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Biomedical subjects

B H Barber

Publications and source records attributed to B H Barber.

At least 55 records · Page 3Linked to original sources
PubMed1990

The conformational flexibility of class I H-2 molecules as revealed by anti-peptide antibodies specific for intracytoplasmic determinants: differential reactivity of beta 2-microglobulin "bound" and "free" H-2Kb heavy chains.

Immunoprecipitation experiments using anti-peptide antisera prepared against exon 6, exon 7 and exon 8-encoded intracytoplasmic regions of the H-2Kb gene product indicated that approximately 1/3 of the H-2Kb heavy chains in a cell surface-labelled glycoprotein fraction from EL-4 cells, or H-2b spleen cells, is not associated with beta 2-microglobulin (beta 2-m). This population of "free" H-2Kb heavy chains failed to react with alloantisera or monoclonal antibodies specific for conventional H-2Kb serological determinants, suggesting that significant conformational alterations were induced in the extracellular domains upon dissociation of beta 2-m. In addition, although antibodies to intracytoplasmic peptide 8 were able to react with both "free" and beta 2-m "bound" heavy chains, the determinants seen by anti-peptide 6 and anti-peptide 7 were only recognized in the "free" heavy chain. These data suggest that the conformational perturbation of the extracellular domains induced by beta 2-m dissociation can be "transmitted" to the intracytoplasmic region of the heavy chain. These results indicate the potential for a class I heavy chain-mediated transmembrane signalling event, and suggest that the "free" class I heavy chain might have a role to play in the major histocompatibility complex (MHC)-restricted presentation of T-cell determinants to cytotoxic T-lymphocytes.

Animals↗
PubMed1990

Characterization of the adjuvant-free serological response to protein antigens coupled to antibodies specific for class II MHC determinants.

Having previously demonstrated that coupling protein antigens to anti-class II major histocompatibility complex (MHC) monoclonal antibodies provides a means of inducing adjuvant-independent IgG responses to the 'targeted' antigen, we have now explored the parameters of this response in more detail. Using as the model system egg avidin coupled to biotinylated anti-class II MHC mAbs, the kinetics of the avidin-specific 1 degrees IgG response, the IgG subclass distribution of the 2 degrees anti-avidin response, and the immunogenicity of anti-class II-avidin conjugates in H-2-recombinant mice have been investigated. In addition, the use of avidin as a bridging molecule in the creation of immunoconjugates involving third-party proteins has been further characterized, and the immunogenicity of defined soluble conjugates in mice bearing the appropriate class II MHC gene products has been demonstrated. These findings extend our understanding of the immunoconjugate approach to adjuvant-independent immunization, and support its exploration as an alternative means of subunit vaccine design.

Animals↗
PubMed1989

Role of beta 2-microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules.

We have examined the requirement for beta 2-microglobulin (beta 2m) in the intracellular transport of murine class I histocompatibility molecules to the cell surface. R1E cells that are defective in the synthesis of beta 2m were transfected with either the class I H-2Kb or H-2Db genes alone, or together with the beta 2m gene. Kb or Db heavy chains synthesized in the presence of beta 2m were transported rapidly through the cell and expressed efficiently at the cell surface. In the absence of beta 2m, no "free" Kb heavy chains were detectable at the cell surface and their intracellular transport was blocked at an early stage. In contrast, a significant quantity of "free" Db heavy chains could be detected at the cell surface as we have reported previously. However, we have shown here that defects in intracellular transport were apparent in that the majority (approximately 70%) of newly synthesized Db heavy chains accumulated intracellularly and were degraded. Therefore, although Kb and Db heavy chains differ in their abilities to be expressed at the cell surface in the absence of beta 2m they both require association with beta 2m for efficient intracellular transport. In addition, R1E cells transfected with a deletion construct of the Kb gene expressed a truncated molecule lacking the alpha 3 extracellular domain (Kb-3) at the cell surface, but, like free Db, most newly synthesized Kb-3 molecules accumulated intracellularly. The free Kb, Kb-3, and Db heavy chains were not recognized by most mAb specific for Kb and Db, respectively. Therefore, even the transported forms of free Db and Kb-3 were not native in conformation, which is surprising given the current view that correct folding is essential for intracellular transport. Interestingly, the free Db and Kb-3 heavy chains that reached the cell surface differed in their detergent binding properties from those retained within the cell. This suggests that the transported heavy chains may have folded differently thus allowing their export to the cell surface.

Animals↗
PubMed1988

Delivery of synthetic peptides by anti-class II MHC monoclonal antibodies induces specific adjuvant-free IgG responses in vivo.

Two synthetic peptides from different regions of the glycoprotein D of herpes simplex virus (HSV) have been used to determine whether or not anti-peptide IgG responses can be generated in the absence of adjuvant. Based on an earlier demonstration (Carayanniotis and Barber, 1987) that protein antigens could be made immunogenic in the absence of adjuvant, if coupled to MAbs specific for the recipient's class II major histocompatibility complex (MHC) antigens, the HSV synthetic peptides were photocoupled to avidin and mixed with biotinylated anti-class II or control antibodies. Peptide-specific IgG responses were obtained when avidin-peptide conjugates coupled to an anti-I-Ak MAb were injected into (H-2k x H-2b) F1 mice, but not when injected into H-2b mice. The same avidin-peptide conjugates were non-immunogenic when coupled to a control anti-influenza virus nucleoprotein antibody or biotinylated bovine serum albumin. These data indicate that targeting synthetic peptides to class II bearing cells in vivo can elicit peptide-specific IgG responses without the need for adjuvant. These findings offer a new approach to the design and delivery of adjuvant-independent vaccine agents based on synthetic peptide antigens.

Animals↗
PubMed1987

Searching for MHC-restricted anti-viral antibodies: antibodies recognizing the nucleoprotein of influenza virus dominate the serological response of C57BL/6 mice to syngeneic influenza-infected cells.

An attempt has been made to generate monoclonal antibodies which recognize the same target structures on influenza-infected cells as those seen by cytotoxic T lymphocyte (CTL) receptors. Such antibodies, if they mimicked the T cell receptor specificity, would be expected to be both virus specific and restricted in their binding by the major histocompatibility complex (MHC) antigens. Approximately 200 hybridomas from C57BL/6 (H-2b) mice primed and boosted with influenza virus (X-31)-infected EL4 (a C57BL/6 T cell lymphoma) were screened for reactivity on infected and uninfected cells of different MHC haplotypes. Of the 10 hybridoma antibodies which were identified as being reactive with X-31-infected EL4, but not uninfected EL4, all reacted equally well with X-31-infected cells of H-2b, H-2d and H-2k haplotypes, indicating a lack of MHC restriction in their recognition of the infected cells. Unexpectedly, 7 of the 10 monoclonal antibodies were found to react specifically with the purified influenza virus nucleoprotein (NP), a predominant viral antigen in CTL recognition of infected cells. Fluorescence-activated flow cytometry confirmed that these antibodies were able to recognize NP serological determinants on the surface of viable, infected cells, but the anti-NP antibodies were unable to block the lytic activity of an NP-specific CTL clone.

Animals↗
PubMed1986

The preparation and characterization of anti-peptide heteroantisera recognizing subregions of the intracytoplasmic domain of class I H-2 antigens.

Peptides corresponding to each of the three intracytoplasmic exons (i.e. exons 6, 7 and 8) of the murine class I H-2Kb gene were synthesized, coupled to bovine serum albumin and used as immunogens in rabbits. In each case the antisera were found to react with the immunizing peptide coupled to a heterologous carrier, and recognized class I heavy chains electrophoretically transferred from SDS-polyacrylamide gels to nitrocellulose. Immunoprecipitation of class I antigens from Nonidet P-40 (NP-40) solubilized EL-4 (H-2b) tumour cells by each of the antisera reflected their ability to recognize the corresponding determinants in non-denatured class I molecules. The same sera were also able to immunoprecipitate class I molecules from NP-40 solubilized RDM-4 (H-2k) and P815 (H-2d) tumour cells, indicating the cross-reactive nature of these antisera for different class I alleles. In addition to reacting with the class I heavy chain in its conventional form as a dimer with beta 2-microglobulin, the antiserum specific for the exon 8 peptide was able to react with "free" (i.e. non-beta 2-microglobulin-associated) class I heavy chains. Thus, a unique set of immunological reagents has been prepared which offer a new approach to studying the structural and functional features of the cytoplasmic domain of class I H-2 antigens.

Antigen-Antibody Reactions↗
PubMed1986

A structural analysis of the carbohydrate side chains on class I and class II histocompatibility antigens of the swine facilitated by heteroantisera specific for the denatured polypeptides.

Rabbit heteroantisera specific for the denatured glycoprotein subunits of swine class I and class II major histocompatibility complex (MHC) antigens have been prepared and utilized to monitor changes in the mobilities of these polypeptides on SDS-polyacrylamide gel electrophoresis subsequent to various deglycosylation procedures. This information, in combination with lectin reactivity patterns for the glycoproteins bound to nitrocellulose, has made it possible to define specific structural features of the MHC antigen-associated carbohydrate side chains. Both the class I heavy (alpha) chain and the class II light (beta) chain bear a single, N-linked, complex-type oligosaccharide which reacts with lentil lectin (LcH), but not concanavalin A (Con A); a reactivity pattern suggesting the possibility of a special triantennary structure. In contrast, the class II heavy (alpha) chains appear to possess two carbohydrate units, one an N-linked, LcH-reactive, complex-type side chain, and the other, an N-linked, Con A-reactive, high-mannose-type of oligosaccharide. The data suggest considerable homology between the swine and human MHC antigens with respect to the structure of their carbohydrate side chains. The analysis also serves to illustrate how antibodies specific for the denatured polypeptide backbone of individual glycoproteins, along with lectin reactivity patterns, can be used to extract structural information about the attached carbohydrate moieties using minimal amounts of partially purified glycoproteins.

Animals↗
PubMed1984

A recommendation for visualizing disulfide bonding by one-dimensional sodium dodecyl sulfate--polyacrylamide gel electrophoresis.

A simple modification of the conventional one-dimensional sodium dodecyl sulfate--polyacrylamide gel electrophoresis technique has been used to visualize inter- and intramolecular disulfide bonding in proteins. The gradient of reducing agent established between adjacent slab gel tracks by electrophoresing identical protein samples next to one another, one containing and the other not containing 2-mercaptoethanol, has been used to visualize the change in mobility of disulfide bond-containing proteins throughout the transition from a reducing to a nonreducing environment. As illustrated by an analysis of immunoglobulin heavy and light chains, the method particularly facilitates the positive identification of proteins containing intrachain disulfide bonds.

Animals↗
PubMed1983

Inter- and intramolecular disulfide bonding among lymphocyte plasma membrane proteins and glycoproteins.

The disulfide bonding characteristics of the pig lymph node plasma membrane (PM) proteins and glycoproteins have been examined by 1- and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Reaction of the purified PM vesicles with N-ethyl maleimide (NEM) prior to detergent solubilization was found to markedly reduce the extent of intermolecular disulfide bonding subsequently observed. Thus the blocking of free sulfhydryl groups with NEM prevented the detergent-induced disulfide bonding of numerous components, including PM-bound actin. The extent of intermolecular disulfide bonding among the NEM-pretreated PM glycoproteins purified by lentil lectin affinity chromatography was found to be relatively limited, with only 3% of the total glycoprotein present as intermolecular disulfide-bonded complexes. In contrast, the degree of intramolecular disulfide bonding revealed by a modified 1-dimensional SDS-PAGE technique was quite striking. Among those polypeptides demonstrating a clearly altered mobility upon reduction was the heavy chain of class I and beta-chain of class II major histocompatibility complex (MHC) antigens. The class II alpha-chain, however, was much less affected. These changes have been compared with those observed for proteins containing intramolecular disulfide-bonded domains of known size and number, and considered in the light of recent information on the structure of MHC antigens.

Animals↗
PubMed1982

A cautionary note on the use of lentil lectin affinity chromatography in the presence of sodium deoxycholate.

When affinity chromatography with lentil lectin (LcH)-Sepharose was carried out in 0.5% (w/v) sodium deoxycholate (DOC), 10 mm Tris, pH 8.2, LcH consistently appeared in the 0.1 M alpha-methyl-mannoside (alpha-MeMan)-eluted fraction. Eluted LcH was recovered in both the control and specific immunoprecipitates with antisera directed against components of the glycoprotein fraction, suggesting that at least a portion of the LcH retained the capacity to interact with protein-bound carbohydrate. We suggest that the presence of functional LcH in the purified glycoprotein fraction is a potential source of artifact which may not be generally appreciated. When the non-ionic detergent. Nonidet P-40 was substituted for DOC in the same type of LcH-Sepharose fractionation, LcH did not appear in the 0.1 M alpha-MeMan-eluted fraction.

Animals↗
PubMed1982

Fractionation and identification of pig lymph node plasma membrane glycoproteins.

Plasma membrane vesicles purified from pig mesenteric lymph node tissue were solubilized in 1% (w/v) sodium deoxycholate and the fractionation of both membrane proteins and glycoproteins assessed by gel filtration on AcA 34. Milligram quantities of the glycoprotein fraction, consisting of 12 distinct bands on SDS-PAGE ranging in apparent mol. wt from 12,000 to 250,000, were purified by affinity chromatography on lentil lectin-Sepharose. Identified among these by specific immunoprecipitation were the major histocompatibility antigen, SLA (band 9), in association with beta 2-microglobulin (band 12); as well as the alpha (band 10) and beta (band 11) subunits of the Ia-like antigens. A tentative identification of the membrane-bound immunoglobulins (IgM, IgG and IgA) was also proposed on the basis of their known affinities for protein A. Thus, the domestic pig represents and inexpensive, abundant source of defined lymphocyte plasma membrane glycoproteins suitable for further structural analysis.

Animals↗
PubMed1982

Structural studies of purified pig lymph node plasma membrane: association of cytoskeletal compounds with the plasma membrane and the effect of detergent solubilization.

The reproducibility of preparation, stability at 4 degrees C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found that 2% (w/v) Nonidet P-40 solubilized 50-60% and 2% (w/v) sodium deoxycholate solubilized 60-70% of the total membrane protein. As judged by 125I-labelled lentil lectin staining of the sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet p-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found that very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 M KCl, or by incubation with DNase I.

Actins↗
PubMed1981

Microtubules and microfilaments during cell spreading and colony formation in PK 15 epithelial cells.

We have studied the distribution of microtubules and microfilaments during the cell spreading and subsequent colony formation in PK 15 pig kidney epithelial cells using indirect immunofluorescence. During the cell spreading on a solid substratum, microtubules grew out from the region around the nucleus, and a collar of microfilament bundles formed around the cell periphery. Although virtually all well-spread cells showed a complex microtubular network, distinctly different patterns of stress fibers were observed. In small colonies, the most commonly observed pattern was a ring of microfilament bundles that appeared to be in register between adjacent cells and encircled the entire colony in a fashion similar to that seen in single cells. In large colonies (more than 50 cells), approximately 60% of the cells displayed clearly stained microfilament bundles, either at the cell periphery or throughout their cytoplasm, whereas in the remaining 40%, no microfilament bundles were observed and only the outline of the cells was delineated by interaction with anti-actin. Such "negative" cells were seen in groups alongside "positive" cells (i.e., cells possessing extensive stress fiber networks) within the same colony. Independent of their stress fiber phenotype, all cells maintained a flattened shape and an extensive network of microtubules. We suggest that dense microfilament bundles are not a uniform feature of well-spread PI 15 cells in culture and that a loss of microfilament bundle occurs in some cells.

Actins↗
PubMed1980

Albumin associated with purified pig lymphocyte plasma membrane.

Plasma-membrane preparations purified from pig lymphocytes contained a major polypeptide component of mol.wt. about 68 000. This component was identified as pig albumin by the following comparisons with authentic pig serum albumin: (a) co-migration when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions; (b) identical isoelectric points; (c) similar "fingerprints" of arginine-containing tryptic peptides; (d) reactivity with anti-(pig albumin) serum. The albumin was bound tightly to the plasma membrane. Biosynthetic labelling of pig lymphocytes under a variety of conditions failed to provide evidence that albumin was synthesized by lymphocytes, suggesting that the plasma-membrane-associated albumin was of extraneous origin. Radiolabelled pig serum albumin, however, failed to bind to the plasma-membrane fraction when added before cell disruption. Although lymphocyte plasma membrane preparations from other species possessed a polypeptide of about 68 000 mol.wt., this was judged not to be albumin on the basis of electrophoretic mobility under non-reducing conditions; also, no polypeptide was precipitated by anti-albumin sera. It is concluded that pig lymphocyte plasma-membrane preparations possess albumin which, although firmly attached, was probably of extraneous origin. This association appeared not to be common to lymphocytes from other species.

Animals↗
PubMed1979

Evidence for two homologous, but nonidentical, Ia molecules determined by the I-EC subregion.

Sequential immunoprecipitation, two-dimensional gel electrophoresis and peptide mapping analyses of B10A(3R), 35S-methionine-labeled, I-EC subregion products were performed. Evidence is presented here for the presence of two structurally homologous, but nonidentical, gene products of the I-EC subregion. These two Ia molecules are independently immunoprecipitable, identical in molecular size and charge, but differ by approximately equal to 20% in their peptides obtained by partial digestion with Staphylococcus aureus protease V8.

Amino Acids↗
PubMed1979

Immunochemical characterization of the Ly-8.2 murine lymphocyte alloantigen: possible relationship to actin.

An immunochemical analysis of the Ly-8.2 antigen was performed. Reactivity of B10-radiolabeled T and B cell lysates with a C3H anti-AKR serum immunoprecipitates a major 43,000 m.w. protein detectable by one-dimensional gel electrophoresis. The protein can be radioiodinated, however, it does not appear to blind to lentil lectin. Two-dimensional gel electro phoresis and peptide-mapping studies indicate that this protein is structurally very similar to lymphocyte actin. The possible molecular relationship between Ly-8.2, other lymphocyte membrane antigens, and actin is discussed.

Actins↗
PubMed1979

The identification of actin as a major lymphocyte component.

Lymphocytes derived from pig mesenteric lymph node and mouse spleen have been shown to contain a major protein conponent which a) co-migrates on one-dimensional (SDS-PAGE with purified pig muscle actin, and b) possesses the same isoelectric point as muscle actin on two-dimensional gel electrophoresis. Further, limited proteolytic digestion of the lymphocyte actin-comigrating band by S. aureus V8-protease revealed extensive primary sequence hemology with purified muscle actin. Based on the virtual identity of molecular size charge, and primary sequence, we conclude that the actin-comigrating band represents lymphocyte actin. The appearance of lymphocyte actin as a nonspecific "contaminant" of immune precipitates has also been discussed.

Actins↗