Microheterogeneous retinol-binding proteins (RBPs) are secreted
by the porcine uterus under the influence of progesterone. Reported
functional capacities of these proteins have included their ability
to bind retinol and immunosuppressive effects on mitogen-induced
T-lymphocytes. In this study, porcine uterine RBPs were isolated,
purified, and tested for effects on mitogen-induced lymphocyte
proliferation and thrombin-induced platelet aggregation. The proteins
were purified and two charge forms were tested for activity. Uterine
flushings were collected on Day 15 after estrus and fractionated
by Sephadex G-75. Subsequent fractionation by Mono P HR 5/20 fast
protein liquid chromatography (FPLC) chromatofocusing yielded
two peaks containing two bands each as determined by native-polyacrylamide
gel electrophoresis (PAGE). Fractionation by Mono Q HR 5/5 anion
exchange column resulted in an identical separation pattern. Slab
isoelectric focusing revealed four bands with a pI range of 4.7
to 5.2. Fractionation of all four proteins with a preparative
isoelectric focusing cell resulted in isolation of two of the
bands to homogeneity as determined by sodium dodecyl sulfate (SDS)-
and native-PAGE. Individually each of the two bands, as well as
the purified set of uterine RBPs, completely inhibited thrombin-induced
human platelet aggregation at physiological levels (13.6 µM).
The uterus of the pig secretes a variety of proteins. Under
the influence of progesterone, a large percentage of the protein
profile consists of a family of low molecular weight (19,000 -
22,000) acidic proteins which have been shown to bind retinol
(1,2). Porcine uterine retinol-binding proteins
(RBPs) are heterogeneous as revealed by native-PAGE (3,4,5)
and two-dimensional-(2D)-SDS-PAGE (6,7). Several
distinct isoelectric variants have been reported ranging in number
from four (3,4,7) to six (5,6).
Microheterogeneity has also been demonstrated to occur with serum
RBPs of the human (8,9), rat (10)
and chicken (11), placental RBPs of the cow
(12), sheep (13) and human
(14), as well as RBPs from the porcine conceptus
(15). Recently it has been reported that all
charge forms of uterine RBPs from the pig were recognized by antiserum
to human serum RBPs on western blots (16).
Two of the more divergent charge forms of uterine RBPs showed
complete amino acid sequence identity with pig serum RBP. It has
been suggested that the uterine RBPs charge forms may be slightly
modified forms of a single protein product corresponding to a
classical form of RBPs (16).
During pregnancy, porcine uterine RBPs are believed to function
to transport retinol from the maternal circulation to the conceptus
(17). The pig has a non-invasive epitheliochorial
placentation. Because of the superficial attachment of the trophoblast,
the conceptus is unable to obtain nurients directly from the maternal
blood supply. Instead, the conceptus must depend on carrier proteins
from the uterine epithelium to supply nutrient requirements. The
proper concentration of retinol is vital to both cell differentiation
and embryonic development (reviewed in (18)).
In addition to the passive carrier role of RBP, studies have
indicated that this protein may also play an active role in cell
regulation. For example, although there is conflicting evidence
(19,20), this protein family has been reported
to inhibit mitogen-induced lymphocyte proliferation (21,22).
Uteroglobin from the rabbit is a protein similar to porcine uterine
RBP. Like uterine RBP, it has been shown to be secreted under
the influence of progesterone and found to be a major protein
component of uterine fluid during early pregnancy at the time
of implantation (23,24). It is a low molecular
weight acidic protein which has been shown to both bind progesterone
(25), as well as effect cell function (26,27,28).
Uteroglobin has been demonstrated to contain anti-inflammatory
properties based on its ability to inhibit both monocyte and neutrophil
chemotaxis and phagocytosis in vitro (26,27).
It has also been shown to inhibit thrombin-induced platelet aggregation
(28). These properties are attributed to its
potent ability to inhibit phospholipase A2 (PLA2)
(29). The aim of this study was to purify porcine
uterine RBPs to homogeniety and to test their effects on thrombin-induced
platelet aggregation and mitogen-induced lymphocyte proliferation.
MATERIALS AND METHODS
Collection of Porcine Uterine Fluid
Uterine flushings were collected from mature cycling pigs
of Yorkshire, Duroc and Landrace breeds on Day 15 after estrus.
The flushings were collected according to the method as described
by Segerson et al.(19). Reproductive
tracts were surgically removed postmortem and each horn was subsequently
flushed with 50 ml sterile 0.01 M PBS (pH 7.4, 4°C) to collect
the accumulated proteins (19). Flushings were
stored at -70°C.
Gel Filtration Chromatography
Each uterine flushing was membrane filtered (0.2 µm)
and concentrated overnight via vacuum concentration using cellulose
dialysis tubing. Concentrated samples were applied to a 72 x 2.6
cm column of Sephadex G-75 (Pharmacia LKB, Piscataway, NJ), equilibrated
in 0.01 M phosphate buffered saline (PBS), pH 7.4 and run with
a mean flow rate of 5 ml/h into 5 ml fractions. Column eluents
were monitored and all protein concentrations were determined
by method of Lowry et al.(30) using
bovine serum albumin (BSA) as a standard. Fractions eluting between
250-300 ml were pooled, membrane sterilized, concentrated by YC05
Amicon ultrafiltration (Amicon, Danvers, MA) and stored at -135°C.
Deionized water was used to prepare all solutions. All purification
procedures were carried out at 4°C.
The presence of RBPs was verified by native-PAGE on a PhastSystem
(Pharmacia LKB) using a PhastGel (8-25%) and separated for 220
Vh. Fractions were analyzed by SDS-PAGE using a PhastGel (8-25%)
and separated for 110 Vh. The SDS-PAGE program on a PhastSystem
was modified using a constant 220 V in replacement of 250 V. Both
native and SDS gels were developed with Coomassie brilliant blue
(PhastGel Blue R) using the staining method for the native-PAGE
PhastSystem program. Molecular weight markers (Sigma, St. Louis,
MO) included BSA (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate
dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen
(24,000), trypsin inhibitor (20,100) and -lactalbumin (14,200).
The isoelectric points of the RBPs were determined on a PhastSystem
using PhastGel isoelectric focusing (IEF) media. The PhastGel
IEF 4-6.5 was prefocused for 75 Vh at 2000 V, 2 mA. Gels were
run at 2000 V limiting, 3.5 W and 5 mA for 654 Vh (~32 min). The
pH gradient was determined using calibrated pI markers pH 2.5-6.5
(Pharmacia LKB). Standards included human carbonic anhydrase B
(6.55), bovine carbonic anhydrase B (5.85), ß-lactoglobulin
A (5.20), soybean trypsin inhibitor (4.55), glucose oxidase (4.15)
and amyloglucosidase (3.50).
Fast Protein Liquid Chromatography(FPLC)
Chromatofocusing was performed on a FPLC Mono P HR 5/20 column
(Pharmacia LKB) equilibrated in 0.025 M bis Tris, pH 6.7 at 20°C.
Equilibrated RBPs Sephadex G-75 aliquots were separated using
a 0.5 ml/min flow rate, a 0.1 AUFS, a 0.5 cm/min chart speed,
and collected in 0.5 ml fractions. The column was eluted with
10% polybuffer 74, pH 5.0 creating a 7-5.0 pH gradient. The absorbance
of column effluent was monitored continuously at 280 nm.
Anion exchange was performed on a FPLC Mono Q HR 5/5 column
(Pharmacia LKB) at 20°C. Pooled Sephadex G-75 fractions containing
RBPs were equilibrated over Superose 12 hr and applied in 500
µl aliquots. The Mono Q HR 5/5 column was equilibrated in
50 mM Tris-HCl, 1 mM dithiothreitol, pH 8.0 and eluted with a
linear gradient of 0-0.5 M NaCl in 30 min. The elution buffer
consisted of the starting buffer containing 1 M NaCl. The column
was run using a 0.5 ml/min flow rate, a 0.1 AUFS, a 0.5 cm/min
chart speed and collected in 0.5 ml fractions. All FPLC column
fractions containing protein were concentrated in a SpeedVac (Savant,
Farmingdale, N.Y.), and extensively dialyzed against 0.01 M PBS.
Preparative Isoelectric Focusing
Final purification of uterine RBP was achieved within a pH
4.5-5.4 ampholyte gradient using a Rotofor Cell (Bio-Rad Laboratories,
Richmond, CA). Samples of up to 15 mg of the G-75 fractionated
RBPs fractions were desalted into deionized water over a PD-10
column (Pharmacia LKB). The RBP, in a 1.1% ampholyte solution
(55 ml) was loaded into the electrofocusing Rotofor Cell and focused
for 4 h at 4°C at a constant 12 W. The final parameters of
1400 V and 8 mA were held for at least 2 h. After focusing, 20
fractions were harvested and immediately concentrated by a SpeedVac
concentrator. Fractions were incubated at room temperature for
30 min in 1 M NaCl and dialyzed against 2 liters 0.01 M PBS (4
changes in18 h). Each sample was then fractionated over a 52 x
2.5 cm Sephadex G-50 (Pharmacia LKB) column equilibrated in 0.01
M PBS at 4°C. Experimental conditions were identical to those
described for G-75 gel filtration.
Lymphocyte Proliferation Assay
Phytohemagglutinin-induced blastogenesis of porcine lymphocytes
in the presence of a dose range of porcine uterine flushings (PUF)
was conducted with minor variations of methods previously described
by Murray et al.(21). Methodological
differences include the following. Lymphocytes were isolated with
Sepracell-MN. Optimal concentrations of lymphocytes (for maximal
blastogenesis) were determined to be 1.0 to 2.5 x 106
cells/ml with phytohemagglutinin at 5 µg/ml. At 48 hours
of culture, 0.5 µCi [3H]-thymidine was added to
each culture well (microtiter plate). Counts were done in Ready
Safe (Beckman Instruments) in a Beckman LS8000 liquid scintillation
counter. Results were expressed in comparison to maximally stimulated
control cultures less background, as a percent of the control.
Platelet Aggregation Assay
Human platelets were used in these studies because of their
ready availability and because of the reliability of our aggregation
assays which were developed for human platelets. Human blood was
collected by venipuncture from healthy volunteers who reported
to be free of medication for at least 10 days prior to donation.
Coagulation was prevented with acid citrate dextrose (85 mM trisodium
citrate, 71 mM citric acid, 111 mM dextrose) in a ratio of 1:6
(v/v). Whole blood was centrifuged at 120 x g for 15 min at room
temperature and the plasma drawn off and recentrifuged at 1100
x g for 15 min. The resulting platelet pellet was resuspended
in 9.5 ml calcium-free modified Tyrode's buffer (pH 6.5) containing
2 mM MgCl2(31). Accumulation of
ADP was prevented by adding 0.1 ml (60 µg/ml) of apyrase
per wash. To remove calcium, EGTA (5.26 mM final concentration)
was added prior to centrifugation of the first wash only. Following
resuspension, the platelets were centrifuged at 1100 x g for 10
min at room temperature. The wash procedure was repeated three
times and the final platelet pellet was resuspended in Tyrode's
buffer (pH 7.4) with 2 mM CaCl2 and 1 mM MgCl2(31). Platelet aggregation studies were performed
using a dual channel aggregometer (Chrong-log Corporation) interfaced
to an Apple II Microcomputer (32). An Epson
FX-80 printer was used to produce continuous recordings of platelet
aggregation curves. All aggregation experiments were performed
in siliconized glass cuvettes containing 0.35 ml platelet suspension
(3 x 108 platelets/ml). Uterine RBPs were preincubated
with platelets for 1 min at 37°C under constant stirring
at 1000 rpm. Parallel control aggregation experiments were conducted
in the absence of RBPs but with 0.35 ml of 0.01 M PBS buffer to
maintain volume equivalence in control and treated assays. An
empirically determined amount (0.014 U/ml) of
thrombin (Sigma, St. Louis, MO) was added to induce an approximately
60% aggregation response in the control. The reaction was measured
over four min. Aggregation was quantitated by light transmittance
and reported as percentage of maximum transmittance. Two controls
were used in each assay: PBS and RBP dialysate. Dialysate was
obtained by removing RBPs from the purification buffer by amicon
filtration and was used as a control in every experiment. Sample
protein was also heat inactivated (56°C for 30 min) and tested
for its effect on thrombin-induced platelet aggregation.
Gel Filtration Chromatography
Porcine uterine secretions were chromatographed by Sephadex
G-75 into three protein fractions (Figure 1).
Uterine RBPs eluted in fractions 50-59 (250 and 300 ml) as determined
by native-PAGE. A total of up to 30 mg of protein was recovered
in this set of fractions (designated peak 3) obtained from flushings
of the uterus of a single Day 15 cycling gilt. The recovery of
RBPs from this first step represented a yield of 22-30% of total
protein from crude flushings. Peak 3 was concentrated for further
A typical native-PAGE pattern of the uterine starting material
and RBPs following Sephadex G-75 fractionation are shown in (Figure 2),
lane 1 and 2, respectively. The RBPs consistently appeared as
4 bands, although up to 6 bands have been observed in individual
samples. SDS-PAGE analysis of these samples revealed a molecular
weight of 22,000 for RBP.
The isoelectric points (pIs) of RBPs were determined using
a PhastSystem and PhastGel IEF 6-4.5. The isoelectric focusing
pattern of RBPs under non-reducing conditions revealed four bands
focusing between pH 5.2 and pH 4.6 as shown in (Figure 3).
The IEF pattern of RBPs following fractionation by Sephadex G-75
is shown in Fig. 3, lane 2. RBPs which was subsequently fractionated
by the Rotofor cell is shown in Fig. 3, lane 3. The pH gradient
was determined using calibrated low pI standards (lanes 1 and
4), and the pI of the RBPs were determined to be 5.20, 4.96, 4.90
and 4.75 for the four isoforms present in porcine uterine secretions.
Proteins were eluted from a Mono P HR 5/20 FPLC column as
two peaks by employing a pH gradient 7-5.0 as shown in (Figure 4).
Native-PAGE analysis showed peak 1 contained the two least acidic
proteins (Figure 4, lane 1) and peak
2 contained the two most acidic proteins (Figure 4, lane 2).
The least acidic proteins in peak 1 eluted at pH 5.6 and the most
acidic proteins at pH 5.1.
FPLC Anion Exchange Chromatography
Fractionation of pooled Superose-12 FPLC RBPs over a Mono
Q HR 5/5 column revealed similar results. (Figure 5)
shows bound proteins were eluted with two peaks with a linear
gradient of 0-0.5 M NaCl. Peak 1 was eluted at 0.13-0.15 M NaCl
and peak 2 was eluted at 0.23-0.24 M NaCl. Analysis by native-PAGE
are shown in (Figure 6A) which revealed
peaks 1 and 2 contained the two least acidic proteins (lane 1)
and the two most acidic proteins (lane 2), respectively. SDS-PAGE
analysis revealed that peaks 1 and 2 each migrated as a single
band as shown in (Figure 6B) lanes 1
and 2, respectively.
Preparative Isoelectric Focusing
Purification of RBPs by a Rotofor cell resulted in the highest
resolution of the four protein bands. (Figure 7)
shows the pH profile and a native-PAGE analysis of Sephadex G-75
fractionated RBPs (lanes 1 and 8) along with Rotofor fractions
14-9 (lanes 2-7, respectively). Rotofor fractions 16 and 4 consisted
of single bands as determined by native-PAGE as shown in (Figure 8, lane 1 and 3),
respectively. The homogeneous band in fraction 16 was eluted at
pH 5.2 and fraction 4 was eluted at pH 4.8. Refractionation of
fractions 5-15 (Figure 8, lane 2) at
a narrow pH range of 4.8-5.2 did not result in resolution of the
central bands. Single band homogeneity of fractions 16 and 4 had
been achieved as shown in lane 1 and 3, respectively.
In one run, a total of 13.6 mg of G-75 fractionated RBPs was
loaded on the Rotofor. A recovery of 2.38 mg in fractions 5-15
was obtained consisting of only four RBPs bands. This represents
a yield of 17.5% of the RBPs from the Rotofor and 3.9% from the
original flushing from one porcine uterus. The yield of RBPs varied
between Rotofor preparations, but final overall yields of up to
9.5% have been obtained. The fractions exhibiting one homogeneous
band each, contained 0.52 mg in fraction 4 and 0.26 mg in fraction
16 representing a 3.8% and 1.9% yield from a Rotofor fractionation,
respectively and an overall yield of 0.84% and 0.42%, respectively.
Effect of PUF and RBPs on Lymphocyte Proliferation
A total of nine blastogenesis experiments with PUF and/or
RBPs derived from as many animals were conducted. In no case did
an inhibition of blastogenesis occur, at any concentration, due
to either PUF or RBP. In spite of intensive effort to duplicate
conditions of the previous experiments (21,22),
we did not find suppression of PHA-induced blastogenesis. In fact,
both PUF and RBPs were consistently stimulatory to blastogenesis
compared with controls. However, the stimulatory activity was
not related to concentration of either PUF or RBPs. Results of
these experiments are summarized in (Table 1).
Platelet Aggregation Assay
A Rotofor preparation of four pooled RBPs and two individual
isoforms of RBP, were tested for their effects on thrombin-induced
human platelet aggregation. The mixture of four RBPs inhibited
thrombin-induced aggregation in washed human platelets in a concentration
dependent manner (Figure 9). Complete
inhibition was achieved with 13.6 µM RBP. The most acidic
(fraction 4) and the least acidic (fraction 16) RBPs both caused
complete inhibition of thrombin-induced platelet aggregation at
13.6 µM as shown in (Figure 10 A and 10B.).
Limited quantities of the RBPs isoforms precluded running dose
response curves for these proteins. Platelet aggregation was not
inhibited in any experiment when platelets were incubated with
70µl PBS alone, 70µl dialysate, or 13.6 µM heat
inactivated sample protein (data not shown).
Porcine uterine RBPs consists of a family of microheterogeneous
proteins of similar molecular weight with four distinct isoelectric
variants. The heterogeneous RBPs family as well as two individual
isoelectric variants were isolated by a three-step-procedure.
Fractionation of uterine secretions by Sephadex G-75 gel filtration
resulted in the isolation of the small molecular weight proteins.
Preparative isoelectric focusing using a Rotofor produced purified
heterogeneous RBPs and allowed isolation of homogeneous preparations
of the most basic and most acidic variants. Yields of 3.9% of
total uterine protein were acheived for the heterogeneous RBPs
and 0.42% to 0.84% for individual isoforms.
Analytical isolation of uterine RBPs from the pig has been
reported using gel filtration, batch treatment with CM-cellulose
and anion exhange chromotography (2). Purification
methods of RBPs involving repetitive chromotographic steps typically
have yielded a low recovery of protein (2,33).
In addition to the high yield of uterine RBPs achieved with the
procedure described here, this is the first report of the purification
of a single, homogeneous porcine uterine RBPs isoelectric variant.
The isoelectric measurements of the four RBPs ranged from
5.2-4.8 as determined by slab gel isoelectric focusing. These
pI measurements were an order of magnitude lower than previously
reported measurements of 6.1 to 6.3 as determined by 2D-SDS-PAGE
(3,7). They are also lower than the isoelectric
points reported for porcine conceptus RBPs of 5.6 to 6.5 also
determined by 2D-SDS-PAGE (15). Subsequent
analysis of the proteins by FPLC anion exchange chromatography
revealed two major peaks which eluted at 0.14 M NaCl and 0.23
M NaCl. Each peak contained two charge variants as revealed by
native-PAGE. These charge variants correspond to the ionic charge
determination reported by Stallings-Mann et al.(16).
In previous studies by the authors (21,22),
PUF and the fraction we now call RBPs were suppressive to porcine
lymphocyte blastogenesis. Other, more recent experiments, have
not supported these observations. For example, Segerson et
al.(19,20) reported suppression of lymphocyte
blastogenesis by a 230 kD protein from PUF but no inhibition by
proteins ranging below 50 kD. Our results in the current study
are in agreement with the more recent reports (19,20),
but differ from them in that we find no suppression by unfractionated
PUF. The reasons for the disparity regarding effect on lymphocyte
blastogenesis between the earlier sets of experiments, those by
Segerson et al.(19,20), and the current
report are not apparent.
Secretion of RBPs by the endometrium during the peri-implantation
period may be important for local transport of retinoids to the
developing conceptus (1). Various effects of
retinoids on tissue and cell function have been demonstrated (18,34,35).
But the effects of RBPs on cell regulation remain to be elucidated.
Several uterine proteins have demonstrated cell regulatory action.
For example, uteroferrin, secreted from the porcine endometrium,
has been shown to be a hematopoietic growth factor (36)
and uteroglobin has been shown to contain anti-chemotactic and
anti-inflammatory activities (26,27). Uteroglobin
has also been shown to inhibit thrombin-induced platelet aggregation
(28). These properties are attributed to their
ability to inhibit PLA2 activity (29).
In this study, porcine uterine RBPs inhibited thrombin-induced
platelet aggregation in a dose dependent mannner. It is unlikely
that these inhibitory effects may be attributed to the action
of retinol or retinol bound to RBP. Retinol has been demonstrated
to stimulate thrombin-induced human platelet aggregation through
the activation of PLA2(37). Inhibition
of platelet aggregation by RBPs was eliminated by a heat treatment
of 56°C for 30 min, and there was no significant difference
between platelet aggregation incubated without sample protein
or with the negative control (RBP dialysate). These results indicate
that the RBP proteins were responsible for the effects demonstrated
and were not caused by an artifact of the buffer.
Furthermore, two RBP variants, purified to homogeneity, individually
caused complete inhibition of thrombin-induced platelet aggregation
at 13.6 µM (Fig. 10). The activity of these individual proteins
appear to be similar to each other and to RBPs as a group. These
results are consistent with the report that the uterine RBP variants
are slightly modified forms of a single protein product corresponding
to serum RBP (16) and that the isoforms are
likely to be functionally similar as well.
The effect of serum RBP on platelet activation in vivo has
not been reported. Normal levels of RBP in human serum range between
approximately 40-60 µg/ml (38) as opposed
to 200 µg/ml (6) in the uterus. This represents
13-20% of the amount of uterine RBPs reported here to completely
inhibit thrombin-induced platelet aggregation. In addition, RBP
is typically bound to the plasma protein, transthyretin, in the
blood (38) and thus may not be available to
express anti-platelet properties in the circulation. In contrast,
RBPs were not found to be complexed with transthyretin in the
porcine uterus (16). The results of this study
indicate that porcine uterine RBPs may potentially function as
an inhibitor of cell action and specifically block platelet aggregation
locally in the uterus. Inhibition of platelet aggregation may
be important during implantation and later in pregnancy to maintain
blood flow in the placental capillary beds.
Porcine uterine secretions contain four isoforms of retinol
binding protein (RBPs). Gel permeation chromatography with Sephadex
G-75 isolated the four RBP isoforms from the other major proteins
of the uterine secretions. Isoelectric points were determined
by isoelectric focusing to be 5.20, 4.96, 4.90, and 4.75, for
the different isoforms. FPLC chromatofocusing resolved the RBPs
into two fractions, one of which was composed primarily of the
less acidic isoforms (pI 5.20 and 4.96) and another which contained
the more acidic isoforms (pI 4.90 and 4.75). Similarly, FPLC anion
exchange chromatography resolved RBPs into two fractions with
similar composition to the fractions resulting from chromatofocusing.
Preparative isoelectric focusing with a narrow pH gradient (pH
4.5 to 5.4) in a Rotofer cell resolved the RBPs into a set of
16 fractions, of which fractions 4 and 16 were nearly homogenous
for isoforms with pIs of 4.75 and 5.20, respectively. Fractions
5 through 15 were mixtures of the four isoforms.
This study did little to clarify the issue of immunosuppressive
activity in porcine uterine secretions. In contrast to studies
of porcine uterine RBPs reporting inhibition of lymphocyte blastogenesis,
e.g., (21,22), and in agreement with
those which have reported no suppression or even stimulation of
blastogenesis, e.g., (19,20), this study
found no suppression and slight stimulation of blastogenesis by
RBPs. However, in contrast to other reports (19,20),
we found that unfractionated PUF was not suppressive. In this
study the effects of unfractioned uterine secretions and Sephadex
G-75 fractionated RBPs on lymphocyte blastogenesis were stimulatory
compared with controls but not in a dose-related manner.
When assayed for effect on human platelet aggregation, RBPs
were inhibitory in a dose-related manner. 13.6 µM RBP completely
inhibited thrombin-induced platelet aggregation. Isolated isoforms
of RBP (13.6 µM) were completely inhibitory to thrombin-induced
platelet aggregation, suggesting that all four isoforms are inhibitory
to platelet aggregation. Inhibition of platelet aggregation may
be an important function of uterine RBPs during implantation and
This research was supported by the College of Arts and Sciences,
the Interdisciplinary Doctoral Program in Molecular and Cellular
Biology, and the College of Osteopathic Medicine, as well as a
research grant from the Ohio University Research Committee.