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Biomedical subjects

M D Lewis

Publications and source records attributed to M D Lewis.

36 records · Page 2Linked to original sources

Peptidomimetic inhibitors of Ras farnesylation and function in whole cells.

The ras protooncogene is involved in regulation of cell growth. Mutations that activate the protein result in uncontrolled cell growth. Ras undergoes a series of posttranslational processing events, the first of which, farnesylation, is crucial for the function of the protein. Inhibitors of the farnesyltransferase enzyme are therefore potential candidates for the development of anticancer drugs. Tetrapeptides have been reported to be good inhibitors of this enzyme in vitro. We have synthesized analogs of the tetrapeptide Cys-Val-Phe-Met by replacement of the amino-terminal amide bonds. One inhibitor, B581, is permeable to the cell membrane. In the cell, it inhibits processing of two farnesylated proteins, H-ras and lamin A, but it does not inhibit processing of a geranylgeranylated protein, Rap 1A. Microinjection of B581 into frog oocytes inhibits maturation induced by activated, farnesylated H-ras but not maturation induced by activated, geranylgeranylated H-ras or by progesterone. These results demonstrate that this peptide mimic inhibits farnesylation selectively in the cell. The inhibition of farnesylation results in inhibition of H-ras function.

3T3 Cells↗

Effects of free fatty acids on somatostatin secretion, content and mRNA levels in cortical and hypothalamic fetal rat neurones in monolayer culture.

The effects of free fatty acids on somatostatin secretion, content and mRNA levels in fetal rat hypothalamic and cortical cell cultures were investigated. Somatostatin secretion and content were quantified by radioimmunoassay. Somatostatin mRNA levels were measured by Northern blot hybridization using a cRNA probe. Treatment with either caprylic acid (5 x 10(-3) M) or oleic acid (5 x 10(-5) M) for 90 min inhibited basal somatostatin secretion in both hypothalamic and cortical cell cultures. In addition, the increase in somatostatin secretion induced by incubation with veratridine (10(-4) M) or carbachol (10(-4) M) for 90 min was significantly reduced by the addition of caprylic acid, but somatostatin release stimulated by 5.6 x 10(-2) M KCl was not affected. Furthermore, treatment with these free fatty acids for 90 min markedly decreased somatostatin mRNA levels in both types of neurone culture. These inhibitory effects were transient, being observed after 90 min, but not after 5 h. These results support the probability that there is a role for free fatty acids in the regulation of somatostatin mRNA levels and somatostatin secretion in both hypothalamic and cortical cell cultures.

Animals↗

Differential effects of in vivo estrogen administration on hypothalamic growth hormone releasing hormone and somatostatin gene expression.

The aim of this study was to investigate the effect of in vivo estrogen administration on hypothalamic growth hormone releasing hormone (GHRH) and somatostatin (SS) gene expression. We found that estrogen administration (estradiol valerate 250 micrograms/every 3 days, subcutaneously) to male rats induced a decrease in both hypothalamic GHRH mRNA levels and GHRH content, that was significant after 3 and 8 days of treatment. In contrast SS mRNA levels were transiently elevated after 1 and 3 days of estrogen administration, returning to normal values after 8 days of treatment. These data suggest that the existence of sexual dimorphism in GH secretion in the rat could be mediated to some extent by gonadal hormones regulating somatostatin and GHRH gene expression in the hypothalamus.

Animals↗

Stimulatory effect of free fatty acids on growth hormone releasing hormone secretion by fetal rat neurons in monolayer culture.

The effects of free fatty acids (FFAs) on growth hormone releasing hormone (GHRH) secretion by fetal rat cortical and hypothalamic neurons in monolayer culture have been investigated. Treatment with caprylic or oleic acids for 90 min, induced a dose-dependent increase in GHRH secretion. The stimulatory effect of caprylic acid was time-dependent, being present after 90 min and 5 h but not after 24 h. Finally, GHRH release induced by 56 mM KCl or by the calcium ionophore A23187 was not modified by 5 x 10(-3) M caprylic acid. These data support a role for FFAs on GHRH secretion.

Animals↗

Motor neurone disease serum and beta-N-methylamino-L-alanine stimulate thyrotrophin-releasing hormone production by cultured brain cells.

We have investigated the effects of serum from patients with motor neurone disease (MND) and of the plant-derived excitotoxin beta-N-methylamino-L-alanine (L-BMAA) on thyrotrophin-releasing hormone (TRH) and somatostatin (SS) production by fetal rat brainstem cell cultures. Compared to age- and sex-matched normal and neurological disease control sera, MND sera produce a 2- to 3-fold increase in TRH content of the cultures with no change in SS levels. L-BMAA produced similar dose-related and stereospecific effects on the cultures increasing TRH levels to 2-fold with no effect on SS levels. These findings may be relevant to the understanding of the aetiology of MND.

Aged↗

The role of calcium and calmodulin in mediating release of thyrotrophin-releasing hormone by cultured hypothalamic cells.

We have developed a fetal rat hypothalamic cell culture system for the study of factors controlling the acute release of TRH. Release of TRH by the cells has been characterized by reversed-phase high pressure liquid chromatography and about 86% of the total immunoreactivity in the medium co-eluted with synthetic TRH. Release of TRH by the cells in response to 56 mmol K+/l increased between days 5 and 9 of culture but reached a plateau thereafter. Cell contents of TRH did not change significantly between days 5 and 14 of culture. Release of TRH from the cells was stimulated by K+ (56 mmol/l), veratridine (100 mumol/l) and ouabain (100 mumol/l) to 550, 480 and 335% of basal release respectively over a 1-h period. Release of TRH was dependent upon calcium in that it was absent when calcium-free medium was used and could be blocked by verapamil (20 mumol/l); however it could not be blocked by nifedipine (50 mumol/l). The calcium ionophore blocked by nifedipine (50 mumol/l). The calcium ionophore A23187 (1 mumol/l) stimulated TRH release to 340% of basal release. Tetrodotoxin (1 mumol/l) completely abolished the release in response to veratridine but had no effect on the release stimulated by K+ (56 mmol/l). The calmodulin antagonists trifluoperazine and triflupromazine (50 mumol/l) inhibited veratridine-stimulated TRH release. This was at a site after calcium influx as they also inhibited A23187-stimulated TRH release. The highly specific calmodulin antagonist W7 (10 mumol/l) also inhibited both veratridine and A23187-stimulated TRH release whereas, at the same concentration, its inactive analogue W5 did not significantly inhibit TRH release in response to either stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Dopamine stimulates release of thyrotrophin-releasing hormone from perfused intact rat hypothalamus via hypothalamic D2-receptors.

We have studied the effect of dopamine together with agonist and antagonist drugs of different specificities on the release of TRH from the perfused, intact hypothalamus of the adult rat in vitro. Dopamine produced a dose-related stimulatory effect on TRH release with maximal effect being achieved at 1 mumol/l (increase over basal, 118 +/- 16.5 (S.E.M.) fmol TRH; P less than 0.001 vs basal). This effect was mimicked by the specific D2-agonist drugs bromocriptine (0.1 mumol/l) and LY 171555 (0.1 mumol/l) (increase over basal values, 137.5 +/- 13.75 fmol and 158.6 +/- 10.7 fmol respectively; P less than 0.001 vs basal), but not by the D1-agonist SKF 38393A. The stimulatory effect of dopamine (1 mumol/l) was blocked in a stereospecific manner by the active (D) but not by the inactive (L) isomers of the dopamine antagonist butaclamol. Similar blockade was achieved with the specific D2-antagonist domperidone (0.01 mumol/l) whereas the D1-antagonist SCH 23390 was only effective when used at a concentration 100 times greater. Lower concentrations (0.01 mumol/l) of this D1-antagonist did not block the stimulatory effect of dopamine. High-performance liquid chromatography characterization of the material secreted within the hypothalamus showed one single peak of immunoreactive material which coeluted with synthetic TRH. These data suggest that dopamine exerts a stimulatory role in the control of hypothalamic TRH release by acting at specific D2-receptors.

Animals↗

Differential production of SRIF 14 and 28 by fetal rat hypothalamic cells enriched by velocity sedimentation.

Dispersed day-17 fetal rat hypothalamic cells have been enriched according to size by velocity sedimentation prior to culture, and the SRIF production by these enriched populations was compared with that of other enriched cell fractions and with mixed-cell cultures. Cultures of mixed cells produced 100-400 pg SRIF/10(6) cells/4 h over a period of 28 days. Total SRIF production by mixed cells was inversely proportional to seeding density over the range 0.25-1 X 10(6) cells/ml/well and SRIF 14 and 28 were secreted in a ratio of approximately 6:1. Although secretory rates by low cell densities remained higher than those by high cell densities, SRIF production decreased with time at all seeding densities (up to 21 days). Dispersed fetal hypothalamic cells were enriched according to size by allowing them to sediment over 4 h through a shallow gradient of BSA in culture medium and subsequent cell fractions developed widely differing morphologies in monolayer cultures. In contrast to mixed-cell cultures, SRIF production at 8 days by both large and small cells were directly proportional to initial seeding density. Furthermore, the smaller cells secreted very much less SRIF than the larger cells (100 pg/10(6) cells/4 h vs. 1,300 pg/10(6) cells/4 h), whereas there was little difference in overall SRIF content (350 pg/10(6) cells vs. 400 pg/10(6) cells). Characterisation by HPLC of the SRIF content and secretion of smaller cells revealed SRIF 14 to 28 ratios of 7:1 and 3:1, respectively. In contrast in the large cells, the ratio was 1:1 for both content and secretion. Therefore, these cell groups contain and secrete different proportions of these 2 molecular forms of SRIF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Characterization and partial purification of the sodium-potassium-ATPase inhibitor released from cultured rat hypothalamic cells.

An inhibitor of sodium-potassium-ATPase has been partially purified from the culture medium obtained from hypothalamic cells maintained in a capillary membrane perfusion system, and some of the properties of this inhibitory factor have been investigated. Gel filtration (Sephadex G-25 Superfine) of heat-treated medium (80 degrees C for 10 min) resulted in elution of inhibitory activity in the post-salt fraction. These fractions inhibited active (i.e. sodium-potassium-ATPase-mediated) sodium transport in intact human erythrocytes, displaced [3H]ouabain from its binding site, and directly inhibited canine kidney sodium-potassium-ATPase as measured by NADH oxidation. High-performance liquid chromatography (on Hypersil ODS) of these fractions after desalting yielded one region which showed inhibitory activity on all three assays. Inhibition of sodium-potassium-ATPase was dose-related and filtered through an Amicon UM10 membrane. Incubation of this material with dispase, carboxypeptidase A, chymotrypsin, and prolidase destroyed inhibitory activity, whereas trypsin and leucine aminopeptidase were ineffective. These studies show that hypothalamic neurones release a low molecular weight heat-stable peptide which inhibits active sodium transport, ouabain binding, and sodium-potassium-ATPase.

Animals↗

Proper identification of anisakine worms.

Luminal, gastric, intestinal and mesenteric forms of anisakiasis are known and can be encountered where raw or undercooked marine fish or squid are eaten. Although the anisakine nematodes which cause infection in humans are usually identified after surgical removal, laboratory personnel should be aware of their similarities to other nematodes. Cases have been reported of detection of larval nematodes in the throats or mouths of patients who have vomited or coughed. When such specimens are submitted to the clinical laboratory, problems in identification can be minimized by proper fixation and clearing. Systems for study involving clearing in phenol-ethanol and dissection to observe presence or absence of a ventricular appendix or intestinal cecum to distinguish Anisakis-, Phocanema- and Contracaecum-type larvae are described. Distinguishing characteristics are illustrated. The recovery of a Phocanema-type larva from a California woman is reported; the presence of the larvae in fish sold for human consumption in San Diego is exemplified.

Animals↗

The promise of dynamic systems approaches for an integrated account of human development.

After decades of theoretical fragmentation and insularity, a converging explanatory framework based on general scientific principles is an important goal for developmental psychology. Dynamic systems approaches may provide such a framework, using principles of self-organization to explain how novel forms emerge without predetermination and become increasingly complex with development. New trends in traditional theoretical families emphasize systemic, emergent processes, and these can now be explicated with principles of self-organization that apply to all natural systems. Self-organization thus provides a single explanation for the multiple facets of development, integrating diverse developmental viewpoints within a larger scientific perspective.

Cognition↗

The Position Paper.

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American Nurses' Association↗