Biomedical subjects

M Segal

Publications and source records attributed to M Segal.

At least 19 recordsLinked to original sources

PubMed1992

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.

Adenylyl Cyclases↗

PubMed1992

AF102B, a muscarinic M1 receptor agonist, mimics some effects of acetylcholine on neurons of rat hippocampus slices.

The putative muscarinic M1 receptor agonist, AF102B, was applied to rat hippocampal slices and the responses of intracellularly recorded pyramidal cells were examined. AF102B mimicked some effects of acetylcholine on these cells as follows: at low concentration, AF102B attenuated a slow after-hyperpolarization in response to a long depolarizing current pulse. This effect was blocked by the M1 antagonist, pirenzepine. At higher concentrations, AF102B also depolarized the cells and caused an increase in their input resistance. AF102B did not affect local excitatory postsynaptic potentials or reactivity to topically applied excitatory amino acid substances. These experiments indicate that AF102B acts as an agonist at some muscarinic M1 receptor subtypes in mammalian brain.

Acetylcholine↗

PubMed1992

Acetylcholine enhances NMDA-evoked calcium rise in hippocampal neurons.

The effects of acetylcholine (ACh) on changes in [Ca]i produced by the glutamate agonist N-methyl-D-aspartate (NMDA) were measured in cultured rat hippocampal neurons loaded with the fluorescent calcium indicator Fluo-3 in a confocal laser scanning microscope. NMDA produced a dose-dependent reversible rise in [Ca]i. ACh had a smaller and less consistent effect on [Ca]i but could cause a marked enhancement of the reactivity of neurons to NMDA. This effect was reversed by the presence of the muscarinic antagonist atropine. AMPA, another glutamate agonist which causes a rise in [Ca]i by activating voltage gated calcium influx was less affected by ACh. Caffeine, which releases calcium from intracellular stores also enhanced reactivity of these neurons to NMDA. It is suggested that ACh can enhance reactivity to NMDA by releasing calcium from internal stores.

Acetylcholine↗

PubMed1992

Acidic amino acids evoke a smaller [Ca2+]i rise in GABAergic than non-GABAergic hippocampal neurons.

Changes in intracellular calcium concentration ([Ca2+]i) in response to topical application of the glutamate agonists N-methyl-D-aspartate (NMDA), or amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were measured in cultured rat hippocampal neurons loaded with Fluo-3 and visualized in a confocal laser scanning microscope. These neurons were subsequently stained for the presence of the enzyme marker for gamma-amino butyric acid (GABA), glutamate decarboxylase (GAD). GAD-positive, putative interneurons were less responsive to NMDA and AMPA than GAD-negative neurons. The time course of the rise and decay of [Ca2+]i was similar in the two groups of neurons. Also, there was no clear difference in the shape and size of these two neuron groups indicating that the difference between them is not due to diffusion distances. These data indicate that interneurons are probably more able to handle a calcium load than other neurons, a difference that may underly their resistance to treatments which cause degeneration of other neurons in the hippocampus.

Animals↗

PubMed1992

Serum beta 2-microglobulin and prediction of progression to AIDS in HIV infection.

Identification of laboratory tests that can help predict progression to acquired immunodeficiency syndrome (AIDS) in people infected with human immunodeficiency virus (HIV) is important for clinical management and counselling. We have assessed the usefulness of CD4 lymphocyte count, serum beta 2-microglobulin concentration, and the presence of p24 antigen as predictors of AIDS. We studied 214 homosexual and bisexual men with well-defined dates of HIV seroconversion. For each participant, we defined the baseline date as the earliest date before the development of AIDS on which the three laboratory tests were done. beta 2-microglobulin concentration at baseline was in all analyses an independent predictor of AIDS, even after stratification by baseline CD4 count, duration of HIV infection, or use of zidovudine before or at baseline. For example, among men with at least 0.5 x 10(9)/l CD4 cells who were negative for p24 antigen, the risks of AIDS at 12 months and 24 months were 1% and 5%, respectively, for those whose beta 2-microglobulin concentrations were below 4.0 mg/l, compared with 17% and 27%, respectively for those with beta 2-microglobulin concentrations above that cut-off point (p less than 0.001). Among men with an estimated duration of infection of 5 years or less, beta 2-microglobulin concentration was the strongest independent predictor of AIDS. Measurement of serum beta 2-microglobulin adds important prognostic information to CD4 count in determining the risk of progression to AIDS in HIV-infected subjects, including those whose CD4 cell count has not yet fallen.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Excitatory inputs to layer V pyramidal cells of rat primary visual cortex revealed by acetylcholine activation.

Cells in layers II-III or VI were activated by microdrop application of acetylcholine (ACh), while monitoring the intracellular response of layer V pyramidal cells. This enabled the tracing of functional connections between the cells of layers II-III or VI with those of layer V. ACh activation of layer II-III or VI cells resulted in a small depolarization of these cells, accompanied by a burst of excitatory postsynaptic potentials (EPSPs) from layer V pyramidal cells. These effects of ACh were blocked by tetrodotoxin (TTX), suggesting the involvement of action potentials in their production. The input resistance of layer V pyramidal cells during and after the EPSP burst was not significantly different from control values, further suggesting an indirect effect of ACh on layer V pyramidal cells. Isolation of the supragranular layer, by horizontal cutting, did not prevent the EPSP burst evoked by ACh application to the lower layer VI, suggesting a direct input from layer VI to layer V pyramidal cells. ACh applied near pyramidal cells in layers II-III, V or VI caused transient hyperpolarization associated with a decrease in input resistance followed by a large depolarization, an increase in input resistance, and action potential discharges. The ACh-mediated hyperpolarization and the train of action potentials of layer II-III pyramidal cells were blocked by TTX. Thus the ACh-activated cells in layers II-III and VI make an excitatory synaptic contact with layer V pyramidal cells, producing the EPSP burst observed in layer V.

Acetylcholine↗

PubMed1992

Calcium dependence of serotonin-evoked conductance in C6 glioma cells.

Whole-cell membrane currents and imaging of intracellular calcium concentrations ([Ca2+]i) were used to investigate the role of calcium in a response to serotonin of C6 glioma cells. Activation of a high-affinity serotonin receptor induced a transient rise in calcium concentration in these cells and activated a predominantly potassium conductance, with a small chloride component. Perfusion of the cytoplasm with an internal solution containing high calcium concentration induced similar but prolonged increase of membrane conductance. The responsiveness of C6 cells to serotonin was negatively correlated with the concentration of the unbound calcium chelator BAPTA when BAPTA-buffered calcium-containing intracellular solutions were used. Responses to serotonin persisted in the absence of external calcium, decreased gradually, and then recovered partially after replenishment of extracellular calcium. These findings substantiate the direct role of intracellular calcium in mediating the serotonin response, and indicate that serotonin-induced release of calcium from intracellular stores is sufficient for the activation of conductance in the C6 glioma cell line.

Brain Neoplasms↗

PubMed1992

Spontaneous recovery of deficits in spatial memory and cholinergic potentiation of NMDA in CA1 neurons during chronic lithium treatment.

The therapeutic action of lithium in affective disorders is still unclear. One effect of lithium is to deplete membrane inositol and consequently to exhaust the phosphoinositide (PI) pathway. Under chronic lithium treatment, rats showed persistent performance deficits in an active avoidance task and in a visually cued maze. The same treatment, however, resulted in only a transient deficit in the performance of rats in a spatial memory task. Lithium treatment caused a similarly transient deficit in the ability of acetylcholine to potentiate responses to N-methyl-D-aspartate (NMDA) in neurons of the hippocampal slice. The authors propose that the development of compensatory mechanisms may account for the lack of severe memory impairments during lithium treatment. It is suggested that the effects of lithium on the PI pathway are not sufficient to explain the behavioral consequences of chronic lithium treatment.

Acetylcholine↗

PubMed1992

Neuropeptidergic innervation of intramuscular hemangiomas.

Intramuscular hemangiomas are idiopathic lesions which are either tumoral or developmental in origin. A close association of abnormal blood vessels with nerve fibers is found and may suggest that nerves have a primary inciting role in the development of these lesions. In the current study, the number of nerve fibers in different zones around the tumors, as well as the type of neuropeptides present in these fibers, was quantitatively assessed by computer-assisted image analysis of immunohistochemical staining of histological slides. The number of nerve fibers as determined by positive staining by anti-protein S-100 antibodies was found to be elevated in the immediate vicinity of the abnormal blood vessels. The density of the nerve fibers rapidly declined with increasing distance from the hemangiomas, reaching normal values at distances of over 2 mm. Furthermore, hemangiomas contain a significantly higher number of calcitonin gene-related peptide (CGRP), substance P, and Met-enkephalin-positive fibers. The most significant rise in number is that of CGRP-positive fibers. This neuropeptide is a known mitogen, which could be responsible for the growth of the hemangiomatous blood vessels. Substance P is a nociceptive neurotransmitter and its presence can explain the pain which often accompanies even tiny intramuscular hemangiomas.

Adolescent↗

PubMed1992

The inositol 1,4,5-trisphosphate pathway mediates cholinergic potentiation of rat hippocampal neuronal responses to NMDA.

1. The cellular mechanism by which acetylcholine (ACh) potentiates neuronal responses to N-methyl-D-aspartate (NMDA) was investigated in CA1 neurones of hippocampal slices using current- and voltage-clamp techniques. 2. Loading cells with 5'-guanylylimidodiphosphate (GppNHp) caused a gradual increase in response to NMDA. Pulses of ACh accelerated this increase. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) blocked the potentiating effect of ACh on responses to NMDA. 3. Acute LiCl caused a gradual decrease in the potentiating effect of ACh, while the potentiation was completely prevented by 3 day chronic 6 mequiv/kg (I.P.) LiCl treatment and restored by acute treatment with 10 mM-inositol. 4. Loading cells with a general protein kinase inhibitor, H-7, enhanced the potentiating effect of ACh on responses to NMDA and blocked the effect of ACh on the after-hyperpolarization (AHP). 5. Ultraviolet irradiation of cells loaded with a photolabile inositol 1,4,5-trisphosphate (InsP3) caused a transient increase in responses to NMDA, while penetrating cells with active InsP3-containing pipettes caused a gradual BAPTA-sensitive increase in responses to NMDA. 6. Reducing the rate of InsP3 metabolism, with 2,3-diphosphoglyceric acid (DPG), caused an increase and prolongation of the potentiating effect of ACh, while blocking the InsP3 receptor with heparin prevented the cholinergic potentiation. 7. NMDA, by itself, potentiated subsequent responses to NMDA, an effect that was blocked when [Ca2+]i was chelated with BAPTA. NMDA and ACh were also found to compete in potentiating responses to NMDA. Finally, the cholinergic potentiation was blocked when cells were loaded with BAPTA. 8. We propose that activation of the InsP3 branch of the phosphoinositide pathway potentiated responses to NMDA and that InsP3 exerted this effect by elevating [Ca2+]i.

Acetylcholine↗

PubMed1992

Confocal microscopic imaging of [Ca2+]i in cultured rat hippocampal neurons following exposure to N-methyl-D-aspartate.

1. The confocal laser scanning microscope (CLSM) was used in conjunction with the calcium indicator dye Fluo-3 to record changes in free intracellular calcium concentration ([Ca2+]i) in cultured hippocampal neurons in response to superfusion of N-methyl-D-aspartate (NMDA). 2. NMDA caused a rapid rise in [Ca2+]i in all parts of the neuron. The rise in [Ca2+]i was dependent on activation of an NMDA receptor, was enhanced by the removal of Mg2+ and addition of glycine to the superfusion medium, and was dependent on normal [Ca2+]o. 3. The rise of [Ca2+]i was seen first near the membrane. A wave of elevated [Ca2+]i moved centripetally at a rate of 117 microns/s. 4. Dantrolene pre-incubation caused a significant reduction in the efficacy of the NMDA-induced rise in [Ca2+]i, indicating that at least part of the rise is caused by intracellular release of calcium. 5. The replacement of calcium by barium caused a reduction in the response to NMDA, but a significant response was still present in these cells, supporting the assumption that NMDA causes release of calcium from intracellular stores. 6. The removal of sodium from the superfusion medium prolonged the [Ca2+]i rise in response to NMDA indicating that the Na-Ca antiporter is instrumental in reducing [Ca2+]i. 7. These studies demonstrate the multiplicity of regulating mechanisms of [Ca2+]i following activation of NMDA receptors.

Animals↗

PubMed1992

Activation of protein kinase C suppresses responses to NMDA in rat CA1 hippocampal neurones.

1. The effects of 1-oleoyl-2-acetylglycerol (OAG), an activator of protein kinase C (PKC), on NMDA receptor-mediated responses were investigated in CA1 neurones of hippocampal slices using current- and voltage-clamp techniques. 2. Topical application of OAG caused a suppression of the slow, voltage-sensitive, NMDA receptor-mediated component of excitatory postsynaptic potentials (EPSPs) evoked by stimulating the schaffer-collateral commissural afferents and had no effect on the fast, voltage-insensitive, quisqualate/kainate component. 3. OAG suppressed the amplitude of inward current responses to NMDA down to about one-third of control responses. OAG could also increase the duration of the responses to NMDA by up to twofold. The effect of OAG on the duration but not on the amplitude of the response to NMDA was blocked by pre-loading cells with the K+ channel blocker, Cs+. Topical application of OAG had no significant effect on current responses to quisqualate. 4. An OAG isomer, which does not activate PKC, had no effect on responses to NMDA. Intracellular application of the kinase inhibitor, H-7, completely blocked the effect of OAG on the amplitude and duration of responses to NMDA, as well as on the slow EPSP. Finally, topical application of another activator of PKC, phorbol 12-myristate 13-acetate (PMA), also suppressed responses to NMDA. PMA reduced the slow component of synaptic responses in about half of the cells tested. 5. We propose that activation of PKC in CA1 hippocampal neurones suppresses NMDA receptor-mediated responses.

Action Potentials↗

PubMed1992

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Adenylyl Cyclases↗

PubMed1992

Raphe grafts in the hippocampus, but not in the entorhinal cortex, reverse hippocampal hyperexcitability of serotonin-depleted rats and restore their responsiveness to fenfluramine.

We compared the effects of embryonic raphe grafted into either the hippocampus or the entorhinal cortex, on the responsiveness of dentate granule cells to stimulation of the perforant path. Raphe grafts in the hippocampus reversed the hyperexcitability of granule cells, resulting from depletion of the serotonergic innervation. Such grafts also restored the responsiveness of the granule cells to application of a serotonin releasing drug, fenfluramine (FFA). In contrast, hyperexcitability was not reversed when the graft was placed in the entorhinal cortex. Furthermore, although some increase in population spike size was observed in these rats after application of FFA, this increase had a response profile which was different from that of control and of lesioned rats that were grafted in the hippocampus. These results suggest that the serotonergic innervation, within the hippocampus and not in the entorhinal cortex, modulates granule cells excitability.

Animals↗

PubMed1992

A parametric family of correlation structures for the analysis of longitudinal data.

In epidemiological settings, we are often faced with numerous short time series, and a parsimonious parametrization of the correlation structure is desired in order to optimize the efficiency of the estimation procedure. We propose a damped exponential correlation structure for modeling multivariate Gaussian outcomes. The correlation between two observations separated by s units of time is modeled as gamma s theta, where gamma is the correlation between elements separated by one s-unit, and theta is a damping parameter. For (theta = 0), (theta = 1), and theta----infinity), the correlation structures of compound symmetry, first-order autoregressive, and first-order moving average processes are obtained. Although the AR(2) dependency structure, and the combination of random effects and AR(1) errors are not special cases of the proposed parametric family, these structures can be well approximated within the family for short time series. Maximum likelihood methods for parameter estimation and interpretations of intermediate models (0 less than theta less than 1) are discussed in the context of modeling pulmonary function in an adult population in The Netherlands and T-cell subsets in homosexual men infected with human immunodeficiency virus Type I.

Adult↗

PubMed1991

Restoration of serotonergic innervation underlies the behavioral effects of raphe grafts.

It has been previously demonstrated that an embryonic raphe grafted into a serotonin-depleted hippocampus restores normal serotonin innervation of the hippocampus and behaviors associated with serotonin. To test the possibility that the behavioral effects of these grafts result from non-specific actions of the grafted tissue or the grafting procedure itself, we compared raphe grafts with septal grafts, in serotonin-depleted rats. We also compared the effects of a serotonin synthesis inhibitor, p-chlorophenylalanine, on the behavior of normal, serotonin-depleted and raphe-grafted rats. The results indicate that the bulk of behavioral effects of raphe grafts are due to the serotonergic nature of the graft.

5,7-Dihydroxytryptamine↗

PubMed1991

Independent regulation of calcium revealed by imaging dendritic spines.

The dendritic spine is a basic structural unit of neuronal organization. It is assumed to be a primary locus of synaptic plasticity, and to undergo long-term morphological and functional changes, at least some of which are regulated by intracellular calcium concentrations. It is known that physiological stimuli can cause marked increases in intracellular calcium levels in hippocampal dendritic shafts, but it is completely unknown to what extent such changes in the dendrites would also be seen by calcium-sensing structures within spines. Will calcium levels in all spines change in parallel with the dendrite or will there be a heterogeneous response? This study, through direct visualization and measurement of intracellular calcium concentrations in individual living spines, demonstrates that experimentally evoked changes in calcium concentrations in the dendritic shaft ([Ca2+]d).

Animals↗

PubMed1991

An alpha 2 antagonist, idazoxan, enhances EPSP-spike coupling in the rat dentate gyrus.

The effect of the alpha 2 antagonist, idazoxan (IDA), on the excitability of neurons in the dentate gyrus of the hippocampus was studied. Population field potentials (PS) evoked by stimulation of the perforant pathway were measured before and after drug treatment. IDA enhanced the amplitude of the PS, while decreasing the slope of the EPSP. Neurotoxic destruction of noradrenergic nerve terminals completely abolished the IDA effect, arguing that its mechanism of action is through enhanced release of noradrenaline (NA). It is proposed that NA enhances the EPSP-to-spike coupling component of the PS.

Adrenergic alpha-Antagonists↗