5195. Web. 15 Jun 2010.

8. Knecht, M.R. and Wright, D.W. 2004. Amine-terminated dendrimers as biomimetic
templates for silica nanosphere formation. Langmuir 20:4728-4732.

9. Sumerel, J., Yang, W., Kisailus, D., Weaver, J., Choi, J, and Morse, D. 2003.
Biocatalytically templated synthesis of titanium dioxide. Chem Mater 15(25):4804-480.

10. Luckarift, H., Spain, J., Naik, R. and Stone, M. 2004. Enzyme immobilization in a
biomimetic silica support. Nature Biotechnology [Online] 22:211-213 Biomedical
Reference Collection: Comprehensive. http://web.ebscohost.com (accessed Jun 17,
2011).

11. Nord, F. 1956. On the mechanism of enzyme action. LXI. The self digestion of trypsin,
calcium-trypsin and acetyltrypsin. Archives of Biochemistry and Biophysics 65(1):120.
http://www.sciencedirect.com/science/article/pii/0003986156901825.

12. Walters, T. 2010. Biomimetically encapsulated enzymes as robust catalysts for organic
synthesis. REU Research Report, University of South Alabama, Mobile, AL.
http://www.southalabama.edu/alliedhealth/biomedical/ucur/NSF%20BOOK%202010.pdf

13. Solution digestion of proteins. Proteomics Facility, La Jolla Neuroscience.
http://www.lajollaneuroscience.org/sr/delta.ljcrf.edu/proteomics/solndig.html (accessed
Jun 9 2011).





77



The Role of the NarU Nitrite/Nitrate Transporter in
Nitrate Reductase Z-Dependent Resistance to
Environmental Stresses











Gregory Schalla
Beloit College














Mentor: Dr. Michael P. Spector
Department of Biomedical Sciences






78

Introduction

Salmonella enterica serovars (e.g., Salmonella enterica serovar Typhimurium) are
major food-borne pathogens that are found worldwide as contaminants of eggs, poultry, beef,
pork, milk, raw vegetables, or their by-products, and water. Consumption of Salmonella-
contaminated food or drink can lead to intestinal (e.g., enterocolitis) and/or systemic disease
(e.g., bacteremias and typhoid fever) in both animals and humans. Symptoms associated with
human enterocolitis include diarrhea, vomiting, fever, stomach cramps, and headaches
(Spector & Kenyon, 2011).
What makes Salmonella enterica serovars so dangerous is that they can adapt to and
survive in many different environments. These environments can include ones with extremes
in pH, temperature, osmolarity; and those with limited nutrients, antimicrobial peptides
and/or H
2
O
2
(Foster & Spector, 1995; Spector, 1998; Spector & Kenyon, 2011). It has the
ability to sense and respond to these different environmental changes, which in turn, allow
the bacteria to survive within environments that are less than optimal for growth or are
potentially lethal (Foster & Spector, 1995; Spector & Kenyon, 2011).
In environments where there are limiting levels of available carbon-energy (C)
sources, Salmonella activates the starvation stress response (SSR) (Spector, 1998; Spector &
Kenyon, 2011). The SSR is the genetic and physiological changes that the bacteria undergo
in response to low C-source environments. It allows Salmonella to survive longer in C-
starved environments and provides cross-resistance to a number of other environmental
stresses, e.g., acidic pH or the presence of H
2
O
2
(Foster & Spector, 1995; Spector, 1998;
Spector & Kenyon, 2011). Portions of the SSR of S. Typhimurium are regulated by proteins
called sigma factors (e.g.,
S
and
E
) that alter the specificity of the cells RNA polymerases
for different promoters, resulting in changes in the patterns of gene expression. In addition,
the SSR is globally controlled by at least two cellular signal molecules, cyclic AMP (cAMP)
and guanosine tetraphosphate (ppGpp); both of which accumulate in C-starved cells (Spector
et al., 1988; Spector & Cubitt, 1992; ONeal et al., 1994; Spector, 1998; Spector et al., 1999;
Spector & Kenyon, 2011)
Genes identified as being required for the maximal generation of the SSR in
Salmonella include those that encode for components of nitrate reductase Z (NR-Z),
narZYWV. The inability to produce NR-Z diminishes the long-term carbon starvation
survival and C-starvation-inducible (CSI) cross-resistance to acid pH (pH 3.3), high
temperature (55C), as well as hydrogen peroxide-inducible adaptive H
2
O
2

resistance.(Spector & Cubitt, 1992; Seymour et al., 1996; Spector et al., 1999; O'Neal et al.,
1994; Spector, 1988). Nitrate reductases (NRs) are enzymes that reduce nitrate into nitrite in
order to create a proton motive force (PMF) which can then be used to generate energy for
metabolic processes. In order for the NR-Z to work, electrons must pass through NarY and
NarV. The electrons come from the reduction of the quinones. This reduction produces two
H
+
ions / protons that are pumped into the periplasm creating a PMF across the membrane.
At the same time, two electrons are transferred through NarV and NarY to NarZ. NarZ uses
these two electrons and two hydrogen ions to reduce nitrate to nitrite and water. The
expression of narZYWV is positively regulated by C-, P-, and N-starvation; guanosine
tetraphosphate (ppGpp); and the alternative sigma factor
S
; and negatively regulated by
cAMP -cAMP receptor protein (cAMP-CRP) complex.


79

Another gene of interest expressed during the SSR within Salmonella is the narU
gene encoding for the nitrite/nitrate transporter. The narU gene was determined to be the first
gene of the narUZYWV operon using RT-PCR. The
S
promoter site is located 116 bp
upstream from the narU gene and no additional
S
-promoter has been shown (Chang et al.,
1999; M.P. Spector, unpublished data). The function of the NarU protein is to pump nitrate
into the cell and to pump potentially lethal nitrite out of the cell. No evidence has been
reported indicating that the NarU protein can pump nitrite into the cell, which leads
researchers to believe that NarU could be an antiporter (exchanging intracellular NO
2
-
for
extracytoplasmic NO
3
-
) as well as a nitrate / nitrite transporter. The NarU protein is proposed
to have a single channel that allows for the transportation of nitrate into and nitrite out of the
cell. NarU may use an alternating access mechanism, or rocker-switch, to allow for the
opposite direction of transportation of nitrate and nitrite. The NarU protein has been shown
to increase in abundance during stationary phase of the cell growth and when there are low
levels of nutrients. It has been shown that the NarU protein is more effective of a transporter
than its similar transporter NarK. Further studies surrounding functions of NarU are still
being conducted and researchers continue to gain a more accurate understanding of the NarU
protein (Spector, 1988; Spector & Cubitt, 1992; O'Neal et al., 1994; Seymour et al., 1996;
Spector et al., 1999; Clegg et al, 2002; Clegg et al, 2006).
Based upon the findings that NR-Z is required for different aspects of the SSR and
H
2
O
2
-inducible H
2
O
2
resistance in the absence of an exogenously added nitrate source, we
proposed that NarU is not required for the function of NR-Z in these stress responses. To test
this hypothesis, strains possessing both NarU and NR-Z (U+ Z+; parental strain), lacking
NarU but making NR-Z (U- Z+; narU narZYWV
+
strain), and making NarU but lacking
NR-Z (U+ Z-; narU
+
narZ::MudJ strain) were tested and compared in terms of their ability
develop H
2
O
2
-inducible H
2
O
2
resistance and aspects of the SSR. If NarU is not required then
that would suggest that NR-Zs function as a nitrate reductase is not what is important and
indicate that perhaps it has an alternative substrate that is formed under these stress
conditions, which must be detoxified in order to maintain cell viability.

Materials and Methods

Bacterial strains used
The strains used in this study were all derivatives of the mouse virulent Salmonella
enterica serovar Typhimurium strain SL1344 and are listed in Table 1.


Table 1. Bacteria strains used for H
2
O
2
adaptative H
2
O
2
resistance and Carbon-starvation-inducible cross-resistance
to acid pH.
Strain Relevant Genotype (Phenotype)*
ST41
hisG46; mouse virulent Salmonella enterica serovar Typhimurium strain
SL1344
ST66 SL1344 narZ1::MudJ (lac Km
r
)
SMS902 SL1344 narU6
*Km
r
= Kanamycin-resistance



80

Media and supplements used

The rich medium used for recovery of stress challenged cells in these experiments
was Luria-Bertani (LB) agar (Difco). The minimal media used were MOPS-buffered salts
(MS) media. MS medium contains 22.5 mM potassium phosphate and 10 mM nitrogen (as
ammonium chloride) plus additional salts and trace elements (Spector et al., 1988; Spector &
Cubitt, 1992; ONeal et al., 1994). To produce log phase cells and H
2
O
2
adapted and
unadapted cells, MS medium with 0.4% w v
-1
glucose (MS hiC) was used. To generate the
5-hour and 24-hour carbon (C)-starved cells, MS medium with 0.03% w v
-1
glucose (MS
loC) was employed. Kanamycin (Km) was used at a final concentration of 50 g mL
-1
, as
needed.

H
2
O
2
-inducible adaptive H
2
O
2
resistance assay

To assay for the effect of a narU or narZ null mutation on H
2
O
2
-inducible adaptive
H
2
O
2
resistance, the desired strains were grown in MS hiC medium with Km, as needed, at
37 C with shaking for 18-24 hours (overnight culture or onc). The onc was diluted 1:100
into MS hiC with Km, as needed, and incubated at 37 C with shaking. Growth was
monitored spectrophotometrically at wavelength 600 nm until the optical density (OD
600
)
reached between 0.2-0.3. Once reached, the culture was split into two cultures of equal
volume. To one of the tubes H
2
O
2
was added to a final concentration of 60 M (a non-lethal
dose used to adapt the cells). Both the cultures, adapted (+ H
2
O
2
) and unadapted ( H
2
O
2
),
were incubated at 37 C with shaking for an additional hour. After 1 hour, the cells were
challenged with 15 mM H
2
O
2
(normally lethal level).

H
2
O
2
challenge method

For the hydrogen peroxide challenge, the adapted and unadapted cultures were diluted
1:100 into MS buffer-15 mM H
2
O
2
. The cells were challenged for a total of 60 minutes with
aliquots being removed at 0, 20, 40, and 60 minutes and serially diluted 1:10 in MS buffer.
The last four dilutions were plated, in triplicate, on LB agar with Km, as needed. The plates
were then incubated at 37 C for 18-24 hours. The number of colony forming units per mL
(CFU mL
-1
) were then determined for each time point. The CFU mL
-1
at each time point was
then divided by the maximum CFU mL
-1
at time 0 minutes and that number multiplied by
100 to get the percent maximum survival.

Carbon-starvation-inducible cross-resistance to acid pH assay

To assay for carbon-starvation-inducible cross-resistance to acid pH, an overnight
culture were diluted 1:100 into MS hiC and MS loC media with Km, as needed. The cultures
were incubated at 37 C with shaking and their growth monitored spectrophotometrically at
600nm. MS hiC cultures were grown until the OD
600
reached 0.3-0.4 to generate log-phase
cells. MS loC cultures were grown until OD
600
stopped increasing due to glucose depletion.
These cultures were allowed to continue to incubate at 37 C with shaking for a total of 5
hours (5-h C-starved cells) and 24 hours (24-h C-starved). Log-phase, 5-h C-starved and 24-
h C-starved cells were then challenged at pH 3.3.


81

Acid pH challenge protocol

For the acid pH challenge, log-phase, 5-h C-starved and 24-h C-starved cells were
diluted 1:100 into MS buffer at pH 3.3. Challenge cultures were incubated shaking at 37 C
for a total of 60 minutes. Aliquots were removed at 0, 30 and 60 minutes and then serially
diluted 1:10 in MS buffer pH 7.4. The last four dilutions were plated, in triplicate, onto LB
agar with Km, as needed. The plates were then incubated at 37C for 18-24 hours. The
CFU mL
-1
for each time point was determined, divided by CFU mL
-1
at time 0 minutes and
that number was multiplied by 100 to get the percent maximum survival.

Results

Despite several attempts we were unable to obtain accurate and reproducible results
for either the H
2
O
2
-inducible H
2
O
2
adaptive resistance or CSI cross-resistance to acid pH.
For the H
2
O
2
inducible H
2
O
2
adaptive resistance trials, we saw too much killing of the wild
type adapted bacteria. The percent survivals were inconsistent and not very reproducible.
In contrast, for the CSI acid pH cross-resistance studies there was insufficient and
inconsistent killing of the log-phase wild type cells. Also, there was a contamination issue.

Discussion

The experiments did not yield usable results as we hoped. Problems with the
concentrations of hydrogen peroxide used for adaptation and challenges could be the reason
for the over killing of the adapted bacteria. If the H
2
O
2
concentrations used for the
challenge assays were too high the S. typhimurium would have a tougher time surviving and
the results would show no survival. If the H
2
O
2
concentrations used for adaptation were too
low, the bacteria would not have induced the adaptation functions needed to survive the
higher concentration of H
2
O
2
.
For the CSI acid cross-resistance, the problem could have been with the MS buffer at
pH 3.3. The buffer might not have been at 3.3 like we thought, but instead may have been
higher. If the buffer had a higher pH the S. typhimurium would have had much greater
chance at survival. Also, with contamination of the MS buffer used for dilutions, the results
came back showing no dilution effect on the plates. So the bacterial growth showed up as
smears rather than as single, countable colonies. By remaking the MS buffer, we can avoid
contamination. If these experiments were to be run again, then the H
2
O
2
should be remade
with extreme precision to avoid incorrect concentrations. The MS buffer at pH 3.3 should
also be remade and the pH should be checked to make sure it is in fact 3.3. If remaking the
solutions still yields inconclusive results, then the experiments procedure must be looked at.
Otherwise the experiment must be repeated to determine if the NarU is needed for NR-Z
dependent resistance mechanisms.





82

References

1. Chang, L., Wei, L.I., Audia, J.P., Morton, R.A. and Schellhorn, H.E. 1999.
Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase
dependent and is controlled by RpoS, the alternative vegetative sigma factor. Molecular
Microbiology 34:756-66.

2. Clegg, S.J., Jia, W., and Cole, J.A. 2006. Role of the Escherichia coli nitrate transport
protein, NarU, in survival during severe nutrient starvation and slow growth.
Microbiology 152:2091-2100.

3. Clegg, S., Yu, F., Griffiths, L. and Cole, J.A. 2002. The roles of the polytopic
membrane proteins NarK, NarU, and NirC in Escherichia coli K-12: two nitrate and three
nitrite transporters. Molecular Microbiology 44:143-155.

4. Foster, J. W. & Spector, M. P. 1995. How Salmonella survive against the odds. Annual
Review of Microbiology 49:145-174.

5. Moreno-Vivin, C., Cabello, P., Martnez-Luque, M., Blasco, R. & Castillo, F.
(1999). Prokaryotic nitrate reduction: molecular properties and functional distinction
among bacterial nitrate reductases. Journal of Bacteriology 181:65736584.

6. O'Neal, C.R., Gabriel, W.M., Turk, A.K., Fang, F.C., Libby, S. and Spector, M.P.
1994. RpoS is necessary for both the positive and negative regulation of starvation-
survival genes during phosphate-, carbon-, and nitrogen-starvation in Salmonella
typhimurium. Journal of Bacteriology 176:4610-4616.

7. Seymour, R., Mishra, P.V., Khan, M.A. and Spector, M.P. 1996. Essential roles of
starvation-stress response loci in carbon-starvation-inducible cross-resistance and
hydrogen peroxide-inducible adaptive resistance to hydrogen peroxide challenge in
Salmonella typhimurium. Molecular Microbiology 20:497-505.

8. Spector, M.P. and Kenyon, W.J. 2011. Resistance and survival strategies of Salmonella
enterica to environmental stresses. Food Research International, e-pub -
doi:10.1016/j.foodres.2011.06.056.

9. Spector, M.P., Garcia del-Portillo, F., Bearson, S.M.D., Mahmud, A., Magut, M.,
Finlay, B.B., Dougan, G., Foster, J.W. and Pallen, M.J. 1999. The rpoS-dependent
starvation-stress response locus stiA encodes a nitrate reductase (narZYWV) required for
carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella
typhimurium. Microbiology 145:3035-3045.

10. Spector, M.P. 1998. The starvation-stress response (SSR) of Salmonella. Advances in
Microbial Physiology 40:233-279.


83

11. Spector, M.P. and Cubitt, C.L. 1992. Starvation-inducible loci of Salmonella
typhimurium: Regulation and roles in starvation-survival. Molecular Microbiology
6:1467-1476.

12. Spector, M.P., Park, Y.K., Tirgari, S., Gonzalez, T. and Foster, J.W. 1988.
Identification and characterization of starvation-regulated genetic loci in Salmonella
typhimurium using Mud-directed lacZ operon fusions. Journal of Bacteriology
170:345-351.










84




85




Glucose Consumption and Lactate Production in
Nih-3t3 Fibroblasts Carrying Mutations in
Mitochondrial DNA







Kanika Topiwala
University of Arkansas at Little Rock









Research Advisor: Dr. Mikhail Alexeyev
Department of Cell Biology and Neurosciences








86

Abstract

While characterizing the mutations in mitochondrial DNA, this study was routed into
the new direction. Three concluding statements derived from this study are that lactate
production per cell is dependent on cell density, lactate production per cell is not dependent
on glucose concentration, and lactate production per cell is dependent on lactate
concentration. Also, studying glucose and lactate concentrations is not an efficient way to
characterize these mutant cells. One possible approach to characterize cells is to study the
rate of oxygen consumption by using inhibitors for each complex.

Introduction

Mitochondria are found in all nucleated cells, and they play a crucial role in
generating energy in the form of adenosine triphosphate (ATP). Hence, they are often called
the powerhouse of the cell. Cells use ATP generated by mitochondria to perform their daily
functions. In animal cells, mitochondria and the nucleus are the only organelles that carry
DNA. The mitochondrion consists of four compartments: the outer membrane, the inner
membrane, the intermembrane space, and mitochondrial matrix. Mitochondrial DNA resides
in the matrix. The human mitochondrial genome is 16.6 kb long. It encodes 37 genes 13
polypeptides for its oxidative phosphorylation system, 2 for rRNA, and 22 for tRNA. One of
the main functions of a mitochondrion is cellular respiration. The three major pathways for
generating ATP are glycolysis, Krebs Cycle, and oxidative phosphorylation.
Cellular respiration is a series of metabolic reactions that takes place in all living cells
to convert biochemical energy from nutrients into ATP. Glycolysis is a universal process that
happens in all organisms. As a result of glycolysis, each molecule of glucose produces two
net ATP molecules. The other products of glycolysis are two molecules of pyruvate and two
molecules of NADH. Pyruvate has three fates: 1) in anaerobic conditions, it can be converted
to lactate by the enzyme lactate dehydrogenase. This reaction consumes NADH produced by
glycolysis and generates lactate and NAD
+
. NAD
+
is then returned to glycolysis where it is
reduced to NADH. 2) In anaerobic conditions (happens in yeast and other microorganisms),
pyruvate can also be converted into ethanol and CO
2
. 3) In aerobic conditions, cells can use
pyruvates to undergo oxidative phosphorylation (OXPHOS) and generate large amounts of
ATP. The pyruvate molecules are oxidatively decarboxylated to form acetyl CoA. Citric acid
cycle (Krebs cycle) includes series of redox reactions that results in oxidation (removal of
electrons) of acetyl group of acetyl CoA to form two molecules of CO
2
. The complete cycle
generates one molecule of GTP and reduction equivalents of three molecules of NADH and
one molecule of FADH
2
. These electron carriers donate electrons to ETC which transports
them to O
2
. As electrons move from lower affinity carriers to higher affinity carriers, a
proton gradient is formed between the intermembrane space and matrix. Protons flow back
into the matrix through ATP Synthase (complex V), which harness energy of this flow in the
form of ATP. Oxidation of one mol of NADH produces 2.5 mol of ATP and one mol of
FADH
2
produces 1.5 mol of ATP. Krebs Cycle and OXPHOS take place in the mitochondria,
mitochrondrial matrix and inner membrane, respectively; while, glycolysis takes place in the
cytoplasm.
One of the key studies with mitochondria is the biochemistry of the Electron
Transport Chain. ETC (a.k.a respiratory chain) consists of four complexes. As mentioned


87

above, the main goal of the ETC is to transfer electrons from NADH to O
2
through these
complexes: NADH dehyrogenase (complex I), Q-cytochrome b (complex III), and
cytochrome c oxidase (complex IV). These three main complexes are linked as a chain.
Electron flow through these complexes is coupled to the transport of protons across the inner
mitochondrial membrane. The other complex is called succinate-Q reductase (complex II),
which transports electrons from FADH
2
instead of NADH, and it does not pump any protons
into the intermembrane space. As mentioned above, 13 genes encode mitochondrial
polypeptides that form subunits of the oxidative phosphorylation system: seven subunits in
complex I (ND1-6), one subunit in complex III, three subunits in complex IV, and two
subunits in complex V [1]. When one complex loses activity, all of the successive complexes
can potentially lose activity, and the whole system may stop functioning.
The electron transport is tightly coupled to ATP synthesis, i.e electrons only flow to
O
2
if ADP is simultaneously being phosphorylated to ATP. This coupling between ETC and
OXPHOS is called respiratory control; the rate of OXHPOS is determined by the need of
ATP. The coupling or uncoupling state in mitochondria is a key characteristic in determining
mitochondrial respiratory control. The system can be uncoupled by uncoupling agents. An
example of such is p- trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCCP).
Dissipation of proton gradient by uncoupling agents prevents ATP synthesis. The uncoupler
is also called the gradient-busting chemical, which brings H
+
across the membrane through a
shuttle system. Therefore, ATP synthesis is inhibited, but the ETC still continues to function
by forming H
+
electrochemical gradient. On the other hand, inhibitors are compounds that
bind to some of the components of ETC and block its ability to function. Oligomycin is a
complex V inhibitor, and it stops ETC due to the presence of high proton gradient in the
intermembrane space. Therefore, oxygen consumption decreases. Other respiratory inhibitors
are rotenone and amytal for complex I, antimycin A for complex III, and cyanide and azide
for complex IV.
Nuclear and mitochondrial genetics differ greatly in some aspects such asin the
structure of DNA molecule, in the process of replication, in the inheritance of genetic
information, in the ways mutations can play role, etc. In animals, the mitochondrial
genome consists of multiple copies of circular, double stranded DNA molecules.
Mitochondrial replication is controlled by nuclear genes. Depending on the cells type, each
cell can have hundreds to thousands of mitochondria. Since there are multiple copies of
mtDNA, cells can exist in two types of conditions homoplasmic and heteroplasmic (similar
to homozygous and heterozygous in nuclear genetics). When all of the cells mtDNAs are
identical in their nucleotide sequence, it is called homoplasmy. On the other hand, when cells
contain two or more types of mtDNA nucleotide sequence, it is called heteroplasmy.
While replicating, both nDNA and mtDNA molecules can acquire mutations due to
errors in replication. Mutations in nDNA and mtDNA can also be induced by chemical
and/or physical damage. One example of chemical mutagen is free oxygen radicals, which
can be produced by electron leak from the ETC. These radicals can damage mtDNA and can
lead to point mutations as well as deletions in increasing frequencies [2, 3]. In 1988, it was
found for the first time that mtDNA mutations can cause human disease such as myopathies
[4]. Mutations induced by ROS (reactive oxygen species or oxygen radicals) may play a
significant role in cardiac aging [2]. Subsequently, mtDNA mutations were also associated
with cancer, diabetes mellitus [5], and were also hypothesized to play a critical role in aging
[6]. Some mitochondrial diseases are associated with elevated blood lactate level, and


88

patients suffer from a condition called lactic acidosis, where blood pH decreases [7].
According to the epidemiological studies, mitochondrial diseases are some of the most
common genetic disorders [8].
The majority of mitochondrial diseases are caused by heteroplasmic mutations in
mtDNA. The level of heteroplasmy can vary from less than 1% to greater than 99% [9]. The
severity of the disease is often determined by the percentage of mutated mtDNA present in
the cell. Therefore, the ratio of mutant DNA to wild type DNA, along with some other
factors, will determine the severity of disease in an individual. To define the line between the
healthy and the diseased cells, the concept of threshold was introduced. For example, 30% of
the heteroplasmic mutation (also called mutation load) may be asymptomatic for a specific
disease, while disease may develop at 70% of the mutation load. The inheritance of mtDNA
is almost exclusively maternal. The mitochondrial heteroplasmy is passed from a mother to
an offspring in a very unpredictable way and can shift dramatically with each pregnancy.
Therefore, a mother with mild or no symptoms for disease can have an offspring with severe
symptoms of disease or no symptoms at all. To describe this phenomenon, the bottleneck
theory was developed. During the maturation of mothers egg cell, only a small number of
mothers mtDNA is passed on into a primary oocyte and is then followed by replication of
mtDNA into a mature oocyte. Bottleneck is referred to a random process of the reduction in a
copy number of mtDNA during the formation of primary oocyte. Studies on mitochondria
are a hot topic today in the field of cell and molecular biology. The ultimate goal of the
research is to find cure for mitochondrial diseases.
One approach to study and to find cures for mitochondrial disorders is to create
trasnsmitochondrial mice models (also known as Mito-mice) [10-13]. Mito-mice will carry
mtDNA with mutations and they may develop symptoms similar to those found in human
disease. The goal is to treat these mice with drugs to find cures for certain disorders. To
create a Mito-mice, the first step is to identify a mutation in the cultured mouse cell. Then,
make a cybrid. Cybrids are cytoplasmic hybrids. Cybrids are made by enucleating (removing
the nucleus and creating a cytoplast) the cells containing mutant mtDNA and fusing them
with the mouse embryonic stem cell (MESC). MESC are treated with rhodamine 6G to
inactivate their mitochondria [14]. Both, enucleated cell and a cell with inactivated
mitochondria, by themselves, are nonfunctional. But when they are fused together, the
resulting cell becomes functional and is known as a cybrid. These cybrids can then be
injected into mouse blastocysts, which can then be implanted into a pregnant mouse. When
the blastocyst develops into an embryo, it will contain some of the cells from MESC cybrids.
These mice carry mutant mtDNA and may develop symptoms that are characteristics of
human diseases [10-12]. Currently, the models of mtDNA disease are predominantly cell-
based rather than transgenic mouse models. Even the models that do exist mimic very few
characteristics of diseased mtDNA [7]. The animal models depend on mutant mtDNA mouse
cell lines. The problem lies with the availability of only limited numbers of cell lines that are
synonymous to the mutated mtDNA in human beings and the lack of a systematic approach
to generate these cell lines. This is what our lab is focusing on.
An important technique used in the lab is depletion and repletion of mtDNA to create
homoplasmic mutants. The polymerase gamma (Polexo) with deficiency in exonuclease
activity (Polexo
-
) would induce mutations in mtDNA when introduced into cells. Pol is a
heterotrimeric structure that consists of three subunits. It has DNA polymerase activity to
replicate mtDNA and exonuclease activity that proofreads the sequence. It also has a 5-dRP


89

lyase activity that is required for base excision repair. But in Polexo
-
, it doesnt recognize
the incorrect pairing of the nucleotides and continues replication. The cells of mtDNA are
then depleted with ethidium bromide (EtBr). EtBr inhibits mtDNA replication by
intercalating into DNA. Then, cells were repleted to make homoplasmic mutant cells by
removing EtBr and allowing cells to replicate.
o
cells are made when they are depleted of
their mtDNA through prolonged incubation with EtBr [14].
In this study, we intended to characterize NIH-3T3 mouse fibroblast cells carrying
mutant mtDNA by investigating the effects of these mutations on glucose consumption and
lactate production. The experiments include three different types of cell lines:
o
cells,
mutant cells, and wild type cells.
o
cells do not have mtDNA; therefore, they lack respiration
due to the absence of subunits of ETC which are encoded by mtDNA. Each mutant cell
contains homoplasmic mutation. And wild type cells are cells with no mutations. One way to
characterize these cells is to compare the wild types, mutants, and
o
cells for their growth
and the concentration of glucose and lactate, using YSI-2300, during their growth in daily
intervals. Glucose was measured to observe how much glucose the cells are consuming or
uptaking. Lactate was measured to observe how much lactate the cells are producing. It is
hypothesized that wild type cells will grow faster than mutants and
o
cells . And also that,
mutants and
o
cells will produce more lactate per cell due to their dysfunctional
mitochondria. The experiment shows the difference between cell lines in two aspects
mentioned above, i.e growth curve and lactate production. It also shows the trend between
cell density and lactate production per cell.
Another study will be performed, in the near future, by using Oxygraph-2k high-
resolution respirometry and investigating the rate of oxygen consumption in each of these
cells by using inhibitors for Complex I, II, III, IV, and V. Both of these studies have been
done in the lab. The goal is to test these mutant cells for defects in mitochondrial function.

Materials and Methods

Cell Growth
NIH-3T3 mouse fibroblast cells have been derived from Swiss mouse embryo tissue.
3T3 is abbreviation of 3-day transfer, inoculum 3x10
5
cell.

Inducing mutations
Wild type cells were transfected with Polexo
-
gene that is flanked by loxP. Once
mtDNA accumulates one mutation per mtDNA molecule, Polexo
-
is eliminated from the
cells. Mutants used in these experiments are following:
Mutants Position Mutation Amino Acid Protein

Mut 13 g3652a TgT->TaT Cys->Tyr ND1 (C1)

Mut 15 t15283c tAT->cAT Tyr->His CyTB (C2)

Mut 144D c13870t gGA->aGA
Gly-
>terminator ND6 (C1)

Mut 9i g11204a CgA->CaA Arg->Gln ND4 (C1)

Mut 14 g3652a TgT->TaT Cys->Tyr ND1 (C1)

Mut 39 a122g rRna1

Mutants were provided for the study by the lab.


90

Sequencing
The mt-DNA was sequenced to make sure that the given cell lines contained only one
type of mutation.

DNA Isolation
Total genomic DNA was isolated from all the cell lines by adding 500 L of lysis
buffer. It contains 10 mM Tris-HCl at pH 8.0, 2 mM EDTA, and 0.5% SDS. 10 L of
Proteinase K added. The mixture was swirled properly and was incubated overnight at 37
o
C.

DNA Extraction
50 L of 5 M NaCl was added to the cell lysate. It was mixed with 500 L of
chloroform/isoamyl alcohol (24:1) in eppendorf tubes. The solution was vortexed until it had
milky appearance. The solution was centrifuged for 10 min at 10000-12000 RPM. The top
layer, without disturbing the interface, was transferred into the clean tube. For 2
nd
extraction,
500 L of chloroform/isoamyl was added to this top layer and mixed and centrifuged. Again,
the top layer was collected into a clean tube and 500 L of chloroform/isoamyl was added,
mixed, and centrifuged for 3
rd
extraction.

DNA Precipitation
Once the samples were centrifuged (again it should have three layers), 400 L of top
layer was collected and transferred it into a new tube. 700 L of ice-cold isopropanol was
added and mixed by hands and centrifuged for 10 min at 10000-12000 RPM. At this point,
DNA pellet should be at the bottom of the tube and supernatant was carefully poured out.

DNA Wash
DNA pellet were washed with 500 L of 70% EtOH, centrifuged for 5 minutes.
EtOH was removed using micropipette without touching the pellet. 200 L of de-ionized
sterile H
2
O was added to the pellet and stored in the freezer overnight.

PCR Amplification
To test for the presence of mtDNA
Reaction mixture was prepared to amplify Pol gene and mtDNA. Pol gene is
present in the nuclear genome that encodes a polymerase gamma for replication of mtDNA.
PCR reaction was assembled. For 1x reaction: 5.0 L of 2x master mix (GoTaq Green from
Promega contains polymerase, dNTP, ions), 5.0 L dH
2
O, 1.0 L of DNA template, 0.1 L
100 M forward primer with sequence of ACTGGATGGATATCAGCAGTGCCA, and 0.1
L of 100 M reverse primer with the sequence of ATGTGTTCTGTGCCTCTGTCAGGT.
They were loaded on 96 well PCR plates. The PCR conditions were: initial denaturation at
94
o
C for 30 seconds, 35 cycles of denaturation at 94
o
C for 7 seconds, annealing at 54
o
C for
20 seconds, extension at 72
o
C at 60 seconds, and a final extension at 72
o
C for 3 minutes.
Every sample that contained Pol gene (i.e. every DNA sample had nuclear DNA) and
mtDNA was amplified (include in results and discussion, also include the picture of the dye).
These fragments were separated by Gel Electrophoresis.





91

To confirm the presence of mtDNA mutations
Reaction mixture was prepared to amplify the region where the mutant gene was
located. Our database was used to find the corresponding primers to amplify the specific
fragment. PCR reaction was assembled. For 1x reaction: 5.0 L of 2x master mix (contains
polymerase, dNTP, ions), 1 L of DNA template, 2.5 L of 2 M forward primer and 2.5 L
of 2 M reverse primer. List of primers is shown in the table below. They were loaded on 96
well PCR plates. The PCR conditions were: initial denaturation at 95
o
C for 30 seconds, 35
cycles of denaturation at 94
o
C for 10 seconds, annealing at 54
o
C for 20 seconds, elongation
at 72
o
C at 30 seconds, and a final extension at 72
o
C for 3 minutes. These fragments were
separated by Gel Electrophoresis (Gel picture is not shown in this paper).

Mutant Forward Primer Reverse Primer
Mut 13 10f-CACTATTCGGAGCTTTACGAGC 7r-TAGGTTGGTGCTGGATATTGTG
Mut 15 44f-GGAACAACCCTAGTCGAATGAA 24r-GTTTCACGGAGGATGGTAGATT
Mut 144D 40f-AGCAAATCCATATTCATCCTTCTC 22r-TGGGTGTGTTTTTCGTATGTTT
Mut 9i 32f-ATTTGAAGCAACCTTAATCCCA 18r-TTAGTTCTCGTGTGTGTGAGGG
Mut 14 4f-GCCTACACCCAGAAGATTTCAT 4r-AGACAGTTGGACCCTCGTTTAG
Mut 39 48f-AGAATCATTAGTCCGCAAAACC 2r-TCATTGGCTACACCTTGACCTA

Gel Electrophoresis
The PCR products were separated using Gel Electrophoresis. The gel was prepared
using 1.2 g of GenePure LE mix with 120 mL of 1x Tris Acetate EDTA (TAE) buffer. The
buffer was prepared using 8 mL of 50xTAE buffer and diluted with 400 mL of H
2
O. 3-4 L
of the product was electrophoresed on 1% agarose gel and visualized using ethidium bromide
staining. The gel was run at 70V for 45 minutes or until the bands were three-fourths down
the gel.

Dye-terminator sequencing Reaction
Excess primers and dNTPS were removed with ExoSAP-it clean-up kit. The
remaining PCR products were treated with 2.0 L of Exo-SAP. PCR machine was used to
incubate the mixture. The protocol to incubate in the PCR is: 4 cycles at 37
o
C for 15 minutes,
1 cycle at 80
o
C for 15 minutes, and final hold at 10
o
C. The product is now ready for use in
DNA sequencing.
The sequencing reaction was assembled. A mixture of reagents was prepared using 50
L of Bigdye from Applied Biosystems (it consist of AmpliTaq DNA polymerase, dNTPs,
fluorescent ddNTPs), 175 L of 5x buffer, and 775 L of deionzed H
2
O. ddNTPs are
labeled with flurophores, which emit light when hit with a laser. This method of sequencing
is also called dye-terminator sequencing. The reaction was assembled using 10 L of this
mixture with 1.5 L of primers, and 1.0 L template. List of sequencing primers is shown in
the table below. The PCR conditions were: initial denaturation at 96
o
C for 60 seconds, 35
cycles of denaturation at 96
o
C for 10 seconds, annealing at 50
o
C for 10 seconds, elongation
at 60
o
C at 4 minutes, final extension at 72
o
C for 3 minutes, and rapid ramp to 4
o
C and hold
until ready to purify.





92

Mutant Sequencing primer
Mut 13 11f-GCCCATTCGCGTTATTCTTTAT
Mut 15 45f-CCCGAATGATATTTCCTATTTGC
Mut 144D 41f-AATACCCGCAAACAAAGATCAC
Mut 9i 33f-ATACCCCTTCATCCTTCTCTCC
Mut 14 5f-AATCAACTCGTCTATGTGGCAA
Mut 39 1f-ACAAAGCAAAGCACTGAAAATG

Purification
The samples were purified using dideoxy terminator remover (DTR) plates. The
plates contain granules such as Sephadex and it was centrifuged at 750 rpm for 5 minutes.
Sephadex will form the column. Sequencing products were added to this column and the
products were separated by size. DNA molecule will pass through the granules, while
ddNTPs will get stuck in the pores of granules. At this point, the product is purified and
mailed to Functional Biosciences in Madison, WI.

Cell Culture
Preparation of the medium
NIH-3T3 cells were grown in Dulbeccos Modified Eagles Medium (DMEM)
containing 4.5 g/L glucose, 4 mM L-glutamine, 3.7 g/L of sodium bicarbonate, 110 mg/L of
sodium pyruvate, 50 mg/L of uridine, and 50 mg/L of gentamycin. After filtering the medium
through 0.22 m polyethersulfone membrane, the pH of the medium was between 7.2-7.4.
10% of fetal calf serum (FCS) was added to the medium. FCS contains growth factors, such
as proteins and low levels of antibodies.

Cell Plating
Cells were plated at the density of 10,000 cells in 2 mL of medium per well in 6-well
plates. All of the cell lines (for 7 days, in this experiment) were plated on the same day and
were placed in the cell culture incubator containing 5% CO
2
at 37
o
C.

Trypsinisation
After 15-24 hours, a plate was removed from the incubator. 1 mL of the medium from
each well was collected in the eppendorf tubes, and the rest of the medium was discarded.
Cells were trypsinized using 250 L of Trypsin EDTA. Trypsin EDTA 1x contains 0.05%
trypsin/0.53 mM EDTA in HBSS (Hanks buffered salt solution) with sodium bicarbonate
and without calcium and magnesium. Cells were incubated for 2 minutes and were checked
under the microscope for detached, round figures. 750 L of the fresh medium was added
and cells were resuspended by pipetting and collected for counting.





93

Cell Count
Cells were counted using Coulter Counter ZM. Cells were
diluted in 1:10 ratio in Isoton II diluents (9 mL of Isoton and 1
mL of cells). The diluted cells were counted in 12 sec interval for
4 to 5 times and results were recorded. Average cell growth
during each time interval and cells doubling time were
calculated. Average cell growth was determined using an
antiderivative function (area under the curve). Cells doubling
time was determined using formula below and the trendline
function in Excel.






Glucose and Lactate Assay
Method to record glucose consumption and lactate secretion
Glucose and lactate concentrations were measured using YSI 2300 STAT PLUS:
Glucose & L-Lactate Analyzer. It is designed to provide measurement of glucose in whole
blood, plasma or serum; and of L-lactate in whole blood, plasma, or cerebrospinal fluid. 1
mL of the collected medium was tested with this instrument. It is very easy to use and has an
automated calibration setup. The sample is placed under the aspirator and the aspirator
collects 25 L for each sample. Along with the medium from
each cell lines, standard medium was also being tested to see
the variance between each day. The units for the
concentration were given in mmol/L. Average lactate
concentration and glucose concentration were calculated and
graphed on Excel. The average rate of lactate production and
glucose uptake per cell per hour was calculated by the change
in lactate or glucose concentration between each time interval
divided by the average number of cells in that time interval
divided by the time interval. The units for lactate production
and glucose uptake were pmol/cell/hr.



Results

We tested each type of cell lines to confirm that wild type had not accumulated any
mutation,
o
cell didnt contain mtDNA, and mutants strictly contained homoplasmic
mutations. To test the wild type and
o
cells, two genes were amplified, Pol and mtDNA
fragment. Figure 1 shows that wild type contained both mtDNA and nDNA, while in
o
cell
line only nDNA is present.
Double Time (hrs) --- Nt = No*2^(t/T); where Nt = # of cells at time t
No = # of cells at time 0
t = change from starting time to calculating time
T = doubling time (time it takes for cell to double)


94



Mutations are confirmed to be homoplasmic and they were analyzed by
chromatograms:
DNA sequencing results below shows the presence of mutation in mutant cell lines.
Capillary electrophoresis separates the product by sizes of fragments. Each fragment is hit
with laser beam and fluorescent peak trace chromatograms are recorded on the computer.
The recorded sequences were analyzed in our lab using the software called Seqman. Figure 2
shows the snapshots of each mutant fragments and it verifies that mutation is present in all
cells. NC is the control or the wild type nucleotide sequences. Position number tells on what
position the mutation occurred and the chromatograms tell what the mutation is.




1 2 3 4 5
1.0kb
Name of the clone
Mutation
0.5kb
Figure 1The PCR fragments were separated
on 1.0% Agarose Gel. PCR amplified two
fragments: mtDNA and Pol gene. The top
band shows the presence of mtDNA at about
1.0 kbp. The bottom band shows the presence
of nuclear Pol gene. 1) DNA ladder 2) No
Template Control (NTC) 3)
o
control 4) WT
control 5) WT 420 6)
o
425.
o
cell line does
not contain mtDNA since top band is missing.


95












Some mutants grew better than wild type, while other mutants grew poorer than wild
type.

Different cell types look differently under the microscope. Wild type and mutant cell
lines look similar because of their branched cytoplasm, while
o
cells look round and have
shortened cytoplasm. Cell growth was studied for each cell line. All the cell lines were
treated under the same conditions throughout the experiment. Figure 3 shows the fold
increase in cells throughout 24 hour period during 7 days. Cell doubling time is show in
Table 1.

Figure 2Results of chromatograms: Each chromatogram shows sequence for a different mutant. For
example, mutation in Mut 13 occurred in position 3652 and a g nucleotide changed to an a. And so
forth.


96










0
10
20
30
40
50
60
70
80
0 50 100 150 200
F
o
l
d

I
n
c
r
e
a
s
e
Time(hrs)
WT420
Rho425
Mt13
Mut15
Mut144D
Mut9i
Mut14
Mut39
WT 420
o
425 Mut 13 Mut 15 Mut 144D Mut 9i Mut 14 Mut 39
Doubling
Time (hrs)
34 51 27 32 32 39 39 41
Figure 3 Cell growth in wild type,
o
, and mutant cell lines. Cells were plated at seeding density of 10K in 2 mL of medium.
It was observed that some of the mutants were growing faster (or in lesser time) than wild types. This graph just shows that
the range varies between wild type cells,
o
cells, and mutant cells. And that there is no correlation wild type cell line is shown
in red,
o
cell line is shown in black, and mutant cell lines are shown in gray.
Table 1Doubling time. It was calculated using the formula mentioned above. It tells how the time it takes for cells to
multiple into two.


97

An interesting phenomenon was observed.
Lactate production per cell decreases as cell density increases


The following experiments were done to observe the cause of the pattern

Three factors that are definite changing througout the previous experiments are cell
density, glucose concentration, and lactate concentration. Could any of these
parameter/s be the reason for decrease in lactate production per cell?
Four follow-up experiments were performed to answer the question speculated from
my results. Common procedures were followed throughout these experiments. Cells were
plated in 12-well plates in regular DMEM (same medium as used in previous experiments).
After 24 hours, the medium was replaced with different concentration of glucose and lactate.
And the cells were incubated for 24 hours. The medium was collected to obtain the readings
of glucose and lactate concentrations using YSI 2300 STAT Plus.

Varying glucose concentration
Different cell densities (200K, 100K, 50K) were plated with varying glucose
concentration.

Varying lactate with low-glucose
Higher cell densities (200K) were plated on low glucose medium and varying lactate
concentration.

Varying lactate with high glucose
Same cell densities of 100K were plated on high glucose medium and varying lactate
concentration.

0
1
2
3
4
5
6
75 100 125 150
L
a
c
t
a
t
e

p
r
o
d
u
c
t
i
o
n

(
p
m
o
l
/
c
e
l
l
/
h
r
)
Time (hrs)
WT420
Rho425
Mut13
Mut15
Mut144D
Mut9i
Mut14
Mut39
Figure 4The rate of lactate
production per cell. This graph
shows that at later hours when
cell density is higher, the rate of
lactate production per cell
decreases. This graph is mainly
to show the trendline while
disregarding the types of
mutant. Red colored line shows
WT, black colored line shows

o
cell, and gray colored lines
show all the mutants. The
graph shows the time point
starting at 75 hours.



98

Lactate production per cell is dependent on cell density. Lactate production per cell is
not depended on glucose concentration.
As cell density increases, the production of lactate per cell decreases. This study
strongly correlates with the results from previous experiments with all of cell lines. Also,
different glucose concentration gives the same decreasing trend in lactate production per cell.


Glucose consumption is not dependent on glucose concentration. Lactate production is
dependent on glucose consumption.
Glucose consumption remains constant regardless of the amount of glucose present in
the media. According to my studies, seeding density of 200K cells need at least 9 mM
glucose concentration to be happy in the media. Figure 6 shows this. Lactate production is
dependent on glucose consumption. All of glucose that cells are consuming is being utilized
towards the production of lactate. Table 2 shows the relationship between glucose and lactate
by showing that one molecule of glucose produces two molecules of lactate.
Glucose consumption overall Lactate Production overall
4.9mM 9.29mM 14mM 22.4mM
200K Plated 4.91 8.60 9.32 9.30
100K Plated 4.91 7.74 7.91 7.50
50K Plated 4.46 5.34 5.44 5.00








0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 50 100 150 200 250
L
a
c
t
a
t
e

p
r
o
d
u
c
t
i
o
n

(
p
m
o
l
/
c
e
l
l
/
h
r
)
Numberofcell(x1000)
9.29mMGC
14.0mMGC
22.4mMGC
4.9mM 9.29mM 14mM 22.4mM
200K Plated 9.72 16.85 18.36 19.69
100K Plated 9.62 15.45 16.06 15.89
50K Plated 8.92 10.85 11.26 10.69
Figure 6shows the correlation between glucose consumption and lactate production when glucose was supplemented in the
medium. The green colored blocks are being compared to the orange blocks. Cells at 200K seeding density consumed all of
glucose at 4.9 mM and most of glucose at 9.29 mM glucose concentration. Green chart shows that as the glucose
concentration increases, the consumption remains constant. Orange chart shows that as lactate production remains fairly
constant as well. The ratio of glucose to lactate is 1:2. Table 2 shows this.
Figure 5Lactate production
(per cell) is dependent on cell
density and not dependent on
glucose concentration. This
shows that lactate production
per cell is dependent on cell
density. It strongly
corresponds to the previous
experiments with different cell
lines. It also shows that trend
is not affected with different
glucose concentration.


99








Lactate production is inhibited by the presence of lactate in the medium.
This phenomenon explains why the production of lactate per cell decreases as cell
density increases.



Discussion

Mutants and
o
cells behaved unpredictably.
This particular experiment didnt provide good comparison results between wild type
and mutant cells. Some wild types grew better while others didnt. A possible hypothesis is
that different treatments during generating mutants could make them grow differently.
Generation I cell lines were treated with doxycycline to turn off the Polexo
-
.

Therefore,
Polexo
-
is still present, but just not expressed. Cells from generation II were transfected with
bacteriophage P1, which contains Cre-recombinase. Cre-recombinase catalyzes site-specific
between loxP sites. Polexo
-
is mapped in between two loxP sites. Therefore, Cre-
recombinase cuts Polexo
-
gene which stops the induction of mutation. It has been showed
that the endonuclease activity of cre recombinase is toxic for cells and inhibits cell growth
[15]. Mentioned by [15], the occurrence of Cre-induced DNA damage in mouse embryo
fibroblasts is very rapid. The mechanism for how it damages the genome is not yet clear.
Generation I cell lines include Mut 13, 15, 144D. Figure 1 clearly shows that these mutants
grew better than WT because they do not carry this gene. Generation II cell lines include WT
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
1.5 10 19
L
a
c
t
a
t
e

P
r
o
d
u
c
t
i
o
n

b
y

c
e
l
l

(
p
m
o
l
/
c
e
l
l
/
h
r
)
LactateConcentrationinthemedia(mmol)
LactateProduction
4.91mM 9.29mM 14.0mm 22.4mM
200K plated 1.98 1.96 1.97 2.12
100K plated 1.96 2.00 2.03 2.12
50K plated 2.00 2.03 2.07 2.14
Table 2This shows that the ratio of glucose to lactate is
approximately 2

Figure 7Monitoring
lactate production in high
glucose media during the
presence of different
concentrations of
supplemented lactate. This
figure shows that presence of
lactate in the medium
suppress its production. It
also shows that glucose
consumption remains
constant. Again, this
corresponds to the
observations in previous
studies.


100

420,
o
, mut 14, 9i, and 39. One way to study the growth rate without bias is to make cybrid
cell lines. That is to transfer mtDNA mutations into the same nuclear background.

Glucose is utilized as the primary source for energy.
The original hypothesis, that mutants and
o
would produce more lactate due to their
dysfunctional mitochondria, failed to prove right. All the cell lines behaved very similarly.
Therefore, it can be concluded that all the cell lines were producing majority of their ATP by
utilizing glucose and producing lactate. It has been demonstrated by numerous studies, that
rapidly dividing cells, such as fibroblasts, convert glucose into lactate at very high rate while
very little glucose goes towards oxidation [16]. In L929 cell, utilization of glucose to lactic
acid is 29 times greater than oxidation rate [17]. L929 cells, that are closely related to NIH-
3T3 cells, also show that glucose and glutamine mainly metabolizes in pyruvate, lactate,
alanine, proline, aspartate, and citrate [17]. Another explanation of this phenomenon could be
derived from the calculation of concentration of glucose + concentration of half lactate. If the
ratio stays close to the concentration of initial glucose, then we can assume that all of lactate
is being derived from glucose. When the ratio starts decreasing, then we can assume that
glucose is being consumed for biosynthesis. In the meantime while the glucose is being
utilized for generation of ATP, it is still questionable as to what amino acid is being
consumed for biosynthesis. Further studies need to be done to test the hypothesis of glucose
+ half lactate.

Correlation between cell density, glucose concentration, and lactate concentration to
lactate production.
The three recurring observations show very strong correlation that lactate production
per cell is dependent on cell density and lactate concentration in the medium. The presence of
more cells corresponds to the presence of higher lactate concentration in the medium. And
the presence of higher lactate in the medium suppresses the production of lactate. At higher
concentration of lactate in the medium, the enzyme called lactate dehydrogenase (LDH)
exhibits feedback inhibition, and therefore, the conversion from pyruvate to lactate is
decreased. Usually pyruvate is reduced to form L-lactate and NADH, produced from
glycolysis, is oxidized to NAD
+
.
As shown in Figure 5, even cells residing in different concentration of glucose
medium, act in a very similar fashion. Glucose concentration in the medium does not affect
lactate production per cell. Cells will consume, according to my studies, ~10 mM glucose
even when total of 23 mM is present in the medium. And it is also shown that if not all,
majority of all the glucose consumed, is being converted into lactate, as clearly shown in
Table 2. This idea that all of glucose uptake in cells, including wild types and mutants, is
used to generate ATP is very intriguing. It raised questions such as how are cells creating
building block for synthetic reactions. Couple of possible answers could be that other amino
acids in the medium are playing major role in synthesizing proteins and biosynthetic
products, or maybe lactate is being produced by other amino acids while glucose is used for
biosynthesis. All of these, and many more could be possible answers. And this opens the
door towards further research.





101

Efficient way to characterize mutant cell lines is to study respiration.
The method of measuring glucose and lactate to characterize mutant cell lines does
not prove to be an efficient pathway because, as mentioned above, rapidly dividing cells
mainly tend to generate energy by converting glucose to pyruvate to lactate. Therefore,
another approach to study these cell lines is to study their rate of oxygen consumption by
feeding inhibitors for each complexes and evaluating how rate of oxygen consumption is
affected by different mutations.

Acknowledgments

I would like to give special thanks to the National Science Foundation for its financial
support. Thank you, Dr. Turrens for accepting me into this program and giving me this
opportunity. Also, a very special thanks to my mentor, Dr. Mikhail Alexeyev, for letting me
gain this experience in his lab and exposing me to this new array of research and giving me
different perspective in Science. Dr. Rafik Fayzalin taught me all the lab techniques and
helped me throughout the program. And Michael Perez provided help with statistics. Also
very special thanks to my mentors at my home university for giving me insights on applying
to this program. And lastly, special thanks to my family and friends for their love and moral
support.

References

1. Anderson, S., et al. 1981. Sequence and organization of the human mitochondrial
genome. Nature 290(5806):457-65.

2. Dai, D.F. and P.S. Rabinovitch. 2009. Cardiac aging in mice and humans: the role of
mitochondrial oxidative stress. Trends Cardiovasc Med 19(7):213-20.

3. Harman, D. 1992. Free radical theory of aging. Mutat Res 275(3-6):257-66.

4. Holt, I.J., A.E. Harding and J.A. Morgan-Hughes. 1988. Deletions of muscle
mitochondrial DNA in patients with mitochondrial myopathies. Nature 331(6158):717-9.

5. Suzuki, Y., et al. 1997. Diabetes mellitus associated with 3243 mitochondrial
tRNA(Leu(UUR)) mutation: clinical features and coenzyme Q10 treatment. Mol Aspects
Med 18 Suppl:S181-8.

6. Chen, J.H., C.N. Hales and S.E. Ozanne. 2007. DNA damage, cellular senescence and
organismal ageing: causal or correlative? Nucleic Acids Res 35(22):7417-28.

7. Taylor, R.W. and D.M. Turnbull. 2005. Mitochondrial DNA mutations in human
disease. Nat Rev Genet 6(5):389-402.

8. Schaefer, A.M., et al. 2004. The epidemiology of mitochondrial disorders-past, present
and future. Biochim Biophys Acta 1659(2-3):115-20.



102

9. Thorburn, D.R. and H.H. Dahl. 2001. Mitochondrial disorders: genetics, counseling,
prenatal diagnosis and reproductive options. Am J Med Genet 106(1):102-14.

10. Marchington, D.R., D. Barlow and J. Poulton. 1999. Transmitochondrial mice carrying
resistance to chloramphenicol on mitochondrial DNA: developing the first mouse model
of mitochondrial DNA disease. Nat Med 5(8):957-60.

11. Sligh, J.E., et al. 2000. Maternal germ-line transmission of mutant mtDNAs from
embryonic stem cell-derived chimeric mice. Proc Natl Acad Sci U S A 97(26):14461-6.

12. Pinkert, C.A. and I.A. Trounce. 2002. Production of transmitochondrial mice. Methods
26(4):348-57.

13. McKenzie, M., et al. Production of homoplasmic xenomitochondrial mice. Proc Natl
Acad Sci USA 101(6):1685-90.

14. Trounce, I. and D.C. Wallace. 1996. Production of transmitochondrial mouse cell lines
by cybrid rescue of rhodamine-6G pre-treated L-cells. Somat Cell Mol Genet 22(1):81-5.

15. Loonstra, A., et al. 2001. Growth inhibition and DNA damage induced by Cre
recombinase in mammalian cells. Proc Natl Acad Sci USA 98(16):9209-14.

16. Newsholme, P., S. Gordon, and E.A. Newsholme. 1987. Rates of utilization and fates
of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages.
Biochem J 242(3):631-6.

17. Lanks, K.W. 1987. End products of glucose and glutamine metabolism by L929 cells. J
Biol Chem 262(21):10093-7.