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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↗
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↗
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↗
PubMed1992

An Automated Tactile Tester for evaluation of cutaneous sensibility.

The Automated Tactile Tester (ATT) is a computer-controlled device designed to measure patients' cutaneous perception of touch, vibration, temperature, and pain. The ATT provides repeatable and precise control of the amplitude, rate of application, and duration of stimuli. Threshold values for skin indentation (touch), high- and low-frequency vibration, pinprick (sharpness), warmth, and two-point discrimination were obtained with the ATT from the fingers of 62 normal subjects. Manual monofilament and two-point discrimination tests were also performed on the same subjects. All the tests with the ATT, except pinprick, showed a statistically significant increase in threshold with age. There were no significant differences attributable to the hand or digit tested or the sex of the subject. These data were used to derive age-adjusted criteria for normal sensory function in the glabrous skin of the fingers. Thresholds were found to remain within normal limits when these subjects were retested at various time intervals. We conclude that the ATT provides repeatable and reliable measurements of sensory function in the skin and has potential application in the diagnosis and evaluation of compression and other peripheral neuropathies.

Adolescent↗
PubMed1992

Evaluation of nerve compression with the Automated Tactile Tester.

The Automated Tactile Tester (ATT) was used to measure threshold values for trapezoidal skin indentation (light touch), low- and high-frequency vibration (50 and 150 Hz), pinprick (sharp-dull transition point), warming (temperature awareness), and two-point discrimination in 61 patients with symptoms of median nerve compression at the wrist. We compared these data with values obtained in the same patients with manual monofilament tests, manual two-point discrimination measurements, and electrophysiologic nerve conduction studies. The ATT detected abnormal sensation in 71% of the hands tested, nerve conduction velocity was abnormal in 44% of the cases, and the manual tests indicated abnormality in 42% of the hands. The most indicative single test among those included in the present study for detecting sensory abnormality in these patients was threshold to a 50 Hz vibration administered by the ATT. We conclude that the ATT is a sensitive tool for the diagnosis and evaluation of compressive peripheral neuropathy and may allow objective documentation in a higher percent of patients than do more traditional testing methods.

Adult↗
PubMed1992

A mechanism of glycine and alanine cytoprotective action: stimulation of stress-induced HSP70 mRNA.

Studies done both in vitro and in vivo have shown that glycine and alanine protect kidney cells from stress injury. However, the mechanism(s) of this cytoprotection is unknown. Our aim was to test the hypothesis that the cytoprotective action is in part due to stimulation of gene(s) expression encoding stress protein synthesis. Experiments were carried out using heat shock as a model for stress in the opossum kidney cell line (OK cells). The induction of HSP70 mRNA was evaluated in cell monolayers exposed to 45 degrees C for 15 minutes followed by a recovery period at 37 degrees C for either 0.5, 1, 2, 3, 4, 6 or 24 hours. The results demonstrate that the maximum level of HSP70 mRNA occurred at approximately three hours after heat treatment. Although the mRNA levels declined thereafter, appreciable amounts were still seen even 24 hours after heat-shock. To examine the effect of glycine or alanine on HSP70 mRNA levels and on the synthesis of stress protein, cultures were preincubated for 30 minutes with Krebs-Henseleit buffer, pH 7.4, supplemented with either 1, 2, 5 or 10 mM glycine or alanine, or with no added amino acids. Comparative studies were performed with 10 mM glutamate, aspartate, arginine or leucine. Following preincubation, cultures were heat-shocked (45 degrees C for 15 min) and then reincubated at 37 degrees C for three hours. Both glycine and alanine enhanced the level of HSP70 mRNA and the synthesis of 72, 73 kDa stress proteins, but neither amino acid induced HSP70 mRNA without concomitant heat treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed1992

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

Long-term survival of heart and kidney allografts is threatened by the development of chronic rejection. Analysis of the relationship between reversible acute rejection episodes and actuarial survival at 5 years showed an inverse correlation suggesting that early cellular events may trigger antibody-mediated chronic rejection. In both heart and kidney allograft recipients producing anti-HLA antibodies we found a significant decrease in the 5-year graft survival rate. However, there was heterogeneity among anti-HLA antibody producers with respect to the development of anti-idiotypic antibodies. The actuarial 5-year graft survival was significantly higher in patients with Ab2 compared to patients without.

Actuarial Analysis↗
PubMed1992

Effect of recipient gender and race on heart and kidney allograft survival.

Study of long-term survival of heart allografts shows that AA males and females have lower graft survival rates than those observed in NAC recipients. Primary kidney allografts in AA males, but not females, also display lower 5-year survival rates compared to those observed in the corresponding populations of NAC. Comparison of graft survival in the overall population of male and female recipients of kidney allografts shows that females have higher graft survival rates, probably as a result of better HLA matching. The level of alloantibody activity in posttransplantation sera is similar in the two populations, suggesting that factors other than HLA mismatching may contribute to the higher degree of graft failure in AA recipients.

Actuarial Analysis↗
PubMed1991

The shedding of group A rotavirus antigen in a newly established closed specific pathogen-free swine herd.

A longitudinal study was undertaken in a newly established specific pathogen-free (SPF) swine herd to determine the dynamics of rotavirus antigen shedding in a closed swine facility. Pregnant SPF gilts which populated the herd, and their offspring, were monitored weekly for three consecutive lactations. Fecal samples were assayed for the presence of group-specific viral antigen by a solid phase immunoassay (ELISA). Results indicate that in the week prior to farrow, 35% of samples from gilts/sows contained rotavirus antigen. During nursing, 37% of the gilts'/sows' fecal samples also contained virus antigen. Over the course of three farrowings, every gilt/sow in the herd excreted virus antigen. Virus antigen was present in 25% of the samples tested from nursing pigs and in 70% of the samples tested from pigs in the postnursing period; 95% of the litters excreted virus antigen either while nursing or postweaning. Seasonal incidence in virus antigen excretion was noted with proportionally more suckling pigs virus antigen-positive in summer and proportionally more sows/gilts positive during winter. Diarrhea occurred only rarely in the sampled population. Although piglets shed rotavirus subclinically, ELISA positive feces from piglets of each lactation caused severe disease when fed to neonatal gnotobiotic pigs. Electropherotyping of these passaged viruses indicated minor variation in RNA banding patterns over time.

Animals↗
PubMed1991

Characterization of two neuroblastoma cell lines expressing recombinant nerve growth factor receptors.

In earlier studies, a 75,000-dalton glycoprotein (gp75) has been identified as a component of both low- and high-affinity nerve growth factor (NGF) receptors (NGFRs). Using an amphoteric expression vector, we have introduced the cDNA encoding the human gp75 into two neuroblastoma cell lines. SHEP is a human neuroblastoma cell line that lacks most neuronal characteristics and does not express NGFRs. The transformant line SHEP/NGFR expressed a single affinity class of NGF binding sites, did not display NGF-induced up-regulation of fos oncogene expression, and did not efficiently internalize NGF. LAN5 is a neuroblastoma cell line with neuronal characteristics, including expression of neurofilament and display of short neurites. This cell line expresses a small number of high-affinity NGFRs but no detectable low-affinity sites. The transformant line LAN5/NGFR expressed both high- and low-affinity NGFRs, displayed NGF-induced up-regulation of fos oncogene, and efficiently internalized NGF. The number of high-affinity NGF binding sites was nearly the same for LAN5 and LAN5/NGFR, a finding suggesting that there is a limiting number of some separately coded factor or subunit that is required for high-affinity NGFRs. Because NGF induction of fos oncogene expression correlated with expression of high-affinity NGFRs, the putative second factor may also limit NGF responsiveness.

Binding Sites↗
PubMed1991

Angioma fusiform cells stimulated by conditioned medium from melanoma cells secrete a neomatrix which plays a role in "in vitro metastasis".

The rebuilt tumor model is a three dimensional mass of tumoral cells and angioma fusiform cells in collagen. Rebuilt tumors can give rise to "in vitro metastases" and these metastases depend on the presence of a neomatrix secreted in vitro by rebuilt tumor cells. This study defines the origin of the neomatrix and its role in "in vitro metastasis". Fusiform cells of angioma origin (AF3cells) were stimulated ten-fold by growing them in conditioned medium from a human melanoma cell line (MM2). The stimulated AF3 cells produced a dense neomatrix that was firmly attached to the culture flask. The AF3 cells were removed and MM2 cells were grown on this neomatrix. They gave rise to tumorous nodules very like the "in vitro metastases" produced by rebuilt tumors. The MM2 conditioned medium contained basic fibroblast growth factor, which could account for the angiogenetic activity of the tumoral cells. The fusiform cells of angioma origin that are stimulated by cancerous conditioned medium, are responsible for secretion of the neomatrix which plays a role in "in vitro metastasis".

Culture Media↗