Biomedical subjects
B Gillard
Publications and source records attributed to B Gillard.
Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes.
On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.
Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.
To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.
Molecular characterization of human X/Y translocations suggests their aetiology through aberrant exchange between homologous sequences on Xp and Yq.
Several DNA sequences from two homologous regions, localized on the distal part of the human X chromosome short arm and on the long arm of the Y chromosome, have been hybridized to DNAs from seven human-rodent hybrids containing human X; Y translocation chromosomes. Molecular characterization of the translocated chromosomes has revealed, in all but one case, transfer of the Y cluster of sequences and complete deletion of the corresponding X-chromosomal sequences. The possible role of X/Y homology in the aetiology of X; Y translocations is proposed.
[Presence of a specific binding site of endothelin on cultured smooth muscle cells].
The 21 amino-acids endothelium-derived peptide, endothelin, recently isolated by Yanagisawa et al. (Nature 1988; 33, 411-5) possesses potent vasoconstrictive properties in vivo and in vitro. In the present study, we investigated the binding of endothelin on cultured rat aortic smooth muscle cells using 125I-iodotyrosyl-endothelin labelled by the chloramine T method. 125I-endothelin bound to a single class of hight affinity binding sites in vascular smooth muscle cells. After 2 hours incubation at 37 degrees C, dissociation constant (Kd) was 1.2 +/- 0.3 nM and binding capacity (Bmax) was 59 +/- 11 fmol/10(6) cells (n = 5). 125I-endothelin was displaced by unlabelled endothelin with a inhibition constant (Ki) of 0.2 nM, whereas an absence of competition was observed with 1 microM of vasoactive substances such as angiotensin II, arg-vasopressin, atrial natriuretic factor, histamine, epinephrine and norepinephrine, and with the calcium entry blocks nifedipine, diltiazem and D 600. 125I-endothelin binding was not reversible by addition of unlabelled endothelin (1 microM) and not dissociable by acetic acid (10 mM) or trypsin (0.1 p. 100) treatment of the cells. Furthermore, preincubation of vascular smooth muscle cells with endothelin (1 nM) at 37 degrees C induced a rapid down-regulation of endothelin binding capacity by about 50 p. 100. These data indicate that specific endothelin bindind sites are present in smooth muscle cells, and suggest a tight binding or a rapid captation of endothelin into the cell membrane leading to contractile events.
Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy.
Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.
Isolation and purification of gangliosides from plasma.
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Identification of a novel ganglioside on erythrocytes with blood group Cad specificity.
The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.
A solvent partition method for microscale ganglioside purification.
A simple and rapid method for the purification of gangliosides from the total lipid extract of plasma, cells, or tissue is described. The novel component of the method is the partition of the dried total lipid extract in the three-component solvent system consisting of diisopropyl ether, 1-butanol, and 50 mM aqueous NaCl (6/4/5, v/v/v). Gangliosides partition nearly quantitatively into the lower aqueous phase, and other lipids into the upper organic phase, resulting from the mixture of these three solvents. The ganglioside-containing aqueous phase is then freed of salts and other low-molecular-weight impurities by gel filtration. The thin-layer chromatographic patterns of total gangliosides thus obtained are clear and distinct, even when small samples with very low ganglioside concentrations (e.g., 1-ml samples of plasma) are processed by this method. Thus, this new ganglioside purification method is especially applicable to comparative qualitative studies of gangliosides requiring the analysis of multiple small samples.
Modulation of the immune response by gangliosides. Inhibition of adherent monocyte accessory function in vitro.
Gangliosides are potent inhibitors of lymphoproliferative responses. Selectively greater inhibitory effects of gangliosides on antigen-induced (vs. mitogen-induced) proliferation have been documented; e.g., 50 nmol of highly purified bovine brain gangliosides (BBG)/ml caused greater than or equal to 87% inhibition of proliferative responses of human peripheral blood mononuclear cells (PBMC) to three soluble specific antigens (Candida, streptokinase-streptodornase, and tetanus toxoid) vs. less than or equal to 37% inhibition of responses to three nonspecific mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen). The possibility that BBG interfere with adherent monocyte accessory function, upon which responses to soluble specific antigens are strictly dependent, was therefore considered. PBMC were separated into the adherent and nonadherent subpopulations, exposed to BBG, recombined, and their proliferative responses were measured. Unseparated PBMC preincubated for 48-72 h with 100 nmol BBG/ml and then washed to remove unbound BBG exhibited 73-76% inhibition of subsequent antigen-induced lymphoproliferation. Separate pretreatment of both adherent and nonadherent cell subpopulations in BBG under the same conditions resulted in similar (72-82%) inhibition, which was reproduced by preincubation of only the adherent cells in BBG. Preincubation of only the nonadherent cells in BBG was not inhibitory. Inhibition (a) was independent of whether gangliosides were added in solution or incorporated into liposomes, (b) was abrogated by adding untreated monocytes to cultures containing adherent cells that were preexposed to BBG (excluding the possibility that BBG was inducing suppression mediated by adherent cells), and (c) was reversible by further incubation of BBG-pretreated adherent cells in control medium. Together, these results delineate a mechanism by which gangliosides modulate lymphoproliferative responses--direct, noncytotoxic, and ultimately reversible inhibition of the accessory function of adherent monocytes.
Shedding and immunoregulatory activity of YAC-1 lymphoma cell gangliosides.
YAC-1 lymphoma cells, both when cultured in vitro and when passaged in ascites form in vivo, synthesize gangliosides (means of 22.1 and 14.7 nmol lipid-bound sialic acid isolated per 10(8) cells, respectively) with potent inhibitory effects on mitogen- and antigen-induced lymphoproliferation: 10 to 30 nmol highly purified YAC-1 gangliosides/ml caused greater than 90% inhibition of proliferative responses of murine lymphocytes to concanavalin A, lysozyme (a soluble specific antigen), and allogeneic cells (mixed-lymphocyte response). Measureable quantities of these gangliosides were shed by the tumor cells in vitro and also were recovered from the ascites fluid in vivo. Furthermore, the gangliosides isolated from ascites fluid (mean of 15.3 nmol/ml) had inhibitory activity of a magnitude similar to that of the gangliosides isolated from the tumor cells. Therefore, significant inhibition of normal lymphoproliferative responses by tumor-derived gangliosides occurred at ganglioside concentrations which are actually present in the fluid surrounding the tumor cells in vivo. These results support the hypothesis that shedding of gangliosides may serve to protect tumor cells from host immune destruction.