Biomedical subjects
A L Chiu
Publications and source records attributed to A L Chiu.
Membrane gangliosides modulate interleukin-2-stimulated T-lymphocyte proliferation.
Membrane gangliosides appear to modulate signal transduction by several growth factor receptors. We have investigated the possible regulation of IL-2-induced proliferation signals by gangliosides. Low concentrations of cholera toxin B subunit (CT-B), which binds specifically to GM1 ganglioside, greatly inhibited IL-2-stimulated DNA synthesis in the IL-2-dependent cell line CTLL-2, but had no effect on proliferation of HT-2. GM1 levels proved to be very low in HT-2 compared to CTLL-2. Large increases in membrane-associated GM1 could be achieved in both cell lines by incubation with exogenous GM1, resulting in a high degree of inhibition of proliferation by CT-B for both CTLL-2 and HT-2. Inhibition was blocked by large unilamellar vesicles containing GM1, but not by vesicles of lipid alone. The time course of CT-B inhibition for CTLL-2 synchronized in G0-G1, indicated that the negative growth signal acts relatively early in the IL-2 activation pathway. CT-B did not affect binding of IL-2 to high-affinity IL-2r. The inhibitory effects of CT-B could not be reversed by pertussis toxin, suggesting that a G protein is probably not involved. These results show that CT-B binding to either endogenous or inserted GM1 can modulate IL-2-induced lymphocyte proliferation.
Gangliosides and glycophorin inhibit T-lymphocyte activation.
Increased levels of gangliosides in the serum have been linked to tumour-induced immunosuppression in vivo. Both bovine brain gangliosides and human erythrocyte glycophorin were potent inhibitors of concanavalin A, periodate, and phorbol ester--ionomycin induced activation of murine T-lymphocytes. Structurally complex gangliosides were more inhibitory, while simpler glycolipids caused less inhibition. Lymphocytes exposed to these molecules for up to 24 h could still proliferate after washing. Substantial inhibition was observed only when gangliosides and glycophorin were present during the first 18 h of activation. Studies using Quin-2 showed that gangliosides did not block the initial rapid rise in cytoplasmic Ca2+ following mitogen stimulation. Interleukin-2 (IL-2) production by ganglioside- and glycophorin-treated lymphocytes was unchanged. After treatment with gangliosides for 24 h, lymphocytes proliferated normally in response to added IL-2. These results suggest that the first round of signal transduction in response to mitogen was unaffected by gangliosides. Addition of gangliosides to activated lymphocytes in the presence of IL-2 resulted in complete inhibition of proliferation. Immunosuppression by gangliosides and glycophorin thus appears to occur at the IL-2-dependent stage of proliferation and may be partially due to IL-2 binding to these molecules. However, high levels of IL-2 failed to reverse inhibition and IL-2-dependent cell lines were much less sensitive to ganglioside inhibition than T-lymphocytes, suggesting that more than one mechanism of inhibition likely exists.
Prostaglandin H synthase-dependent mutagenic activation of benzidine in a Salmonella typhimurium Ames tester strain possessing elevated N-acetyltransferase levels.
Watanabe and colleagues (Biochem. Biophys. Res. Commun. 147: 974-979, 1987) have constructed plasmid-containing derivatives of Salmonella typhimurium Ames tester strain TA1538 with high levels of acetyltransferase activities. In this paper, we describe the mutagenic response of one of these strains, TA1538/1,8-DNP6 (pYG 121), to the bladder carcinogen benzidine and other arylamines. Strain TA1538/1,8-DNP6 (pYG 121) was far more sensitive to benzidine than any previous tester strain, following metabolism of the aromatic amine by hamster hepatic S9, ram seminal vesicle microsomal preparation (RSVM), or purified prostaglandin synthase. Therefore, bacterial acetyltransferase-dependent metabolism of a proximate mutagen is implicated in each of these systems. The mechanism of RSVM-dependent activation of benzidine was examined further. The arachidonic acid-independence and indomethacin insensitivity previously noted with strain TA98 were also observed with the new tester strain. We confirmed that prostaglandin H synthase is the enzyme activity responsible for activation of benzidine by RSVM. Purified prostaglandin H synthase holoenzyme, or apoenzyme reconstituted with heme, supported benzidine activation. However, apoenzyme reconstituted with manganese protoporphyrin IX, which yields enzyme having cyclooxygenase activity but not peroxidase activity, was inactive. Addition of catalase inhibited, and addition of exogenous hydrogen peroxide increased, RSVM-mediated benzidine mutagenicity. We propose that hydrogen peroxide released by the tester strain bacteria (rather than arachidonic acid-derived peroxide) is the oxidizing agent which supports prostaglandin H synthase peroxidase activity in Ames test systems.
Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material.
Ram seminal vesicle (RSV) microsomal preparations activate benzidine and other arylamines to mutagenic species in a modified Ames assay. We have examined the mechanism of this activation process in more detail. The mutagenic effect was neither arachidonic acid-dependent nor indomethacin inhibitable. The mutagenic species was stable for at least 30 min in experiments in which addition of bacteria was delayed. Acetylbenzidine was a much more potent mutagen than benzidine in this system. Substitution of the acetylase-deficient tester strain TA98/1,8-DNP6 for strain TA98 markedly reduced the mutagenicity of acetylbenzidine and completely eliminated the mutagenicity of benzidine. Benzidine analogues 3,3'-dimethoxybenzidine (o-dianisidine), o-tolidine and 3,3',-5,5'-tetramethylbenzidine were not mutagenic in the RSV activation system. RSV-dependent activation of all radiolabeled congeners examined resulted in covalent binding to calfthymus DNA. The rank order of binding was: 3,3'-dichlorobenzidine greater than benzidine greater than o-dianisidine greater than acetylbenzidine greater than tetramethylbenzidine. This binding required active enzyme and arachidonic acid or hydrogen peroxide. The reactive species was short-lived: delayed addition of DNA reduced the level of binding nearly to zero. Binding was inhibitable by indomethacin, but this inhibition was incomplete in the cases of dichlorobenzidine and acetylbenizidine. We conclude that the extracellular generation of peroxidase-catalyzed oxidation products does not explain the RSV microsome-dependent mutagenicity observed with these compounds.
A quantitative stability analysis of human monoclonal antibody production by heteromyeloma hybridomas, using an immunofluorescent technique.
We describe a quantitative method of analysis for assessing stability of human monoclonal antibody production by hybridomas. Clones derived from fusion between the SHM-D33 heteromyeloma line and EBV-stimulated human lymphocytes were studied for antibody presence using a fluorescent labelling technique. Frequencies of antibody-negative variants in clonal populations were measured, and measurements on parallel clonal populations were subjected to Luria-Delbrück fluctuation analysis to compute rates of generations of antibody-negative cells. Independent hybridoma clones exhibited a range of stabilities and the corresponding rates varied between 5 X 10(-4) and 6 X 10(-2) cell-1 generation-1. Rates of generation of antibody-negative variants for the more stable heteromyeloma hybridomas compared well with those of 2 established mouse hybridoma lines tested (less than 10(-3) cell-1 generation-1). There was a positive correlation between frequency of antibody-negative variants measured in clonal populations grown to large numbers of cells (greater than 10(7) per culture) and their rate of loss of antibody production. Large variations in frequency of antibody-negative variants were observed in parallel clonal populations, suggesting that loss of ability to produce antibody is due to random, mutation-like events including chromosome loss (Luria and Delbrück, 1943). High frequencies of antibody-negative variants may indicate imminent loss of antibody-producing capacity by a clone growing in suspension culture.