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Biomedical subjects

M Hardy

Publications and source records attributed to M Hardy.

At least 19 recordsLinked to original sources
PubMed1995

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage.

We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.

Animals↗
PubMed1995

The CCK-B antagonist CI-988 increases dopamine levels in microdialysate from the rat nucleus accumbens via a tetrodotoxin- and calcium-independent mechanism.

CI-988, a water-soluble, selective cholecystokinin-B antagonist, was perfused through a microdialysis probe into the anterior nucleus accumbens, posterior nucleus accumbens, or caudate nucleus of anesthetized rats. High concentrations of CI-988 produced three- to fivefold increases in dopamine overflow, at all three sites, that were temporally correlated with the CI-988 perfusion and returned to baseline levels upon cessation of CI-988 perfusion. However, the cholecystokinin-A antagonist CAM-1481, and the relatively inactive enantiomer of CI-988, CAM-1241, also increased dopamine overflow in the nucleus accumbens. Furthermore, the ability of CI-988 to increase dopamine overflow persisted in the absence of calcium in the perfusate and was not sensitive to tetrodotoxin treatment. The mechanism by which locally administered CI-988 increases dopamine overflow appears not to be anatomically specific, not selective for one cholecystokinin receptor subtype, and may be nonvesicular.

Animals↗
PubMed1994

Analysis of carbohydrates on IgG preparations.

Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.

Amidohydrolases↗
PubMed1994

11 beta-Amidoalkoxyphenyl estradiols, a new series of pure antiestrogens.

11 beta-Amidoalkoxyphenyl estradiols, a series of new antiestrogens, have been prepared and compared with tamoxifen (TAM) and 4-hydroxytamoxifen (OH-TAM). In vitro, these compounds were up to 20 times as active as OH-TAM on estradiol (E2)-stimulated MCF-7 cells. Unlike TAM or OH-TAM which were inactive, they displayed potent growth inhibitory effects on MCF-7 cells stimulated by a cocktail of epidermal growth factor and platelet derived growth factor. One of the most active compounds, 5e, was tested in vivo for its antiuterotrophic and antitumoral activities: it proved to be fully antiuterotrophic at 3 mg/kg subcutaneously in mice while being devoid of any uterotrophic activity. It inhibited the E2-induced growth of MCF-7 tumors implanted in nude mice and prevented the partial agonistic activity of TAM on MCF-7 tumor growth in ovariectomized mice. Moreover, on MCF-7 variant tumors, 5e, unlike TAM, did not display any proliferative activity, but inhibited the TAM-induced growth. Overall, these results show that this new series of compounds displays an improved activity profile compared with that of TAM, on tests relevant to human breast cancer treatment.

Animals↗
PubMed1994

Expression of non-NMDA glutamate receptor channel genes by clonal human neurons.

Treatment of the human teratocarcinoma line NTera2/c1.D1 (NT2) with retinoic acid induces terminal neuronal differentiation. In a previous study, we found that the neurons obtained in this way express functional N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptor channels. We now show by reverse transcriptase-polymerase chain reaction and Southern blotting that these neurons transcribe each of the nine known non-NMDA glutamate receptor genes (GluR1-7, Ka-1, and Ka-2) and that four of these genes (GluR2, GluR6, GluR7, and Ka-1) are also transcribed by undifferentiated NT2 cells. Patch clamp studies demonstrate that individual non-NMDA glutamate receptor channels are readily isolated from NT2-derived neurons and that these channels are potently modulated by the desensitization blocker cyclothiazide. NT2-derived neurons are susceptible to kainate excitotoxicity but are not injured by prolonged exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. We expect that the NT2-derived human neuronal culture system will facilitate studies of human neuronal non-NMDA glutamate receptor channels and of the pathophysiology of neuronal excitotoxicity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line.

Glutamate receptor (GluR) channels are responsible for a number of fundamental properties of the mammalian central nervous system, including nearly all excitatory synaptic transmission, synaptic plasticity, and excitotoxin-mediated neuronal death. Although many human and rodent neuroblast cell lines are available, none has been directly shown to express GluR channels. We report here that cells from the human teratocarcinoma line NT2 are induced by retinoic acid to express neuronal N-methyl-D-aspartate (NMDA) and non-NMDA GluR channels concomitant with their terminal differentiation into neuron-like cells. The molecular and physiologic characteristics of these human GluR channels are nearly identical to those in central nervous system neurons, as demonstrated by PCR and patch clamp recordings, and the cells demonstrate glutamate-induced neurotoxicity.

2-Amino-5-phosphonovalerate↗
PubMed1993

Effect of glutamine on heat-shock-induced mRNA and stress proteins.

Our aim was to delineate the effect of glutamine on the level of heat shock-inducible mRNA and synthesis of stress protein(s) in cultured kidney cells. Experiments were carried out using opossum kidney (OK) cells. The induction of HSP70 mRNA as well as the synthesis of 72,73 kDa stress proteins was evaluated in cell monolayers exposed to 45 degrees C for 15 minutes followed by a recovery period at 37 degrees C for 3 hours. Incubations were performed in Krebs buffer supplemented with 0, 2, 5, or 10 mM glutamine. A separate series of experiments was performed in the presence of glutamine metabolites, such as NH4Cl, glutamate, or aspartate. Glutamine without preincubation at 37 degrees C remarkably increased the steady-state level of HSP70 mRNA as well as the production of 72,73 kDa stress proteins in a dose-dependent manner. The production of stress protein(s) in the presence of glutamine was associated with decreased percent LDH efflux, suggesting cytoprotective action of glutamine in cultured kidney cells. However, when OK cells were preincubated for 1 hour at 37 degrees C with 10 mM glutamine, there was an approximately fourfold decline in level of HSP70 mRNA compared with experiments in the presence of 10 mM glutamine without preincubation. In addition, metabolites of glutamine, i.e., ammonia and glutamate decreased the level of heat-inducible HSP70 mRNA. Furthermore, aspartate or NH4Cl had little effect on LDH release compared with heat shock experiments, without addition of amino acids. These observations suggest that metabolites of glutamine may blunt the steady-state level of glutamate or HSP70 mRNA. The decreased level of HSP70 mRNA in the presence of NH4Cl may explain the role of ammonia in renal injury and brain toxicity, as well as glutamate excitotoxicity.

Ammonium Chloride↗
PubMed1993

Electrophysiologic and molecular properties of cultured enteric glia.

Enteric glia, the support cells of myenteric ganglia, have been widely studied with respect to their morphology and immunohistochemical phenotype, but little is known about their functional properties. We developed a method for the amplification of enteric glia from newborn guinea pigs to further characterize these cells. Treatment with a combination of basic fibroblast growth factor and the adenylate cyclase activator, cholera toxin, permitted expansion of enteric glial cultures to confluence and serial passage for up to 8 months. The long-term cultured cells retained expression of 1) S100 protein, 2) GD3 ganglioside recognized by the monoclonal antibody LB1, and 3) the gene encoding glutamine synthetase. The electrophysiologic properties of cultured enteric glia were studied under whole-cell patch clamp conditions. Most cells expressed "delayed rectifier"-type potassium currents, and some also demonstrated tetrodotoxin-sensitive sodium currents. Other subsets of voltage-dependent potassium currents, calcium currents, and glutamate-gated currents were not demonstrable.

Animals↗
PubMed1993

Anti-idiotypic antibodies specific for HLA in heart and kidney allograft recipients.

Chronic rejection is the major threat to both heart and renal allograft survival. We have explored the possibility that some patients with anti-donor HLA antibodies (Ab1) develop specific anti-idiotypic antibodies (Ab2) which suppress the production of Ab1, and subsequently, the progression of chronic rejection. Analysis of Ab2 in sera obtained from Ab1 producers showed that 22% of heart and 18% of kidney recipients produced Ab2. The 4- and 5-year actuarial graft survivals in Ab2 producers were 100% and 83%, respectively, compared to 57% in patients who formed Ab1 but not Ab2 (p < 0.004). Patients carrying the DR2 alleles, DRB1*1501, *1502 or *1601 were at a lower risk of producing anti-donor HLA antibodies.

Alleles↗
PubMed1993

Adverse events temporally associated with immunizing agents.

In Canada during 1990, a total of 2832 reports of adverse events temporally associated with the administration of immunizing agents were received by the Childhood Immunization Division of the Laboratory Centre for Disease Control. This paper summarizes the data collected, describes the surveillance system, and demonstrates that, with more than 12,000,000 doses of vaccines distributed during 1990, the incidence of adverse events reported is very low.

Adverse Drug Reaction Reporting Systems↗
PubMed1993

Effect of anti-HLA antibodies on the long-term survival of heart and kidney allografts.

Study of anti-HLA antibodies in a population of 238 primary renal and 199 primary heart allograft recipients showed significant association between development of anti-HLA antibodies and that of chronic allograft rejection. The 5-year renal allograft survival was 70% in recipients without antibodies and 53% in recipients who developed anti-HLA alloantibodies during the first year following transplantation. Heart allograft survival at 5 years was 91% in patients without and 78% in patients with antibodies during the first 12 months posttransplantation. Development of antibodies is associated with acute rejection episodes and probably with the release of soluble HLA antigens.

Antibodies, Anti-Idiotypic↗
PubMed1992

Platelet-derived growth factor and regulation of Schwann cell proliferation in vivo.

To examine the role of platelet-derived growth factor (PDGF) in the in vivo regulation of Schwann cell proliferation, steady-state levels of mRNAs encoding PDGF A and B chains, and PDGF alpha and beta receptors were measured in immature and adult rat sciatic nerves and in cultured rat Schwann cells. PDGF B chain and PDGF beta receptor mRNAs are present in immature rat sciatic nerves and to a lesser extent in adult rat nerves. Short-term cultures of neonatal rat Schwann cells express PDGF beta receptor mRNA, but not PDGF B chain mRNA, and are stimulated to synthesize DNA by addition of PDGF BB to the medium. These data indicate that PDGF BB is a developmentally regulated paracrine growth factor for rat Schwann cells. Very long-term cultures of rat Schwann cells, which have lost normal dependence on exogenous growth factors, express PDGF B chain mRNA as well as mRNAs encoding the PDGF alpha and beta receptors, suggesting that, under these circumstances, PDGF BB also act as an autocrine growth factor. PDGF A chain mRNA is present in both immature and adult rat sciatic nerves and is expressed by primary and secondary cultures of rat Schwann cells as well. However, because the abundance of PDGF alpha receptor mRNA is very low in rat Schwann cells, PDGF AA is not likely to be a significant autocrine growth factor for rat Schwann cells.

Animals↗
PubMed1992

Vasopressin biosynthesis in rodent Leydig cells.

Local biosynthesis of the peptide hormone vasopressin is demonstrated in vitro in Leydig cells derived from rat and mouse testis. Cycloheximide-sensitive production of the nonapeptide was shown for rat Leydig cells in primary culture. A polymerase chain reaction technique demonstrated the presence of functionally constituted vasopressin mRNA in rat and mouse testis, primary mouse Leydig cells and in rat and mouse Leydig tumour cell lines (MA10, R2C). Stimulation of cells with gonadotropins, however, had no effect either on peptide production or on levels of specific mRNA. Similarly, treatment of the MA10 cell line with a phorbol ester, or with rat atriopeptin, which activate other second messenger pathways, had no influence on vasopressin mRNA levels. The results are discussed in terms of an autocrine regulatory system which would provide the cell with information about its microenvironment.

Animals↗