(The FASEB Journal. 1999;13:S121-S127.)
© 1999 FASEB
Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity
MILLIE HUGHES-FULFORD1 and
VICKI GILBERTSON
Laboratory of Cell Growth and Differentiation, Veteran's Affairs Medical Center and University of California Medical Center, San Francisco, 94121
1Correspondence: Millie Hughes-Fulford, Ph.D., Director, Laboratory of Cell Growth (151F), Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. E-mail: milliehf{at}spacedu.com
 |
ABSTRACT
|
|---|
The well-defined osteoblast line, MC3T3-E1 was used to examine
fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN
matrix accumulation after growth activation in spaceflight. These
osteoblasts produce FN extracellular matrix (ECM) known to regulate
adhesion, differentiation, and function in adherent cells. Changes in
bone ECM and osteoblast cell shape occur in spaceflight. To determine
whether altered FN matrix is a factor in causing these changes in
spaceflight, quiescent osteoblasts were launched into microgravity and
were then sera activated with and without a 1-gravity field. Synthesis
of FN mRNA, protein, and matrix were measured after activation in
microgravity. FN mRNA synthesis is significantly reduced in
microgravity (0-G) when compared to ground (GR) osteoblasts
flown in a centrifuge simulating earth's gravity (1-G)
field 2.5 h after activation. However, 27.5 h after
activation there were no significant differences in mRNA synthesis. A
small but significant reduction of FN protein was found in the
0-G samples 2.5 h after activation. Total FN protein
27.5 h after activation showed no significant difference between
any of the gravity conditions, however, there was a fourfold increase
in absolute amount of protein synthesized during the incubation. Using
immunofluorescence, we found no significant differences in the amount
or in the orientation of the FN matrix after 27.5 h in
microgravity. These results demonstrate that FN is made by
sera-activated osteoblasts even during exposure to microgravity. These
data also suggest that after a total period of 43 h of spaceflight
FN transcription, translation, or altered matrix assembly is not
responsible for the altered cell shape or altered matrix formation of
osteoblasts.—Hughes-Fulford, M., Gilbertson, V. Osteoblast fibronectin
mRNA, protein synthesis, and matrix are unchanged after exposure to
microgravity.
Key Words: growth activation • spaceflight • mRNA expression
|
INTRODUCTION
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THE UNIQUE ENVIRONMENT of spaceflight places
unusual stress on, and causes many physiological changes in, organisms
that evolved in a 1-G environment (1-3)
. Some
of the basic physiological changes include muscle atrophy and loss of
calcium and mineralized bone. The bone loss that accompanies
spaceflight is one of the most serious health hazards associated with,
and impediments to, long-term manned missions (4
,
5
). Biomedical studies of manned spaceflight have
consistently indicated a continuous and progressive loss of calcium and
weight-bearing skeletal bone. Evidence from animal studies suggests
that the bone loss occurring in spaceflight is due to a decrease in
bone formation (6)
. The decrease in bone formation and
osteoblast growth in microgravity is likely due to both indirect
effects such as systemic or hormonal changes in the body and direct
effects such as cellular response to the lack of mechanical stress.
Proper cell attachment to the extracellular matrix
(ECM)2is required for normal cell growth (7)
. Previous studies
have demonstrated that bone exhibits altered ECM assembly of collagen
(8-9)
, actin (10)
, and altered cell
morphology and nuclear shape of osteoblasts (9-12)
in
microgravity. The cell surface adhesion protein, fibronectin (FN),
mediates the attachment of a variety of cell types to other ECM
components such as type I collagen, heparin, and proteoglycans. Cell
binding to FN is mediated by integrins, which recognize the
arginine-glycine-aspartic acid (RGD) sequence on the FN molecule. The
integrins on the cytoplasmic surface bind talin and 
-actinin,
forming a link between ECM components and the cytoskeleton. Binding of
FN can also be mediated by RGD-independent interactions such as
4ß1 and proteoglycan receptors including
CD44 and syndecans (13)
. FN is known to be a factor in
cell shape and growth regulation (14
, 15
). It
has been demonstrated that FN adhesion, resulting in the ability of
epithelial cells to spread over a planar surface, can promote cell
cycle progression from G0 to S phase (16)
.
Although the mechanisms are still under investigation, FN has also been
shown to increase mechanochemical signal transduction intermediates in
fibroblasts (17)
. FN is present during all stages of
matrix-mediated bone formation with synthesis rates being high during
cell proliferation and attachment (18)
. In addition,
blocking FN attachments has been shown to inhibit osteoblast
differentiation and in mature bone cultures resulted in apoptosis
(19)
.
For these reasons we were interested in determining what effect
microgravity would have on the FN message, protein, and matrix
organization. To define the role of FN in the altered cell shape and
altered ECM matrix formation we performed space shuttle experiments in
Biorack hardware in the following gravity environments: ground (GR),
0-G flight (0-G), and 1-G flight
(1-G) conditions. Early passage MC3T3-E1 osteoblasts were
used because they have been shown to exhibit osteoblast behavior during
both proliferation and differentiation stages of bone mineralization
(20
, 21
). We have demonstrated previously
that these osteoblasts change cell shape in microgravity and that
osteoblast genes are induced by application of gravity and vibration
forces (22-24)
. In this study, we asked whether FN plays
an important role in microgravity-induced changes in cell shape and
matrix formation. Therefore, analysis of FN gene induction, protein
synthesis, and matrix formation was done on samples collected under
different gravity conditions during spaceflight. Our results
demonstrate that extended exposure (>24 h) to microgravity does not
cause changes in FN synthesis and matrix.
|
MATERIALS AND METHODS
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Cell culture
The MC3T3-E1 osteoblast cell line is clonally derived from
embryonic mouse calvaria (17
, 18
) and was
kindly provided to us by Dr. M. Kumegawa (Josai Dental University,
Japan). The cell line was maintained at low passage. Cells were grown
in alpha minimal essential medium (-MEM) with 10% fetal calf serum
(Hyclone Labs Inc., Logan, UT) supplemented with 2 mM
L-glutamine (Sigma, St. Louis, MO), 25 mM HEPES, and
antibiotic-antimycotic solution (100 U penicillin/ml, 0.01 mg
streptomycin/ml, 0.25 mg Amphotericin B/ml). Cells were grown in a
37°C incubator with 5% CO2. They were fed three times a
week and passed when the cells reached confluence. For flight
experimental samples, 120,000–200,000 cells were plated onto
non-coated, sterile 11 x 22-mm glass coverslips (Thomas
Scientific, Swedesboro, NJ) placed in six-well plates, and grown in
10% serum containing -MEM overnight. Cell-coated coverslips were
transferred into the plungerbox units in 2% serum-containing medium
for flight. In cooperation with NASA flight rules, the plungerbox units
were held for 17 h in the shuttle at mid-deck temperature before
launch. This, combined with low serum-containing media placed the
osteoblast in a quiescent state before space shuttle launch. Many
previous culture experiments sent into space have been actively growing
at the time of launch. Moreover, lack of refrigeration has previously
limited on-board sample preservation and therefore many experiments
were terminated after landing on earth. Even for the few experiments
that had the opportunity for on-board collection, the data were
compromised by flight-imposed limitations including the following: lack
of sufficient sample numbers, addition of supplements directly
affecting gene expression, lack of fresh media changes, and lack of
on-board 1-G controls. To achieve our goal of studying FN
metabolism in microgravity, we launched cells that were in a quiescent
condition and were not sera activated until on-orbit in the
microgravity environment. There were four samples for each time point
in 0- and 1-G flight samples and ground samples.
Refrigerator and freezer space was available for reliable sample
storage after collection in microgravity. In contrast to some previous
spaceflight experiments, media supplements such as dexamethasone,
ß-glycerol phosphate, and ascorbic acid were not added to our media
because these agents are known to directly affect gene expression and
cell morphology of the osteoblast.
Biorack facility and osteo hardware
Biorack is a multi-user facility that consists of incubators
with variable gravity centrifuges, a cooler, a freezer, and a sealed
glovebox. Two identical Biorack modules were used: one remained on
earth and the other was integrated into SpaceHab and flown on the space
shuttle. Biorack has an important advantage over other microgravity
facilities in that it provides a small-radius (78 mm) slow-rotating
(107.0 ± 0.5 rpm) centrifuge. The centrifugal force results in a
1-G on-board control. Because of proximity, both the
0-G and 1-G samples experience identical launch
vibrations, accelerations, cosmic radiation, and other unknown
conditions of flight. The only difference between the 0-G
and 1-G flight groups is the gravity parameter. In addition,
an identical experiment was performed in the Biorack module on earth
with a 2-h delay from in-flight procedures.
The "Osteo" experimental flight hardware was designed according to
European Space Agency specifications for use in the Biorack facility
and constructed by Centrum voor Constructie Mechatronica (CCM, Neuenen,
The Netherlands). The hardware consisted of the CCM plunger box and its
Type I container developed for spaceflight cell culture. The Type I
container provided a second level of fluid containment. The plunger
boxes were designed to provide a sterile environment for cell growth
activation and fixation in a microgravity condition. The plunger box is
composed of two independent culture chambers that each hold two 11 x 22-mm glass coverslips. Each culture chamber was a separate sample.
For each condition a sample size of n = 4 was used.
Each culture chamber has series of compartments filled by either 10%
serum-containing -MEM or fixative, which can be exchanged into and
out of the cell culture compartment by manually releasing a
spring-loaded plunger.
Experimental time line
Quiescent cells were stored in the mid-deck locker until 18 h after launch. The astronauts then transferred the samples into a
37°C incubator (either in the 1-g centrifuge or
0-g static rack) for 1 h before stimulating the cells
to grow by changing their media from 2 to 10% serum-containing medium
(t = 0). Zero-G and 1-G samples
were fixed during flight with a modified guanidinium thiocyanate
solution (for RNA preservation) t = 0, 2.5 h, and
27.5 h or 3.7% formaldehyde solution (for morphology) at
27.5 h. Samples fixed with the guanidinium thiocyanate solution or
the formaldehyde solution were stored at -20°C or +5°C,
respectively, for the remainder of the space shuttle mission. There
were four separate samples for each gravity condition at each time
point.
RNA isolation, reverse transcription (RT), and polymerase chain
reaction (PCR)
RNA from cultured MC3T3-E1 osteoblast cells was extracted by a
modified guanidinium thiocyanate method, which was based on the
protocol previously described by Chomczynski and Sacchi
(25)
. Linear RT-PCR was performed as previously described
(9
, 19
, 20
,
23-26
). Because sample size was so small (200,000 cells)
RNA content was held constant and linear RT-PCR was accomplished by
varying the number of PCR cycles. PCR conditions were established so
that the amplification reaction was stopped in the linear range and
reaction products could be accurately quantified and compared.
Oligonucleotide primers were designed to span at least one intron in
order to detect any contaminating genomic DNA. FN primer sequences were
designed from Genebank sequences by MHF: sense, 5'-CCC CAG TGA TGT TAG
CAG ACC; anti-sense, 5'-GAT GGC AAA AGA AAG CAG AGG. Operon
Technologies, Inc. (Alameda, CA) synthesized the oligonucleotides. PCR
bands were identified by agarose gel electrophoresis and photographed
with a direct screen instant camera DS-34 (Polaroid Corp., Cambridge,
MA). For quantification, the bands were scanned at 400 dpi with a LaCie
Silver Scanner III (LaCie, Portland, OR) and stored as TIFF files. The
area and density of each band were determined using Sigma Gel (SPSS,
Inc., Chicago, IL). The total number of pixels was corrected to the
micrograms of RNA used for each RT-PCR. Sample size for all conditions
was n = 4. Descriptive statistics and unpaired
Student's t test analysis was performed with Sigma Stat v.
2.0 (SPSS, Inc.).
Protein isolation
Protein remaining in the guanidinium thiocyanate solution after
the RNA extraction was precipitated out of each sample using a method
similar to that previously described (27)
. Briefly, 2.5 ml
of isopropanol was mixed with the remaining organic phase from the
RNA extraction, incubated at room temperature for 15 min, and
centrifuged at 12,000 g for 10 min at 4°C. The
resulting protein pellet was washed three times in 4 ml of 0.3 M
guanidine hydrochloride in 95% ethanol. For each wash samples
were incubated in the wash solution at room temperature for 20 min
followed by centrifugation. Protein was resuspended in 2% sodium
dodecyl sulfate (SDS). Total protein was quantified for each sample in
triplicate by DC Protein Assay (Bio-Rad Laboratories, Hercules, CA)
according to the manufacturer's protocol and read on a Dynatech MR5000
96-well plate reader. The standard curve and protein quantification
results were interpreted with BioLinx 2.0 software (Dynatech
Laboratories, Chantilly, VA).
Western blots
Five micrograms of total protein for each sample were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE;
7.5% polyacrylamide). Two molecular weight markers were also loaded
onto the gel: a prestained molecular weight marker (Bio-Rad) was
used to track the migration of the samples through the gel and a Cruz
MarkerTM (Santa Cruz Biotechnology, Santa Cruz, CA), which is
compatible with the horseradish peroxidase-conjugated secondary
antibody and can be visualized by chemiluminescence, and was recorded
directly onto the film. Proteins were transferred onto nitrocellulose
filters with 100 volts for 1 h. No detectable protein, as measured
by staining, remained in the gel after transfer. Filters were blocked
with 5% blotto (20 mM Tris, 150 mM NaCl, pH 7.6, with 5% nonfat milk
and 0.02% Tween-20) for 2 h. Primary antibodies for FN were
purchased from DAKO (Carpinteria, CA). Molecular weight markers and
secondary antibodies were purchased from Santa Cruz Biotechnology.
Proteins were visualized by enhanced chemiluminescence (Amersham Life
Sciences Inc., Arlington Heights, IL) and recorded on Kodak XAR-5 film.
To ensure that the Western blot signal was within a linear range, the
amount of protein loaded on the gel, primary and secondary antibody
concentrations, and film exposure length were all optimized. Films were
digitized with an Epson Expression 836XL scanner into Adobe Photoshop
(Adobe Systems Inc., Mountain View, CA). Density analysis was performed
with the Sigma Gel program (SPSS, Inc.). All densitometry readings were
normalized to nanograms of total protein loaded onto the gel. Sample
size for each condition was n = 4. Statistical analysis
was performed in Sigma Stat and graphed with Sigma Plot (SPSS, Inc.).
Cell morphology
Osteoblasts were fixed on coverslips with 3.7%
formaldehyde in phosphate-buffered saline (PBS) during flight and
stored at 5°C for 1–2 weeks before staining. There was no visible
difference between control samples that were immediately stained and
samples stored in formaldehyde at 5°C. For FN visualization,
coverslips were blocked with 10% goat serum in PBS, incubated with
anti-FN (rabbit) primary antibody (DAKO), and then incubated with
anti-rabbit secondary labeled with Texas Red. Dried osteoblast
coverslips were mounted onto slides and photographed with a Zeiss
Axioscope microscope at x40 and x100 magnification. Slides were
processed at the same time under identical conditions. Photographs were
taken at identical exposure times and conditions. Photographs were
taken at pre-determined areas of the samples. Slides were digitized
with an Epson Expression 836XL scanner into Adobe Photoshop for
computer analysis with NIH image. Controls not shown were incubated
with protein blocker, in the absence of FN antibody, and then with the
secondary antibody for the appropriate incubation times.
|
RESULTS
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FN matrix
To compare the organization of the FN portion of the ECM under
different gravity conditions, osteoblast cells were launched on the
space shuttle in a semi-quiescent state and were sera activated 19 h after reaching orbit. Twenty-seven and one-half hours after
activation, the osteoblasts, which were grown on untreated glass
coverslips in microgravity (0-G) or in a 1-G
centrifuge on board the space shuttle (1-G) or ground
controls (GR), were fixed with formaldehyde for visualization of the FN
matrix on conclusion of the flight.
Samples were treated with a polyclonal antibody that selectively
recognizes cellular FN to immunolocalize the FN matrix. The majority of
the FN matrix had a diffuse distribution throughout the ECM. After
approximately 1 day in microgravity there were no apparent changes in
the FN matrix orientation between the 0-G and the
1-G or GR samples (Fig. 1
). Quantification of total fluorescence from randomly selected
slides revealed no significant differences among the different gravity
conditions (Table 1
). As an additional control, in samples without primary antibody,
there was no FN matrix seen (image not shown).
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Figure 1. Immunofluorescence localization of FN in MC3T3-E1 osteoblast cultures
grown on earth (GR), in microgravity (0 G), or in an
on-board 1-g centrifuge (1 G). Cells were seeded
on non-treated glass coverslips and grown in 10% fetal calf serum
overnight. Cells were sera deprived before launch and sera activated in
microgravity 19 h after launch. Twenty-seven and one-half hours
after activation, while still in microgravity, cells were fixed with
formaldehyde. Cells were stained with an anti-FN primary antibody and
then with anti-rabbit secondary antibody labeled with Texas Red.
Photographic exposure times and conditions were identical using a x100
objective.
|
|
FN protein synthesis
The Western blot shown in Figure 2
shows representative samples of the relative amount of FN per cell
because proteins were extracted from samples plated with an equal
number of cells. Most of the immunoreactive material migrated to the
position of authentic FN, although some lower molecular mass was also
seen. In control blots performed without primary antibody present,
these bands were not seen (data not shown). At the 2.5 h timepoint
there was a small but significant reduction of FN protein in the
0-G sample when compared to GR samples (Fig. 3
).This difference was lost at the later timepoint, where there was no
significant difference in total FN protein between the various gravity
conditions. FN protein content is evident 27.5 h after activation.
FN protein is shown to have increased by approximately fourfold during
the 25 h between the two time points.
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Figure 2. Representative data from FN Western blot analysis. Osteoblast cultures
were sera activated in microgravity. Total protein extracts were
obtained from osteoblast grown at GR (lane 1), 0-G (lane 2),
and 1 G (lane 3). Five micrograms of total protein was
loaded per lane. Sample size was n = 4 for each
condition.
|
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Figure 3. FN protein quantification. Western blot films from 2.5 h and 27.5
h samples were digitized and quantified as described in Materials and
Methods. For each condition the samples size was n = 4
and presented as total pixels ± SD. In the 2.5 h
samples there was a significant difference between the 0 G
and GR samples (P < 0.030). There was approximately a
fourfold increase in FN protein in the 27.5-h samples compared to the
2.5 h samples with no significant difference between the gravity
conditions.
|
|
FN mRNA
To determine the level of regulation at which FN synthesis is
controlled, FN mRNA synthesis was measured using RT-PCR (Fig. 4
).A specific band corresponding to the expected FN PCR product was in
high copy number relative to most of the other genes analyzed. This
increase was more pronounced at the 27.5 h time point where we
reduced the total amount of RNA in the RT-PCR reaction in order to
remain in a linear amplification range. Final densitometry
quantification was corrected to micrograms of RNA so both the 2.5 h and
the 27.5 h time points could be directly compared.
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Figure 4. RT-PCR results for the amplification of FN mRNA. RNA was extracted from
osteoblast sera activated on ground (GR) or in microgravity (0
G) or in an on-board 1-G centrifuge (1
G). Results are representative of four samples for each
condition.
|
|
As shown in Figure 5
,2.5 h after initiation of osteoblast growth in microgravity, there
was a small but significant change in the total FN mRNA synthesis
between the 0-G and 1-G samples, with mRNA
synthesis for FN in the 0-G samples lower than 1
G. There was a trend for the GR samples to be higher, but it
was not significant due to outlying data points. In contrast, at the
27.5 h time point, there is no significant difference in FN mRNA
synthesis between samples grown in microgravity and those grown in a
1-G environment. In addition, at this later time point (27.5
h) the FN message was over twofold higher than at the 2.5 h timepoint.
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Figure 5. Quantification of FN RT-PCR. PCR bands from 2.5 h and 27.5 h
samples were digitized and quantified as described in Materials and
Methods. Total pixel density was corrected for the amount of RNA added
to the RT reaction. For each condition the sample size was
n = 4 and presented as total pixels ±
SD. In the 2.5 h samples there was a significant
difference between the 0-G and 1-G samples
(P < 0.001). There was approximately a twofold
increase in FN message in the 27.5 h samples compared to the 2.5-h
samples with no significant difference between the gravity
conditions.
|
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|
DISCUSSION
|
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The loss of bone in spaceflight is thought to be due to a lack of
osteoblast activity and possibly to a defect in morphological assembly
of the ECM. Previous work has demonstrated a cell shape change in
osteoblasts exposed to microgravity after 15 or more parabolic
weightless events or as long as 4 days. Change in cell shape is
heterogeneous, with a portion of the cells having nuclear elongation
and a smaller area of contact with the surface (9
,
10
). Experiments on STS-56 demonstrated that osteoblast
cells had a change in cell shape after 4 days of weightlessness
(9)
and, more recently, experiments by Kacena et al.
(11)
demonstrated a significant change in osteoblast cell
shape in microgravity as early as 3 h and as late as 3 days. None
of these experiments had the convenience of an on-board 1-G
centrifuge. We have recently published evidence (12)
that
nuclear morphology as well as the cell morphology was altered in
microgravity when compared to on on-board 1-G control in
these Biorack experiments. The changes in actin cytoskeleton and the
nucleus were heterologous in 0-G with a portion of the cells
showing nuclear elongation. In this study, in an attempt to explain the
changed osteoblast cell shape, we examined the ECM FN on the Biorack
samples.
FN plays a crucial role in growth and differentiation of osteoblasts
(28)
and is crucial for normal growth and cell spreading
(15)
. We considered FN as a potential moiety for
controlling the altered cell shape seen in microgravity. This study was
undertaken to assess the role of FN in alterations of cell shape and
cell matrix under microgravity conditions. By launching quiescent
osteoblasts and activating and collecting them in microgravity, we were
able to analyze gene activation and FN metabolism under microgravity
conditions. In addition, for the first time, a total study of cell
regulation of protein message, polypeptide synthesis, and
immunolocalization of matrix was accomplished in spaceflight samples.
Because protein and RNA were extracted from the same samples, we were
able to analyze both the transcriptional and translational control of
FN. Although there was a slight and significant change in both
messenger RNA and protein 2.5 h after sera activation, at later
timepoints FN was not regulated differently in microgravity. We do not
know why the earlier time points show significantly lower levels of
both FN mRNA and protein synthesis but it is possible that a slow
response to sera activation could be causing a lag in response since it
is known that growth factor-induced signal transduction is sensitive to
gravity (28)
. The presence of de novo synthesis
of FN by MC3T3-E1 osteoblast is supported by results that show an
accumulation of both FN message (more than twofold) and protein (more
than fourfold) during the 25-h incubation period between the early and
late collection times. This finding is consistent with reports of
others which show that FN protein, unlike other osteoblast matrix
molecules, is synthesized throughout osteoblast growth and
differentiation (29)
. These data suggest that
microgravity-induced cell morphology alterations are not caused by an
aberration in the FN portion of the ECM. Extrapolation of our findings
also suggests that changes in FN are not a causal factor in the loss of
osteoblast growth in spaceflight. However, it is possible that FN
binding and receptor-mediated signaling may be affected in microgravity
and play a role in other altered responses.
The analysis of FN matrix from parallel samples fixed in flight for
immunofluorescence localization studies confirmed the Western blot
analysis showing that FN protein synthesis was not affected by
microgravity. In addition, these data reveal that FN protein had been
synthesized and exported out of the cells. The results also demonstrate
that typical organization of the matrix was occurring. Finally, our
data shows that 1 day after sera activation in 0-G, the
samples have an overall regulation of FN that is indistinguishable from
1-G on-board controls or ground controls. Taken together, it
is likely that changes in bone matrix formation, cell shape, and loss
of bone growth previously observed in microgravity are not dependent on
changes in FN regulation.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by NASA Grant NAG-2-1086 and the Department
of Veteran's Affairs. We thank Kimberly Gasuad for preparation of the
fibronectin immunofluorescence and Wayne Sezto and Tephillah Chi for
their technical assistance at Hanger L. We thank the space shuttle
flight crew from STS-84, especially cosmonaut/astronaut Elena V.
Kondakova for her excellent execution of the experiment in flight. We
appreciate the support of the Bionetics crew at Kennedy Space Center,
the NASA team, and the ESA Biorack team.
|
FOOTNOTES
|
|---|
2 Abbreviations: FN, fibronectin; ECM,
extracellular matrix; RGD, arginine-glycine-aspartic acid; GR, ground;
-MEM, -minimal essential medium; RT-PCR, reverse
transcriptase-polymerase chain reaction; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered
saline.
Received for publication September 16, 1998.
Revision received February 8, 1999.
|
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ABSTRACT
INTRODUCTION
