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Biomedical subjects

C J Barnstable

Publications and source records attributed to C J Barnstable.

At least 109 records · Page 6Linked to original sources
PubMed1979

The structure and evolution of the HLA--Bw4 and Bw6 antigens.

The HLA--Bw4 and Bw6 antigenic determinants have been shown to co-migrate with HLA--B determinants on gel-filtration, sucrose density gradient centrifugation and affinity chromatography, using Lens culinaris lectin and antibody against human beta 2 microbulin. These and other published data imply that the HLA--Bw4 and Bw6 determinants reside on the same polypeptide chain as other HLA--B locus determinants. The implications of this in terms of the evolution of cross reacting groups of antigens at the HLA--B locus are discussed.

Biological Evolution↗
PubMed1978

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.

Antibody-secreting hybrid cells have been derived from a fusion between mouse myeloma cells and spleen cells from a mouse immunized with membrane from human tonsil lymphocyte preparations. Hybrids secreting antibodies to cell surface antigens were detected by assaying culture supernatants for antibody binding to human tonsil cells. Six different antibodies (called W6/1, /28, /32, /34, /45 and /46 were analyzed. These were either against antigens of wide tissue distribution (W6/32, /34, and /46) or mainly on erythrocytes (W6/1 and W6/28). One of the anti-erythrocyte antibodies (W6/1) detected a polymorphic antigen, since blood group A1 and A2 erythrocytes were labeled while B and O were not. Antibodies W6/34, /45 and /46 were all against antigens which were mapped to the short arm of chromosome 11 by segregation analysis of mouse-human hybrids. Immunoprecipitation studies suggest that W6/45 antigen may be a protein of 16,000 dalton, apparent molecular weight, while W6/34 and /46 antigens could not be detected by this technique. Antibody W6/32 is against a determinant common to most, if not all, of the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. This was established by somatic cell genetic techniques and by immunoprecipitation analysis. Tonsil leucocytes bound 370,000 W6/32 antibody molecules per cell at saturation. The hybrid myelomas W6/32 and W6/34 have been cloned, and both secrete an IgG2 antibody. W6/32 cells were grown in mice, and the serum of the tumor-bearing animals contained greater than 10 mg/ml of monoclonal antibody. The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens. In particular, this study highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.

Antibody Formation↗
PubMed1978

The genetic control of HLA-A and B antigens in somatic cell hybrids: requirement for beta2 microglobulin.

The lymphoblastoid cell line Daudi lacks both HLA-A and B antigens and beta2 microglobulin. Somatic cell hybrids derived from a fusion between this line and D98/AH--2 were shown to express four HLA antigens not detectable on either parent cell, A1, A10(Aw26), Bw16(Bw38, Bw17. The initial definition by direct cytotoxicity assay was confirmed by absorption of reactions against target T lymphocytes, thus avoiding problems due to contaminating Ia antibodies, and by blocking the reactions by pretreatment with a chicken anti-human beta2 microglobulin serum. That the new specificities were due to the Daudi HLA region was confirmed by the finding that interspecific hybrids between Daudi and A9L, containing a single human chromosome 6, expressed A10 and Bw17. This also defined the haplotypes of Daudi as A10(Aw26), Bw17 and A1, Bw16(Bw38). The re-expression of the Daudi HLA-A and B antigens in two independent sets of hybrids indicates that it does not carry a mutation in the HLA region. It has previously been reported that somatic cell hybrids with Daudi, which contain chromosome 15, do not express human beta2 microglobulin. These results suggest that the reason for the lack of HLA-A and B antigens on Daudi is a secondary effect due to the mutation(s) in the beta2 microglobulin gene.

Beta-Globulins↗
PubMed1977

Molecular structure of human histocompatibility antigens: the HLA-C series.

The HLA-CW2 antigen of the B lymphoblastoid cell line BRI 8 is structurally homologous to the HLA-A and B antigens as judged by various criteria. Each antigen comprised a glycosylated polypeptide of 43 000 molecular weight that is noncovalently associated with beta2-microglobulin (beta2m). Some small differences in molecular parameters were, however, revealed. Thus, the deoxycholate-solubilized HLA-CW2 antigen sedimented at the same rate as the HLA-A antigens but at a slightly faster rate than the HLA-B antigens. This variation is apparently is apparently due to different amounts of bound deoxycholate. Also, whereas essentially all of the HLA-A and B antigens and about half of the HLA-CW2 antigen were adsorbed strongly by Lens culinaris lectin-Sepharose, the remaining HLA-CW2 antigen was bound much more weakly and did not require sugar for elution. This difference reflects some structural heterogeneity in the carbohydrate moiety of the HLA-CW2 antigen. The results of various studies suggest that the HLA-CW2 antigen is expressed to a lower extent than the HLA-A or B antigens and that essentially all of the beta2m of the BRI 8 plasma membrane is associated with the HLA-A, B and C alloantigenic polypeptides.

Cell Line↗
PubMed1977

Cellular distrubtion, purification, and molecular nature of human Ia antigens.

Human Ia antigens were extensively purified (1390-fold increase in specific activity) in 32% yield from BRI 8 cells, a lymphoblastoid B-cell line. Purification was monitored by using allogeneic antisera arising by foetal-maternal stimulation. The product, a glycoprotein fraction, contained the Ia antigens, the HLA-A and -B antigens, and a glycoprotein of unknown function. The glycoprotein fraction was composed of four glycosylated polypeptides with molecular weights of 43,000, 39,000, 33,000, and 28,000, and beta2-microglobulin; no polypeptide was linked to another by disulphide bridges. The A and B antigens only were absorbed by antibody against beta2-microglobulin. The Ia antigens comprised one each of the 33,000 and 28,000 molecular weight glycosylated polypeptides noncovalently linked together. Thus, only these chains were absorbed by xenogeneic anti-Ia antisera and were cross-linked by dimethyl-3-3'-dithiobispropionimidate dihydrochloride. The dimeric molecule bound deoxycholate (0.26 g/g of protein) and, when solubilized in deoxycholate, has a molecular weight of 77,000. The Ia allo- and xeno-antigenic activities were labile to heating and proteolysis and are probably determined by the polypeptide structure. Xenogeneic specific anti-Ia antisera were raised in rabbits and mice by immunizing with the glycoprotein fraction. These antisera reacted with B lymphocytes and monocytes but not T lymphocytes and fibroblasts. Their Fab fragments blocked the cytotoxicity of the allogeneic antisera for B lymphocytes and were potent inhibitors of the mixed lymphocyte reaction.

Antigens↗
PubMed1976

Expression of HLA system antigens on placenta.

The levels of HLA-A and -B antigens expressed by placenta have been assessed relative to parental and other A and B antigen types that were not shared by the foetus. A purified preparation of placenta plasma membrane was used to estimate the antigen activities. The results indicate that maternally and paternally inherited A and B antigen activities and beta2-microglobulin are expressed to similar extents but at much lower levels than in spleen lymphocytes (less than 5%). The possibility that the amounts detected were caused by contamination with blood or maternal tissue was ruled out. The low levels of A and B antigens may account for the lack of a cellular immune response to the other polymorphic cell surface antigens of the trophoblast. No evidence was obtained for the expression of a significant level of Ia antigens.

Cell Membrane↗
PubMedDate not supplied

A gradient molecule in developing rat retina: expression of 9-O-acetyl GD3 in relation to cell type, developmental age, and GD3 ganglioside.

In the embryonic and postnatal rat retina a cell surface antigen that is detected by monoclonal antibody JONES is distributed in a dorsoventral gradient. Biochemical analysis has determined that the antigen is a modified ganglioside, 9-O-acetyl GD3. In the present study, the distributions of 9-O-acetyl GD3 and its possible precursor GD3 in developing rat retina were compared immunocytochemically using specific monoclonal antibodies JONES and R24. On embryonic day 13 (E13) immunoreactivity to JONES was localized to central retina; however, R24 stained throughout the retinal epithelium. By E20, when JONES binding was distributed in a gradient along the dorsoventral axis, R24 again stained dorsal and ventral regions with uniform intensity. Analysis of freshly dissociated retinal cells further revealed that GD3 and 9-O-acetyl GD3 expressions do not necessarily coincide. At E15 and postnatal day 2 (PN2), the majority of cells (78 and 92%, respectively) were immunolabeled by antibody to GD3, while between E15 and PN2 the percentage of cells immunolabeled by antibody to 9-O-acetyl GD3 rose from 19 to 68%. By PN4, labeling decreased for both molecules; however, the rate of decline in 9-O-acetyl GD3 labeling was more pronounced. Regulation of the numbers of JONES-positive cells does not appear to depend on interaction with the extraretinal environment, for in neural retina explanted at E15 the proportions of 9-O-acetyl GD3-bearing cells was found to be similar to the percentage observed in neural retina developing to an equivalent age in vivo. Experiments to identify the retinal cell types bearing 9-O-acetyl GD3 revealed that it is expressed by both neurons and glia growing in monolayer cultures of rat perinatal neural retina. Examination of freshly dissociated retinal cells following simultaneous labeling for some specific cell types and 9-O-acetyl GD3 demonstrated that the latter determinant is present on photoreceptor, amacrine, and ganglion cells. For each neuronal cell type, however, not all of the cells were immunoreactive with JONES. We conclude that the differences in the percentages of JONES- and R24-positive cells, and in particular the different rates at which JONES and R24 staining declined with age, indicate that the expression of the JONES epitope is regulated with some independence from parent ganglioside GD3.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMedDate not supplied

Characterization and regulation of the protein binding to a cis-acting element, RET 1, in the rat opsin promoter.

RET 1 is a binding site for retinal nuclear proteins located at -136 to -110 bp in the rat opsin promoter, as defined by DNase protection assays. A similar sequence is found in the upstream flanking regions of many other photoreceptor genes in mammals and other species, including Drosophila. A 7-base consensus sequence, CAATTAG, is found in these genes and has the binding activity of the longer RET 1 element. A 40-kDa protein that binds to RET 1 has been purified over 2 x 10(5)-fold to apparent homogeneity by affinity chromatography. The RET 1 binding activity is first detectable at E18 and increases during the first two postnatal weels, At embryonic ages the retarded bands show an altered mobility and at early postnatal ages two bands are detected, with the adult band increasing and the embryonic band decreasing in intensity. Treatment of early postnatal retinas with bFGF increased the binding activity in nuclear extracts and caused a shift in migration of the retarded band to a position characteristic of the embryonic form of the complex. The results support the hypothesis that RET 1-like elements play an important role in rod photoreceptor development.

Animals↗
PubMedDate not supplied

A molecular view of vertebrate retinal development.

Immunological probes have begun to identify molecules that delineate cell layers and cell types during the formation of the retina and other parts of the optic cup. Within the developing retina, cell-type-specific monoclonal antibodies have been used to show that differentiation occurs before cells reach their final laminar position. Cell surface molecules have been found expressed in position-dependent gradients across the retina. These molecules may convey positional information to the retinal cells and their topographic connections. One such molecule is a modified carbohydrate group on a ganglioside, suggesting that such groups may play a role in neural development. A variety of molecules that are expressed by rod photoreceptors at defined stages of their differentiation have been characterized. These molecules have been used to show the development of subcellular compartments within rods. In vitro studies have suggested that photoreceptor molecules expressed at different times are under different forms of regulation. Some of these cell-specific molecules have been shown to be under transcriptional control and thus defined cell interactions seem to be linked to changes in gene expression during retinal development.

Aging↗
PubMedDate not supplied

Developmental expression of intracellular targets of cGMP in rat visual cortex and alteration with dark rearing.

We describe the temporal pattern of mRNA expression of some of the molecular components of the NO/cGMP second messenger system in the developing rat visual cortex and the effect of dark rearing on their expression levels using semiquantitative RT-PCR. mRNA expression for these molecules was altered by dark rearing in one of three ways: (1) no change--rod, olfactory, and cone/testis CNG channels, nonselective cation channels gated by cyclic nucleotides and highly permeable to Ca2+; (2) decrease--cyclic nucleotide phosphodiesterases which regulate cyclic nucleotide levels, and soluble guanylyl cyclase, the key synthetic enzyme producing cGMP and potently activated by nitric oxide; and (3) increase--cGMP kinase 1, a key enzyme activated by cGMP to phosphorylate a variety of intracellular proteins including cytoskeletal elements. These data suggest important and distinct roles for the cGMP system in both early and late developmental events in the rat visual cortex.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMedDate not supplied

Developmental expression of the rat rod photoreceptor cGMP-gated cation channel.

The appearance of cGMP-gated cation channel protein in the postnatal rat retina has been studied by fluorescence immunocytochemistry of radial retinal sections and immunoblots of retinal membrane proteins. Channel immunoreactivity was first detectable with RCNGC1-7H2 monoclonal antibody at postnatal day 7 (PN7) by both methods. Immunocytochemical label in retinal sections was localized to the outer segments, and immunoreactivity increased with increasing age. We also compared the developmental appearance of the cGMP-gated cation channel to that of other phototransduction proteins and developmental markers. RET-P2, a monoclonal antibody recognizing the 39-kDa rds/peripherin disc protein, first labeled outer segments at PN7, coincident with cGMP-gated cation channel expression. Double labeling of the same section of PN7 rat retina with RET-P2 and R309 (a polyclonal antiserum against the rod cGMP-gated cation channel) revealed identical patterns of labelling. Similarly, double labeling with RCNGC1-7H2 and an antibody against the rod cGMP-phosphodiesterase gave coincident labeling, suggesting coordinate expression mechanisms of phototransduction proteins with each other and with outer segment structural proteins.

Animals↗