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A M Edwards

Publications and source records attributed to A M Edwards.

At least 109 records · Page 6Linked to original sources
PubMed1993

Role of Ca2+ in stimulation of DNA synthesis by epidermal growth factor and tumor promoters in cultured rat hepatocytes.

This study examines the effects of extracellular Ca2+ concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+ levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]o in the range 25-1,800 microM, although the peak of DNA synthesis occurred 5-10 h earlier with 1.2 mM Ca2+ than with 25 microM Ca2+. Complete removal of extracellular Ca2+ using EGTA blocked DNA synthesis if Ca2+ was removed on the second day after EGF addition but not if Ca2+ was absent only on day 1. Treatment of cultures in 1.2 mM Ca(2+)-containing media with Ca(2+)-ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o > 0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters alpha-hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor-promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]o but in combination with EGF, stimulated DNA synthesis at [Ca2+]o > 0.4 mM, suggesting that Ca2+ may have some role in mediating or modulating the stimulatory effects of these agents.

Animals↗
PubMed1993

RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability.

The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB7 was isolated and sequenced. RPB7 is a single copy gene whose sequence predicts a 19,000 Dalton protein of 171 amino acids. RPB7 is known to dissociate from RNA polymerase II as an RPB4/RPB7 subcomplex in vitro. RPB7 also appears to interact with RNA polymerase II in a manner dependent upon RPB4, since RNA polymerase II purified from cells lacking RPB4 also lacks RPB7. Previous results have demonstrated that deletion of the RPB4 results in slow growth and cold- and temperature-sensitivity. In contrast, deletion of the RPB7 gene revealed that it is essential for cell growth and viability. Loss of both the RPB4 and the RPB7 genes causes lethality. These results suggest that RPB7 contributes to the function of RNA polymerase II in the absence of RPB4 either in a manner independent of its association with the enzyme or by directly binding to the enzyme in a manner independent of its association with RPB4.

Amino Acid Sequence↗
PubMed1993

A model of the sodium dependence of dopamine uptake in rat striatal synaptosomes.

Initial velocity of uptake of dopamine (DA) has been measured in rat striatal synaptosomes as a function of both [DA] and [Na]. Carrier mediated uptake is totally dependent on external sodium. The data were fitted to a rapid equilibrium model which has been found in previous studies to fit, with appropriate simplification, uptake data for glutamate, GABA, and choline in several brain regions under varying conditions. This model also gives a good fit to the dopamine data. The minimal best fit simplification of this model allows for DA uptake along with two sodium ions and predicts that apparent maximal velocity of uptake should increase with [Na], while the Michaelis-Menten constant should decrease. The minimal best fit model for DA, and a number of kinetic parameters which quantitate the model, are compared to those for the GABA, glutamate, and choline transporters. The results are consistent with a symmetrical, rapid equilibrium model, which has been presented previously for other neurotransmitters and precursors (18). This model offers a unifying basis for understanding the sodium and membrane potential dependence of neurotransmitter transport and the possible participation of transporters in depolarization induced release throughout the CNS.

Animals↗
PubMed1993

The effect of cocaine on membrane potential, on membrane depolarization by veratridine or elevated [K]o and on sodium/potassium permeability ratios in synaptosomes from the limbic cortex of the rat.

Effects of cocaine on the synaptosomal membrane potential (Em), on membrane depolarization induced by veratridine or elevated [K]o and on sodium/potassium permeability ratios (pNa/pK), have been measured in buffer containing either low or high [Na]. Fluorescence of the dye rhodamine 6G was used to measure the membrane potential. Cocaine began to reduce the Em (depolarized) at concentrations between 10(-6) and 10(-5) M in low [Na] buffer and between 10(-5) and 10(-4) M in high [Na] buffer. Maximum depolarization (with 10(-3) M cocaine) was 21 mV in low [Na] buffer and 11 mV in high [Na] buffer. Cocaine also reduced the depolarization caused by veratridine or elevated [K]o; the effective concentration of cocaine in reducing the response to veratridine was one-tenth that necessary to reduce the response to elevated [K]o. The antagonism by cocaine of the response to veratridine was similar to that found by other investigators; however, this action would tend to oppose depolarization and thus cannot explain the depolarizing effect of cocaine alone. The antagonism by cocaine of the depolarization caused by elevated [K] was consistent with a reduction in pK; such a change in pK could explain the observed reduction in Em caused by cocaine alone. The effect of cocaine (10(-3) M) on the Em was also measured as a function of [K]o at low and high [Na]o. Cocaine caused membrane depolarization at all [K]o's (3.9-19.2 mV), an effect that was somewhat greater in the low [Na] medium. These measurements of Em were fitted to the Goldman equation and the ratio of pNa/pK estimated. The presence of cocaine increased the estimate of pNa/pK by 45.7%, presumably by reducing pK.

Animals↗
PubMed1993

Dopamine uptake in five structures of the brain: comparison of rate, sodium dependence and sensitivity to cocaine.

The rate of uptake of dopamine (DA) per microgram protein and the sensitivity of uptake to sodium ([Na]o) and cocaine, have been measured in synaptosomes from five structures in the brain of the rat: striatum, nucleus accumbens, neocortex, limbic cortex and thalamus. Probably reflecting the number of DA terminals, there was a wide variation in the rate of uptake in the different structures: uptake was far greater in the striatum and nucleus accumbens (10-20-fold) than in the neocortex, limbic cortex or thalamus. Uptake in all structures was inhibited by cocaine. With high [Na]o, the IC50's varied from 1.10 microM for the thalamus to 3.32 microM for the nucleus accumbens. Maximum percentage inhibition varied from 76.0 for limbic cortex to 96.8 for nucleus accumbens and 97.7 for striatum; thus most uptake was carrier-mediated. With low [Na]o, IC50 for the nucleus accumbens was unchanged, while the IC50's for the striatum and limbic cortex were less. Maximum percentage inhibition was similar to that found for high [Na]o. Although the sensitivity to [Na] varied, synaptosomes from all areas showed Na-dependent uptake; lowering [Na] in the incubation medium from 133.2 to 9.5 mM reduced the uptake by a minimum of 36.9% in the neocortex to a maximum of 78.0% in striatum. Neither the rate of uptake nor the Na-dependence correlated precisely with the sensitivity to cocaine but the two structures which showed the greatest rate of uptake (nucleus accumbens and striatum) also showed the greatest sensitivity to [Na]o. Generally, the rate of uptake correlated with the density of DA terminals and number of cocaine binding sites as reported by others.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Quantitative localization of AMPA/kainate and kainate glutamate receptor subunit immunoreactivity in neurochemically identified subpopulations of neurons in the prefrontal cortex of the macaque monkey.

Excitatory amino acid transmission has been proposed as the principal synaptic mechanism for distribution of information through corticocortical and thalamocortical pathways. The following study utilized a double labeling paradigm, using antibodies that recognize non-NMDA ionotropic glutamate receptor subunits and other neuronal markers, to further define, quantitatively, the subclasses of neurons that contain immunoreactivity for the AMPA/kainate and kainate receptor subunits in the monkey prefrontal cortex. Double labeling with an antibody that recognizes common epitopes in AMPA/kainate subunits GluR2 and GluR3 (GluR2/3) in combination with an antibody that recognizes the kainate receptor subunits GluR5, GluR6, and GluR7 (GluR5/6/7) demonstrated that immunoreactivity for these two receptor classes was highly colocalized in a great majority of the pyramidal neurons in this region but present in only a minority of neurochemically identified subclasses of GABAergic interneurons. Furthermore, GluR2/3 immunoreactivity had principally a somatic distribution whereas GluR5/6/7 labeling was predominately found in the perikarya and/or particular dendritic domains. In contrast, intense GluR1 labeling was observed in a small subpopulation of interneurons and low GluR1 immunoreactivity was present in many other cortical neurons. These results demonstrate that there is a high degree of specificity in the distribution of AMPA/kainate and kainate receptor-class proteins to subclasses of neurons within the neocortex. A neuron's combination of excitatory amino acid receptor subunits may regulate its response to excitatory inputs and further defines the role of identified subclasses of neurons in the complex circuitry of the cerebral cortex and may also indicate the basis for the apparent cellular selectivity of excitotoxic degenerative processes.

Alzheimer Disease↗
PubMed1993

The clinical efficacy of inhaled nedocromil sodium (Tilade) in the treatment of asthma.

Inhaled nedocromil sodium, an anti-inflammatory agent for the maintenance treatment of asthma, has been the subject of many clinical trials but only a proportion have been published. This paper provides an overview analysis of all known placebo-controlled, double-blind, randomized therapeutic trials. Both single centre and multicentre trials are included. A comparison has been made between the treatment effects of nedocromil sodium and placebo, using six efficacy variables (day and night asthma, cough, mean daily peak expiratory flow rate, inhaled bronchodilator use, clinic lung function (forced expiratory volume in one second) and global opinion of treatment efficacy. One hundred and twenty seven trial centres, involving 4,723 patients, have been included. The results demonstrate the efficacy of inhaled nedocromil sodium in a variety of patients with asthma. Optimal effects are evident in patients with moderate asthma, currently receiving treatment with inhaled and oral bronchodilators.

Administration, Inhalation↗
PubMed1992

Riboflavin-photosensitized anaerobic modification of rat lens proteins. A correlation with age-related changes.

When rat lens homogenate or its soluble protein fractions are irradiated in the presence of riboflavin, a photo-adduct is obtained between this vitamin and the lens proteins. Irradiation of these proteins in the presence of riboflavin also leads to a modification in the chromatographic elution pattern with an increase in the high-molecular-weight fraction. In an aging study with rats, it was shown that the proportion of the high-molecular-weight protein fraction significantly increased with age, whereas the proportion of the low-molecular-weight protein fraction concomitantly decreased. It is postulated that aging produces an increase in the accessibility of the tryptophan residues of the lens proteins, as established by iodide fluorescence quenching experiments.

Aging↗
PubMed1992

Effect of fatty acids on rat liver nuclear T3-receptor binding.

The effects of selected fatty acids (linoleic, oleic, and palmitic) on triiodothyronine (T3)-receptor binding were compared in isolated rat hepatocytes, rat liver nuclei, and receptor protein. Scatchard analysis indicated that the inhibition of T3-receptor binding by fatty acids was characterized by an increase in Kd and no change in maximum binding capacity (MBC). In isolated receptors, the rank order of potency for inhibition was linoleic acid greater than oleic acid greater than palmitic acid. The Ki for oleic acid in isolated receptors was the same as that for whole nuclei (15.4 +/- 1.3 v 16.3 +/- 1.9 mumol/L, respectively), indicating that the inhibition of nuclear T3 binding is probably at the level of the receptor protein itself. In isolated hepatocytes, linoleic acid was more potent than oleic acid in inhibiting T3 binding to nuclear receptors. Cell-associated T3 was not affected by the presence of fatty acids, implying that cellular uptake of T3 was not inhibited. High concentrations of fatty acids were necessary for inhibition of T3-receptor binding in isolated hepatocytes, with linoleic acid being one to two orders of magnitude less potent in isolated hepatocytes compared with isolated receptors (Ki, 179 +/- 12 v 4.4 +/- 0.5 mumol/L, respectively). It is concluded that the inhibitory effect of fatty acids on T3-receptor binding in isolated rat hepatocytes probably occurs at the level of the nuclear receptor, and does not involve an inhibition of the access of T3 to the receptor. However, in vivo it seems unlikely that fatty acids will have access to the nuclear receptors in sufficiently high concentrations to affect T3-receptor binding in liver cells.

Animals↗
PubMed1991

Two-dimensional and epitaxial crystallization of a mutant form of yeast RNA polymerase II.

A mutant form of yeast RNA polymerase II that lacks the fourth and seventh largest subunits, referred to as pol II delta 4/7, crystallized on positively charged lipid layers. Both single-layered (two-dimensional) crystals and several multi-layered crystal forms were obtained. The two-dimensional crystals, preserved in negative stain, diffracted strongly to about 1/20 A-1 and more weakly to 1/13 A-1 resolution. A projection map computed from averaged Fourier transforms revealed four pol II delta 4/7 complexes per unit cell and further revealed a cleft on the surface of the complex similar to that previously observed in the structure of Escherichia coli RNA polymerase. One of the multi-layered crystal forms, preserved in negative stain, diffracted strongly beyond 1/15 A-1 resolution. Coherent diffraction from the multi-layered crystal is indicative of protein-protein interactions between layers and ordering in the third dimension.

Crystallization↗
PubMed1991

Three-dimensional structure of yeast RNA polymerase II at 16 A resolution.

The structure of yeast RNA polymerase II has been determined by three-dimensional reconstruction from electron micrographs of two-dimensional crystals at approximately 16 A resolution. The most prominent feature of the structure is an arm of protein density surrounding a channel about 25 A in diameter, similar to that found previously for E. coli RNA polymerase. The 25 A-diameter channel bifurcates on one face of the protein, connecting with a 25 A-wide groove and with a channel about half as wide. The 25 A channel and groove, and the narrow channel, may bind double- and single-stranded nucleic acids, respectively. A finger of protein density projecting from the molecule adjacent to the arm-like feature may represent the C-terminal domain of the largest subunit. These results provide a structural basis for analyses of the transcription process and its regulation.

Microscopy, Electron↗
PubMed1991

Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.

RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.

Cell Nucleus↗
PubMed1991

A light-induced tryptophan-riboflavin binding: biological implications.

We review here the covalent photo-binding induced by visible light between the essential amino acid tryptophan and the vitamin riboflavin. We discuss the biological implications of this photoadduct in relation to the hepatotoxic and cytotoxic effect associated to parenteral nutrients and to culture media exposed to the action of light, respectively. We also analyze the formation of a photo-binding between riboflavin and the residues of tryptophan present in the proteins of the eye lens, a tissue which is permanently exposed to visible light.

Animals↗
PubMed1990

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase.

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.

1-Propanol↗