PubMed Health⌕ Search

Biomedical subjects

B S Dunbar

Publications and source records attributed to B S Dunbar.

At least 19 recordsLinked to original sources
PubMed1992

Molecular analysis of the antigenicity and immunogenicity of recombinant zona pellucida antigens in a primate model.

The studies reported here are the first to demonstrate that recombinant zona pellucida (ZP) proteins will elicit a humoral immune response that recognizes native ZP proteins. Three cDNAs encoding rabbit ZP protein antigens expressed in bacteria were used to immunize cynomolgus monkeys. Four groups of six monkeys each were immunized with bacterially expressed cro-beta-galactosidase recombinant proteins encoded by a full-length cDNA (rc55) encoding the 55-kDa rabbit ZP recombinant protein (rec55), two partial cDNAs (rc75a and rc75b) encoding two recombinant peptides (rec75a and rec75b) of the 75-kDa rabbit ZP protein, and the plasmid-encoded cro-beta-galactosidase control protein. Initial immunizations with these fusion proteins using the muramyl dipeptide adjuvant did not elicit significant levels of antibodies to native or recombinant ZP proteins. Further immunizations were therefore carried out using recombinant ZP proteins conjugated to either protein A or keyhole limpet hemocyanin. Antibodies were detected in the groups immunized with the rec55 and rec75a; however, no antibodies were generated against the rec75b protein. These antibodies have been characterized by two-dimensional PAGE immunoblotting and shown to recognize antigenic domains associated with two of the native rabbit ZP proteins. Reprobes of these immunoblots with sheep anti-total native rabbit ZP proteins, affinity-purified on pig ZP, further demonstrate that a fourth distinct rabbit ZP antigen may be present. The characterization of species-conserved antigenic domains of mammalian ZP proteins is important for studies of the functional regions of ZP proteins and is critical for the design of safe and effective contraceptive vaccines.

Animals↗
PubMed1992

Localization of a carbohydrate antigen associated with growing oocytes and ovarian surface epithelium.

We used a monoclonal antibody (PS1) to a carbohydrate antigen to study the development of the oocyte and follicle during early stages of differentiation in several mammalian species. This antigen has been shown to localize within the cytoplasm of oocytes in primordial follicles as well as in growing oocytes. It is also localized within distinct layers of the zona pellucida (ZP) of developing follicles. Although this antibody was made against a specific ZP glycoprotein, the antigen also appears to be abundant in cells of the ovarian surface epithelium (OSE). The localization of this carbohydrate moiety has been observed in ovaries of rabbits of different ages as well as in the ovarian surface epithelium of other mammalian species including cat, cynomolgus monkey, baboon, and human. These studies demonstrate that there is an abundant carbohydrate antigenic determinant which is associated with both the mammalian oocyte and the ovarian surface epithelium but which is not apparent in other ovarian cell types or in non-ovarian secretory epithelium. This antibody probe should provide a valuable tool for studying the development and differentiation of the ovary, since this antigen is associated with two highly differentiated but distinct cell types.

Animals↗
PubMed1992

Characterization of equine zona pellucida glycoproteins by polyacrylamide gel electrophoresis and immunological techniques.

This study was designed to explore the composition of the equine zona pellucida (EZP) by one- and two-dimensional polyacrylamide gel electrophoresis (1D- and 2D-PAGE), silver staining and immunoblotting techniques. Antral follicles palpable on frozen-thawed equine ovaries were aspirated with a needle and syringe, and the resultant follicular fluid, cellular material and oocytes were pooled. Oocytes were placed in Petri dishes, moved by narrow-bore pipette to droplets of phosphate-buffered saline (PBS) and mechanically cleaned of cumulus cells. The EZP from these collected oocytes was solubilized, and then analysed by 1D- and 2D-PAGE. Silver stained 2D-PAGE of the EZP revealed the presence of three EZP glycoprotein families of apparent molecular mass ranges of 93-120 kDa, 73-90 kDa and 45-80 kDa. Immunoblot analysis of EZP glycoproteins resolved by 2D-PAGE using rabbit antisera against pig zonae pellucidae (R alpha HSPZ) confirmed the presence of three EZP glycoprotein families and established the existence of common epitopes between equine and porcine ZP glycoproteins. Further immunodetection using 2D-PAGE-separated glycoproteins illustrated that the 45-80 kDa family is recognized by the monoclonal antibody R5, developed against the porcine ZP glycoprotein of molecular mass 55-120 kDa. Guinea-pig antiserum against endo-beta-galactosidase-treated rabbit ZP 55 kDa glycoprotein (R55K), which specifically recognizes the rabbit ZP glycoprotein with the lowest molecular mass, also recognized the EZP 45-80 kDa glycoprotein family. Guinea-pig polyclonal antisera developed against total heat-solubilized rabbit ZP (GP alpha HSRZ) recognized the 73-90 kDa EZP glycoprotein family exclusively. After heat solubilization and treatment of EZP with endo-beta-galactosidase to remove polylactosaminoglycans, silver stained 1D-PAGE again demonstrated the presence of three glycoproteins with apparent molecular masses of 60, 75 and 90 kDa. The partially deglycosylated 60 kDa equine glycoprotein is recognized on immunoblot by the monoclonal antibody R5; the 75 kDa EZP glycoprotein is recognized by GP alpha HSRZ; and all three EZP glycoproteins separated by 1D-PAGE are recognized by R alpha HSPZ. These data add further support to the concept of cross-species zona pellucida glycoprotein antigenicity.

Animals↗
PubMed1990

Mouse phenylalanine hydroxylase. Homology and divergence from human phenylalanine hydroxylase.

The laboratory mouse represents an important model for the study of phenylalanine metabolism and the pathochemistry of phenylketonuria, yet mouse phenylalanine hydroxylase (PAH) has not been extensively studied. We report the cloning and sequencing of a mouse PAH cDNA, the expression of enzymic activity from the mouse PAH cDNA clone and the identification of mouse PAH and human PAH by two-dimensional PAGE of liver samples. These data confirm the expected homology of mouse PAH and human PAH and suggest differences in the primary sequence and the phosphorylation state of the two enzymes.

Amino Acid Sequence↗
PubMed1990

Identification of extracellular proteins in the rat cumulus oophorus.

We have examined the proteins associated with the mucous matrix of the rat cumulus oophorus and compared them to the composition of rat serum, follicular fluid, ampullary fluid, and oocyte-cumulus cell extract. The cumulus matrix was dispersed using Streptomyces hyaluronidase, and the proteins were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis and compared with proteins of the serum, proestrous follicular fluid, and postovulatory ampullary fluid and extracts of oocytes and cumulus cells. In addition to albumin and transferrin, which were common to all the fluids analyzed, the cumulus material contained many proteins in common with the follicular fluid and the ampullary fluid. However, the protein extract of the cumulus matrix also contained four major proteins not present in the other fluids analyzed. Two of these proteins were acidic and heterogenous in charge and size (MW approximately 81,000 and 100,000). The other two proteins were more basic and occurred at MW approximately 90,000 and 150,000. Our results show that the extracellular matrix of the cumulus contains proteins that are not present in the fluids that surround the oocyte.

Animals↗
PubMed1990

Effects of extracellular matrix on the expression of specific ovarian proteins.

A unique ovarian follicle cell culture system has been established to analyze the effects of extracellular matrix (ECM) on early granulosa cell differentiation. Primary and early secondary follicles isolated from ovaries of sexually immature rabbits were grown on poly-D-lysine or Englebreth-Holm-Swarm basement membrane biomatrix substrata (EHS) in serum-free, hormonally defined medium. Granulosa cells from these follicles were examined for growth pattern characteristics and for secretory protein synthesis by two-dimensional (2D) PAGE. Whereas some proteins were synthesized by cells on either matrix, the expression of other secreted proteins was markedly affected by the ECM used. Secretion of zona pellucida (ZP) proteins was demonstrated by ELISA assays and immunoblots of one-dimensional (1D) and 2D-PAGE separations of secreted proteins probed with monoclonal and epitope-selected antibodies. Expression of two ZP proteins was altered by ECM: 55-kDa endo-beta-galactosidase (EBGD)-treated ZP glycoprotein (55-kDaEBGD) was secreted by cells grown on either ECM, but a greater amount of 75-kDaEBGD was secreted by cells grown on poly-D-lysine. These studies are the first to show that granulosa cells from early-stage follicles express ZP proteins in vitro in the absence of oocytes, although proper post-translational modification may not occur. They also demonstrate the dramatic effect of ECM on the expression of these and other secretory proteins.

Animals↗
PubMed1990

The heterogeneity of porcine 32,000 Mr inhibin alpha-subunit: a gel electrophoresis and immunoblot study.

Porcine 32,000 Mr inhibin is a glycoprotein with one asparagine-linked glycosylation site on the alpha-subunit. The presence of carbohydrate on the alpha-subunit was visualized by periodate-Schiff (PAS) staining. This stain for carbohydrate also verified that the beta-subunit of 32,000 Mr porcine inhibin does not contain carbohydrate. When analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) under reducing conditions, the inhibin alpha-subunit consistently existed as a doublet, and by the PAS stain, both bands of the doublet were glycosylated. Analysis by two-dimensional (2D) PAGE further revealed the presence of charge isoforms of the alpha-subunit. The alpha-subunit of inhibin could be deglycosylated by N-glycanase, but not by endoglycosidase F, endoglycosidase D, or endoglycosidase H. When the N-glycanase-treated inhibin was analyzed by either 1D-PAGE or 2D-PAGE, the molecular size of the alpha-subunit was reduced by 3500 Mr. Each doublet band observed with reducing conditions in 1D-PAGE or 2D-PAGE for the alpha-subunit became a single band (spot) in the deglycosylated alpha-subunit. However, the charge heterogeneity detected by 2D-PAGE was retained, indicating that only a portion of this heterogeneity is attributable to the carbohydrate moiety. The in vitro biological activity of the deglycosylated inhibin was not different from the control sample. The composition of the carbohydrate in inhibin was investigated with the Dionex carbohydrate analyzer. Inhibin contains fucose, glucosamine, galactose, mannose, and glucose. Colorimetric analysis revealed the presence of sialic acid. Taken together, this implies some aspect of the peptide portion of the molecule is involved in charge heterogeneity. Inhibin may have an unusual carbohydrate component, as evidenced by the detection of glucose in inhibin samples. The absence of glucose in the carbohydrate moiety of another glycoprotein fraction that accompanied the inhibin through all the same fractionation procedures argues against the artifactual introduction of glucose in the fractionation medium per se.

Amidohydrolases↗
PubMed1989

Necessity of adjuvants for inducing effective antibody response to zona pellucida antigens.

Rhesus monkeys evoked a vigorous antibody response to a single heteroimmunization dose of zona pellucida antigens, when these were administered along with complete Freund's adjuvant. The antisera recognized all the three major porcine zona glycoprotein families and the animals were rendered amenorrheic after such immunization. Monkeys immunized with zona without adjuvant, however, failed to show any anti-zona antibody response and had normal menstrual cycles. Zona pellucida glycoproteins are thus not effective immunogens unless administered along with a powerful adjuvant.

Adjuvants, Immunologic↗
PubMed1989

Use of a synthetic peptide adjuvant for the immunization of baboons with denatured and deglycosylated pig zona pellucida glycoproteins.

Baboons were immunized using a synthetic peptide adjuvant with two purified pig zona pellucida glycoproteins. The major zona pellucida glycoprotein (ZPI) was purified by preparative isoelectric focusing, and the 80 K deglycosylated zona pellucida protein (ZPIII) was purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis. The immunogenicity as well as the antigenicity of these proteins were evaluated by characterizing antibodies using the enzyme-linked immunoassay and by immunoblotting of zona pellucida proteins separated by high-resolution two-dimensional polyacrylamide gel electrophoresis. Both groups of animals developed antibodies that recognize the major zona pellucida glycoprotein, (ZPI) and immunoblotting procedures provide evidence that two of the major porcine zona pellucida glycoprotein families (ZPI and ZPIII) contain shared antigenic determinants. The animals immunized with ZPI showed decreased levels of estrogen throughout their menstrual cycles, and two of the animals ceased ovulation. All animals in the group immunized with ZPIII had a significant reduction in the numbers of antral follicles as compared with control animals. Although ovarian cyclicity was not altered significantly within a few months after immunization, two of the five animals in this group became amenorrheic by 8 months. Histologic analysis of ovarian tissue shows that follicles were absent or frequently abnormal in animals of both groups following long-term immunization. These studies demonstrate that the synthetic adjuvant is effective in inducing antibodies (to purified zona pellucida glycoproteins) that recognize antigenic determinants to either denatured or deglycosylated zona pellucida glycoproteins, and that some of these antibodies may interfere with normal ovarian function.

Adjuvants, Immunologic↗
PubMed1989

Intestinal lymphatic flow during portal venous hypertension.

We used a circuit-model technique to analyze the flow from intestinal lymphatics. Postnodal lymph vessels from the small intestine were cannulated in five halothane-anesthetized dogs. We measured the flow rate from the cannula (QL) as we held the outflow end of the cannula at several heights above the site of cannulation. At each cannula height we calculated the pressure at the outflow end of the lymphatic (Po) as Po = QL X cannula resistance + height of the outflow end of the cannula above the cannulation site. Then we determined a best-fit regression line for QL vs. Po. The effective lymphatic resistance (RL) was estimated from the regression line as -delta Po/delta RL and the effective pressure driving lymph flow (PL) was taken as the Po at which QL = 0. At base line, RL = 0.039 +/- 0.018 (SD) cmH2O.min.microliters-1 and PL = 5.2 +/- 2.4 cmH2O. When we raised the portal venous pressure from 11.4 +/- 4.0 cmH2O (base line) to 34.4 +/- 9.0 cmH2O, RL decreased by only 26 +/- 11%, but PL rose by 160% to 13.6 +/- 8.7 cmH2O (P less than 0.05). Thus our data indicate that increases in portal venous pressure to 34.4 +/- 9.0 cmH2O cause intestinal lymph flow to increase primarily because of increases in the effective PL.

Animals↗
PubMed1988

A simple, inexpensive method for the salt-free concentration of proteins for analysis by two-dimensional gel electrophoresis.

A rapid, inexpensive method for the salt-free concentration of small quantities of proteins for analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been developed. Proteins adsorbed to diatomaceous earth are subsequently removed using either sodium dodecyl sulfate or urea solubilization reagents for 2D-PAGE analysis. This procedure has been found to concentrate proteins having wide ranges of molecular weight and charge. It is also valuable for the concentration of large numbers of small samples from cells cultured in vitro.

Animals↗
PubMed1988

Identification and characterization of an abundant ovarian interstitial gland protein associated with sexual maturity in rabbits.

An abundant ovarian protein with a relative mol wt (Mr) of 37K and an apparent pI of 8, associated with the onset of sexual maturity in the rabbit, has been identified. Ovaries from sexually mature (greater than 6 months old) rabbits contain large quantities of this 37K protein, while none can be detected in ovaries of immature (1 and 2 months) animals. Analysis of polyacrylamide gel electrophoresis (PAGE) gels of mature ovarian homogenates demonstrates that this protein is more abundant than actin in these preparations. It appears to be tissue specific, since it was not detected in 16 other rabbit tissues tested. Autoradiographic analysis of proteins labeled with 35S in ovarian organ culture demonstrates that a protein of identical Mr and charge to the 37K protein is synthesized in this tissue. Polyclonal sheep antiserum has been produced to the two-dimensional PAGE-purified protein. Immunoblotting of two-dimensional PAGE gels shows specific recognition of this protein and two slightly more acidic proteins of the same Mr by this antiserum. These three protein species also stain identical colors with a silver-based color stain, further suggesting that these are charge variants of the same protein. This protein is not present in corpora lutea isolated form sexually mature ovaries and is present in interstitial cell-enriched ovaries of rabbits which have been actively immunized with zona pellucida proteins. Immunocytochemical localization studies further demonstrate that this protein is localized in the interstitial gland cells. These findings suggest that this 37K protein is not associated with either follicular or luteal cells, but, rather, is linked with the 20 alpha-hydroxyprogesterone-secreting interstitial gland cell population.

Animals↗