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Biomedical subjects

R A Harris

Publications and source records attributed to R A Harris.

At least 199 records · Page 11Linked to original sources
PubMed1995

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

Animals↗
PubMed1995

Chemical modification and site-directed mutagenesis studies of rat 3-hydroxyisobutyrate dehydrogenase.

Rat 3-hydroxyisobutyrate dehydrogenase shares sequence homology with the short-chain alcohol dehydrogenases. Site-directed mutagenesis and chemical modifications were used to examine the roles of cysteine residues and other residues conserved in this family of enzymes. It was found that a highly conserved tyrosine residue, Y162 in 3-hydroxyisobutyrate dehydrogenase, does not function catalytically as it may in other short-chain alcohol dehydrogenases. Of the six cysteine residues present in 3-hydroxyisobutyrate dehydrogenase, only cysteine 215 was found to be critical to catalysis. C215A and C215D mutant enzymes were catalytically inactive but produced CD spectra identical to wild-type enzyme. C215S mutant enzyme displayed a lowered Vmax than wild-type enzyme, but Km values were similar to those of wild-type enzyme. The C215S mutant enzyme was inactivated by treatment with phenylmethanesulfonyl fluoride but was not inactivated by treatment with iodoacetate, whereas the wild-type enzyme was inactivated by treatment with iodoacetate but not inactivated by treatment with phenylmethanesulfonyl fluoride. The present data suggest that 3-hydroxyisobutyrate dehydrogenase differs in mechanism from other short-chain alcohol dehydrogenases studied to date and that cysteine 215 has a critical function in catalysis, possibly as a general base catalyst.

Alcohol Oxidoreductases↗
PubMed1995

Regulation by physical training of enzyme activity and gene expression of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle.

We examined the effects of short-term (5 weeks) and long-term (12 weeks) physical training on actual and total activities, protein content and mRNA abundance of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle. The actual and total activities were significantly increased approximately 60% and approximately 40%, respectively, by long-term training. No effects of short-term training on activities were observed. The increase in the total activity corresponded to increased protein content of the E1 alpha and E2 components of the complex. On the other hand, mRNA abundance for E1 alpha and E2 were not affected by the training, but that for E1 beta was slightly, but significantly increased by both short-term and long-term trainings. These divergent alterations of the message levels for the subunits of the complex suggest that posttranslational regulatory mechanisms determine the amount of the complex in skeletal muscle. Since the complex is located in the mitochondrial matrix space, mitochondrial biogenesis in response to the training was examined by determining the content of mitochondrial DNA in the muscle. The mitochondrial DNA was proportionally increased with the total activity as well as the protein content of the complex, suggesting that expression of branched-chain 2-oxo acid dehydrogenase complex in skeletal muscle in response to physical training is associated with mitochondrial biogenesis.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

Dietary control and tissue specific expression of branched-chain alpha-ketoacid dehydrogenase kinase.

The branched-chain alpha-ketoacid dehydrogenase complex, catalyst for the rate-limiting step of branched-chain amino acid catabolism, is controlled by a highly specific protein kinase (branched-chain alpha-ketoacid dehydrogenase kinase) that associates tightly with the complex. The activity state (proportion of the enzyme in its active, dephosphorylated state) of the complex varies dramatically in different rat tissues. The activity state of the complex in the liver is greater than that in any other tissue, and liver contains the lowest amount of kinase protein and kinase mRNA. However, protein malnutrition, a condition under which the complex is largely phosphorylated and inactive, resulted in a three- to fourfold increase in hepatic kinase activity with an accompanying increase in amounts of kinase protein and mRNA. Refeeding a 50% protein diet restored the normal activity state and the original levels of kinase protein and mRNA. The amount of kinase protein associated with the complex rather than changes in specific activity of the kinase appears responsible for observed differences in activity states of the complex in several rat tissues tested. Accordingly, the levels of kinase protein and mRNA measured are highest in tissues with greatest kinase activity (heart > kidney > liver), correlating reasonably well inversely with activity state of the branched-chain alpha-ketoacid dehydrogenase complex in the respective tissues. These observations suggest that the amount of kinase protein expressed in various tissues and in response to dietary protein deficiency is an important factor determining the activity state of the complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

A new family of protein kinases--the mitochondrial protein kinases.

Molecular cloning has provided evidence for a new family of protein kinases in eukaryotic cells. These kinases show no sequence similarity with other eukaryotic protein kinases, but are related by sequence to the histidine protein kinases found in prokaryotes. These protein kinases, responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase complexes, are located exclusively in mitochondrial matrix space and have most likely evolved from genes originally present in respiration-dependent bacteria endocytosed by primitive eukaryotic cells. Long-term regulatory mechanisms involved in the control of the activities of these two kinases are of considerable interest. Dietary protein deficiency increases the activity of branched-chain alpha-ketoacid dehydrogenase kinase associated with the branched-chain alpha-ketoacid dehydrogenase complex. The amount of branched-chain alpha-ketoacid dehydrogenase kinase protein associated with the branched-chain alpha-ketoacid dehydrogenase complex and the message level for branched-chain alpha-ketoacid dehydrogenase kinase are both greatly increased in the liver of rats starved for protein, suggesting increased expression of the gene encoding branched-chain alpha-ketoacid dehydrogenase kinase. The increase in branched-chain alpha-ketoacid dehydrogenase kinase activity results in greater phosphorylation and lower activity of the branched-chain alpha-ketoacid dehydrogenase complex. The metabolic consequence is conservation of branched chain amino acids for protein synthesis during periods of dietary protein deficiency. Two isoforms of pyruvate dehydrogenase kinase have been identified and cloned. Pyruvate dehydrogenase kinase 1, the first isoform cloned, corresponds to the 48 kDa subunit of the pyruvate dehydrogenase kinase isolated from rat heart tissue. Pyruvate dehydrogenase kinase 2, the second isoform cloned, corresponds to the 45 kDa subunit of this enzyme. In addition, it also appears to correspond to a possibly free or soluble form of pyruvate dehydrogenase kinase that was originally named kinase activator protein. Assuming that differences in kinetic and/or regulatory properties of these isoforms exist, tissue specific expression of these enzymes and/or control of their association with the complex will probably prove to be important for the long term regulation of the activity of the pyruvate dehydrogenase complex. Starvation and the diabetic state are known to greatly increase activity of the pyruvate dehydrogenase kinase in the liver, heart and muscle of the rat. This contributes in these states to the phosphorylation and inactivation of the pyruvate dehydrogenase complex and conservation of pyruvate and lactate for gluconeogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

Tyrosine kinase phosphorylation of GABAA receptors.

Phosphorylation of purified bovine brain GABAA receptors by the tyrosine kinase, pp60v-src was examined. pp60v-src phosphorylated two bands of 54-62 kDa and 48-51 kDa that migrated to approximately the same position as bands recognized by antisera against the beta 2 and gamma 2 GABAA receptor subunits, respectively. Bacterially expressed proteins containing the putative large cytoplasmic loops of the beta 1 and gamma 2L subunits were phosphorylated by pp60v-src, indicating that the phosphorylation sites are located in these subunit domains. The tyrosine kinase inhibitors, genistein and the tyrphostins B-42 and B-44, inhibited muscimol-stimulated 36Cl- uptake in mouse brain membrane vesicles (microsacs). magnitude of the tyrphostin B-44-induced inhibition of muscimol-stimulated 36Cl- uptake was significantly reduced in microsacs that were lysed and resealed under conditions that inhibit phosphorylation. GABA-gated Cl- currents were also inhibited by genistein and tyrphostin B-44 in Xenopus oocytes expressing alpha 1 beta 1 and alpha 1 beta 1 gamma 2L subunits. Consequently, protein tyrosine kinase-dependent phosphorylation appears to be another mechanism of regulating the function of GABAA receptors.

Animals↗
PubMed1995

Video light microscopy of 670-kb DNA in a hanging drop: shape of the envelope of DNA.

Although its conformation has not been observed directly, double-stranded DNA in solution is usually assumed to be randomly coiled at the level of the DNA double helix. By video light microscopy of ethidium-stained DNA at equilibrium in a nonturbulent hanging drop, in the present study, the 670 kb linear bacteriophage G DNA is found to form a flexible filament that has on average 17 double helical segments across its width. This flexible filament 1) has both asymmetry and dimensions expected of a random coil and 2) has ends that move according to the statistics expected of a random walk. After unraveling the flexible filament-associated DNA double helix near the surface of a hanging drop, recompaction occurs without perceptible rotation of the DNA. Both conformational change and intermolecular tangling of the DNA are observed when G DNA undergoes nondiffusive motion in a hanging drop. The characteristics of the G DNA flexible filament are explained by the assumption that the flexible filament is a random coil of double helical segments that are unperturbed by motion of the suspending medium.

Bacteriophages↗
PubMed1995

Aortoiliac segment examination with colour flow Duplex--a pilot study using Klean Prep. to improve the image quality.

OBJECTIVES: Currently colour flow Duplex examination of the iliac arteries is at best 84-92% sensitive. In an attempt to find a technique to improve on this sensitivity we have studied the effect of Klean Prep, an iso-osmotic bowel preparation, on the Duplex image and Doppler signal obtained when scanning iliac arteries. METHODS: Twenty iliac segments in 10 arteriopaths were scanned by a blinded observer, after either starving for 12 h or having Klean Prep bowel preparation. Grey scale image, colour mapping and Doppler signal to noise ratios were scored on a linear analogue system. Each patient was subsequently rescanned after the other method of preparation and was once again scored by a blinded observer. The two sets of scores were then compared. RESULTS: We found significant improvements in the linear analogue scoring of grey scale images, colour mapping and Doppler signal to noise ratios, when using Klean Prep as opposed to starving the patient prescan. CONCLUSIONS: Preparing patients with Klean Prep before iliac Duplex examination improves the visualisation of these arteries.

Aorta, Abdominal↗
PubMed1995

The induction of apoptosis in human mammary luminal epithelial cells by expression of activated c-neu and its abrogation by glucocorticoids.

The effects of expressing neu-T, a mutated constitutively activated form of c-neu, have been examined in the non-transformed conditionally immortalised human mammary luminal epithelial cell line, HB4a. A variant cell line, N4.1, which expressed neu-T, showed evidence of transformation, including partial loss of growth factor dependence and acquisition of anchorage-independent growth, but failed to give rise to tumours in nude mice, indicating that expression of neu-T alone was probably insufficient to cause tumorigenic progression to a full malignant phenotype. During characterisation of the N4.1 cell line, it was observed that under conditions of serum deprivation, it underwent apoptotic cell death, as demonstrated by light microscopy, flow cytometry and DNA gel electrophoresis. The induction of apoptotic cell death in the N4.1 cell line by serum deprivation was abrogated specifically by the addition of steroids with glucocorticoid activity but not any peptide growth factors studied. This study shows the induction of apoptosis by serum deprivation, and its abrogation by glucocorticoids occurring in human mammary luminal epithelial cells transformed by expression of neu-T, and implicates the involvement of receptor protein tyrosine kinases in an apoptotic signalling pathway in this cell type.

Apoptosis↗
PubMed1995

Mammalian alpha-keto acid dehydrogenase complexes: gene regulation and genetic defects.

Recent application of the tools of molecular biology has provided insight with respect to the structure, regulation, and defects of the genes encoding the PDC and the BCKADC. cDNAs for the alpha-KGDC have only recently been cloned. Evidence for long-term regulation of the expression of the subunits of the PDC and the BCKADC has been obtained. Detailed analyses of the promoter-regulatory regions of the genes encoding the individual subunits of these complexes have provided leads as to molecular mechanisms involved. Evidence for coordinated regulation of expression of the subunits of the complexes has been obtained under some conditions but not others. Except for the PDC E1 alpha (X) promoter, the other promoters of the genes encoding PDC and BCKADC components are TATA-less with characteristics of housekeeping genes. Most cases of PDC deficiency, which produces lactic acidosis, are caused by defects in the coding region of the E1 alpha subunit. In contrast, gene defects that cause maple syrup urine disease are more heterogeneous, involving errors in any one of the subunits of the BCKADC. Alpha-KGDC deficiency has been reported but specific mutations have not been identified. Future work is expected to include detailed studies of the genes encoding the protein kinases and phosphatases that specifically regulate the PDC and the BCKADC by covalent modification.

Animals↗
PubMed1995

Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition.

Molecular cloning of cDNAs coding for ligand-gated ion channel subunits makes it possible to study the pharmacology of recombinant receptors with defined subunit compositions. Many laboratories have used these techniques recently to study actions of agents that produce general anesthesia. We review the effects of volatile and intravenous anesthetics on recombinant GABAA, glycine, AMPA, kainate, NMDA, and 5HT3 receptors. Evidence for and against specific ligand-gated ion channel subunits as targets responsible for anesthesia or the side effects of anesthetic agents is discussed for each type of receptor. Subunit specific actions of some of the agents suggest that construction and testing of certain chimeric receptor subunits may be useful for defining the amino acid sequences responsible for anesthetic actions.

Anesthetics↗
PubMed1995

Ethanol increases GABAA responses in cells stably transfected with receptor subunits.

Ethanol enhancement of GABAA receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., 36Cl- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk-) cells stably transfected with alpha 1 + beta 1 or alpha 1 + beta 1 + gamma 2L GABAA receptor subunit DNAs and compared 36Cl- flux with whole-cell, patch-clamp measurements of GABAA receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the gamma-subunit and was maximal at a concentration of 10 mM. Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing alpha 1 beta 1-gamma 2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 mM. Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABAA receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a gamma-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.

Animals↗
PubMed1995

GABAA receptor function and binding in stably transfected cells: chronic ethanol treatment.

Effects of chronic ethanol exposure on GABAA receptors may contribute to tolerance and dependence to alcohol. Ethanol treatment of mice and rats can produce alterations of GABAA receptor binding, function, and subunit mRNA and protein levels. We treated a cell line (PA3 cells) that stably expresses GABAA receptors chronically with ethanol. Expression of bovine alpha 1, beta 1, and gamma 2L GABAA receptor subunits genes in these cells is controlled by a dexamethasone-sensitive promoter, and this provides an excellent system to study the regulation by chronic ethanol treatment of receptors with a defined subunit composition. The actions of the GABA agonist muscimol on receptor function (36Cl- uptake) were not affected by 100 mM ethanol treatment for 4 days, but the actions of flunitrazepam (1 microM) were decreased in cells treated with ethanol. The functional coupling between benzodiazepine and GABA sites on the receptors was affected by chronic ethanol treatment in a manner consistent with results from mice. Ethanol treatments (50 or 100 mM) for 4 days did not affect the affinity (Kd) or receptor density (Bmax) of [3H]flunitrazepam binding, or the levels of alpha 1 subunit mRNA, or alpha 1 or beta 1 subunit proteins. These results demonstrate that the regulation of the stably expressed GABAA receptors by chronic ethanol, in the absence of neuronal receptor gene promoters, is posttranscriptional and likely posttranslational.

Alcoholism↗
PubMed1995

Iso-osmotic bowel preparation improves the accuracy of iliac artery colour flow duplex examination.

Colour flow duplex ultrasonography is currently the non-invasive method of choice for investigating the iliac arteries. However it is only 84-92% sensitive in the best hands when compared with biplanar angiography. Bowel gas and faeces overlying the iliac arteries obscure the vessels and prevent a good ultrasound image. We have previously shown that preparation of the bowel with an iso-osmotic bowel preparation (Klean Prep) improves the clarity of duplex image of the iliac arteries. This is caused by a volume effect which flushes out gas and faeces, leaving a fluid-filled bowel transparent to ultrasound. The aim of this study was to investigate whether this enhanced image increased the diagnostic accuracy of duplex examination. We performed iliac duplex examinations on 56 patients with clinically suspected iliac artery disease, initially with the normal preparation of starving the patients for 12 h and subsequently after preparation by Klean Prep. The results from each investigation were compared with the gold standard of biplanar intra-arterial digital substraction angiography. The use of iso-osmotic bowel preparation (Klean Prep) significantly improved the accuracy of iliac duplex ultrasonography over preparation by 12 h starvation, when compared with biplanar angiography.

Angiography, Digital Subtraction↗
PubMed1995

Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes: role of calcium and protein kinase C.

Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors expressed in oocytes are inhibited by ethanol and the sensitivity to ethanol depends on the kainate concentration and the subunit(s) expressed. For example, GluR3 kainate channels are more sensitive to inhibition by ethanol than GluR6 channels in the presence of maximally effective kainate concentrations. To determine if the ethanol inhibition was influenced by the cation permeability (Na+ vs Na+ and Ca2+) of the channels expressed, we compared ethanol inhibition of Ca(2+)-permeable glutamate receptors (GluRs) in oocytes perfused with normal- and high-Ca2+ buffers. The ethanol inhibition was much greater when Ca2+ was the only permeant cation. When Ba2+ was substituted for Ca2+, the ethanol inhibition was reduced, although it was still greater than with normal buffer. The enhanced ethanol inhibition of kainate-stimulated Ca2+ currents was reduced in oocytes injected with the Ca2+ chelator BAPTA, suggesting a role for intracellular Ca2+ in mediating enhanced ethanol sensitivity of kainate channels. The enhanced ethanol inhibition of Ca2+ currents was not due to a direct ethanol inhibition of Ca(2+)-stimulated Cl- currents in the oocyte because ethanol produced no effect on Ca(2+)-stimulated Cl-currents induced by injection of myo-inositol-1,4,5-trisphosphate. Because Ca2+ activates protein kinase C (PKC) and because we found that the PKC activator phorbol 12-myristate 13-acetate inhibits kainate responses (Dildy-Mayfield and Harris, 1994), we examined the role of PKC in mediating the enhanced ethanol inhibition of kainate responses produced by increased Ca2+. Inhibition of PKC by injection of the PKC inhibitor peptide or calphostin C prevented the enhanced ethanol inhibition of kainate-induced Ca2+ responses without altering ethanol inhibition in normal buffer. Thus, ethanol inhibition of kainate channels may involve two mechanisms, one that is independent of PKC and a second type that is due to activation of PKC under conditions of elevated Ca2+, resulting in enhanced inhibition of kainate responses.

Animals↗