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Biomedical subjects

R A Harris

Publications and source records attributed to R A Harris.

At least 307 records · Page 17Linked to original sources
PubMed1991

Molecular defects in the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex that cause maple syrup urine disease.

Maple syrup urine disease (MSUD) results from an inborn metabolic error caused by a deficiency of the branched-chain alpha-ketoacid dehydrogenase complex (BCKDC). cDNA clones encoding the E1 alpha subunit of BCKDC from rat and human liver have been isolated and characterized. The chromosomal location of E1 alpha on chromosome 19q13.1-13.2 has been determined using complementary methods. The etiology of MSUD has been studied by determining the enzyme activity, protein mass and mRNA level of BCKDC in fibroblasts from a human family and Polled Hereford calves, both with classic MSUD. A TACTyr to AACAsn substitution at residue 394 of the E1 alpha subunit was identified in the human patient by using enzymatic amplification of mRNA followed by DNA sequencing. Amplification of both mRNA and genomic DNA, in combination with allele-specific oligonucleotide hybridization, demonstrated that the patient was a compound heterozygote, inheriting an allele with a structural mutation from the father, and an allele from the mother containing a presumably cis-acting defect in regulation that abolished the expression of one of the E1 alpha alleles. The results revealed for the first time that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit. Recent studies by others have demonstrated that the same structural mutation as is found in this patient is responsible for the high incidence of MSUD in the Philadelphia Mennonite population.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Genetic differences in the ethanol sensitivity of GABAA receptors expressed in Xenopus oocytes.

Animal lines selected for differences in drug sensitivity can be used to help determine the molecular basis of drug action. Long-sleep (LS) and short-sleep (SS) mice differ markedly in their genetic sensitivity to ethanol. To investigate the molecular basis for this difference, mRNA from brains of LS and SS mice was expressed in Xenopus oocytes and the ethanol sensitivity of gamma-aminobutyric acid A (GABAA)- and N-methyl D-aspartate (NMDA)-activated ion channels was tested. Ethanol facilitated GABA responses in oocytes injected with mRNA from LS mice but antagonized responses in oocytes injected with mRNA from SS animals. Ethanol inhibited NMDA responses equally in the two lines. Thus, genes coding for the GABAA receptor or associated proteins may be critical determinants of individual differences in ethanol sensitivity.

Animals↗
PubMed1990

Premature translation termination of the pre-E1 alpha subunit of the branched chain alpha-ketoacid dehydrogenase as a cause of maple syrup urine disease in Polled Hereford calves.

Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain alpha-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain alpha-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E1 alpha mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1 alpha subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1 alpha subunit revealed a single base substitution at codon -6 (CAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1 beta protein stability on the co-expression of the E1 alpha.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate.

An enzymatic spectrophotometric end-point assay has been developed for determination of S-3-hydroxyisobutyrate in biological fluids. The assay measures NADH production at 340 nm after initiation of the reaction with rabbit liver 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). The assay is not affected by R-3-hydroxyisobutyrate, lactate, malate, 3-hydroxybutyrate, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 3-hydroxy-n-valerate, 2-methyl-3-hydroxy-valerate, and 3-hydroxypropionate. The assay does measure 2-ethyl-3-hydroxypropionate, a minor metabolite produced by catabolism of alloisoleucine. Application of the method to measure S-3-hydroxyisobutyrate in plasma obtained from normal, 48-h starved, and mildly and severely diabetic rats gave levels of 28, 42, 112, and 155 microM, respectively.

Alcohol Oxidoreductases↗
PubMed1990

Purification and partial characterization of branched-chain alpha-ketoacid dehydrogenase kinase from rat liver and rat heart.

Branched-chain alpha-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart. The initial step was the purification of rat liver and heart branched-chain alpha-ketoacid dehydrogenase complex with high kinase activity by a modification of a method described previously. Preservation of high kinase activity during purification of the complex required the presence of fresh dithiothreitol throughout the procedure. The kinase was released from the complex by oxidation of dithiothreitol with potassium ferricyanide and purified by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography. Both kinase preparations gave only one polypeptide band with a molecular weight of 44,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex by the purified kinase was inhibited by alpha-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex. The kinase did not exhibit autophosphorylation and does not correspond to the same protein as pyruvate dehydrogenase kinase. The kinase phosphorylated histone (type II-S), but this reaction was slow relative to the phosphorylation of the branched-chain alpha-ketoacid dehydrogenase complex and was not inhibited by alpha-chloroisocaproate.

Animals↗
PubMed1990

Factors affecting actions of ethanol on GABA-activated chloride channels.

Effects of ethanol in vitro on membrane vesicles (microsacs prepared from mouse cerebral cortex) were evaluated by monitoring 36Cl- influx. Different assay parameters were tested to determine increased or decreased action of ethanol on GABA-activated chloride channels. The ability of 30 mM ethanol to augment 36Cl- flux was seen at 0 degrees C, in the absence of GABA ("direct" action of ethanol), and at 34 degrees C in the presence of GABA, using two different assay procedures. Picrotoxin blocked the direct effects of ethanol (at 0 degrees C) suggesting GABAa involvement. Endogenous GABA in the medium surrounding the microsacs was assayed at different temperatures both in the presence and absence of GABA and ethanol. The direct effect of ethanol did not appear to involve the action of endogenous GABA. In addition to temperature effects on the assay, time of membrane storage also influenced ethanol action. Microsacs stored on ice for 2 hours or more lost their ability to respond to ethanol but not to GABA, pentobarbital or flunitrazepam. When these drugs were tested on membranes from mice that had been sacrificed by cervical dislocation as opposed to decapitation, ethanol did not augment GABA-stimulated chloride flux. The method of sacrifice did not influence the response to GABA, pentobarbital or flunitrazepam.

Animals↗
PubMed1990

Regulation of the branched-chain alpha-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease.

The hepatic branched-chain alpha-ketoacid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is converted to the inactive, phosphorylated state to conserve branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for dietary protein, results in activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex. The activity state of the complex is regulated in the short term by the concentration of branched-chain alpha-ketoacids (inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase) and in the long term by alteration in total branched-chain alpha-ketoacid dehydrogenase kinase activity. cDNAs have been cloned and the primary structure of the mature proteins deduced for the E1 alpha subunit of the human and rat liver branched-chain alpha-ketoacid dehydrogenase complex. The cDNA and protein sequences are highly conserved for the two species. Considerable sequence similarity is also apparent between the E1 alpha subunits of the human branched-chain alpha-ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex. Maple syrup urine disease is caused by an inherited deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. The molecular basis of one maple syrup urine disease family has been determined for the first time. The patient was found to be a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele which is not expressed from the mother. The only known animal model for the disease (Polled Hereford cattle) has also been characterized. The mutation in these animals introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in premature termination of translation. Two thiamine responsive patients have been studied. The deduced amino acid sequences of the mature E1 alpha subunit and its leader sequence were normal, suggesting that the defect in these patients must exist in some other subunit of the complex. 3-Hydroxyisobutyrate dehydrogenase and methylmalonate-semialdehyde dehydrogenase, two enzymes of the valine catabolic pathway, were purified from liver tissue and characterized.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Activation of branched-chain alpha-keto acid dehydrogenase complex by exercise: effect of high-fat diet intake.

The effect of exercise on the activity of branched-chain alpha-keto acid dehydrogenase complex in liver and muscle was studied in rats fed a high-fat (FAT) or a high-carbohydrate (CHO) diet. Both diet groups of rats were offered isoenergetic diets by a meal-feeding method and were trained by treadmill running. On the final day of the experiment, half of the rats in each diet group were exercised by 2 h of running just before they were killed. The activity state of the enzyme complex was elevated maximally by exercise in liver of rats fed the FAT diet but not in liver of rats fed the CHO diet, suggesting that catabolism of branched-chain amino acids in rat liver during exercise was enhanced by the FAT diet. The activity state of the enzyme complex in muscle was enhanced by exercise in both groups of rats, but a significant difference was not observed between the groups. The concentration of branched-chain amino acids was elevated in liver and muscle by exercise in both groups of rats, but the elevated levels in liver were lower in rats fed the FAT diet than in those fed the CHO diet. Serum branched-chain amino acid concentrations were significantly lower in rested rats fed the FAT diet than in those fed the CHO diet, and the leucine and isoleucine concentrations in the former were elevated by exercise, but the serum concentrations in the latter were not significantly affected by exercise. ATP and ADP concentrations in muscle were not significantly affected by either diet or exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. I. Acute effects of ethanol.

Acute exposure to ethanol was found to enhance the ability of a benzodiazepine (BZ) inverse agonist, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), to reduce muscimol-activated 36Cl- uptake by membranes isolated from mouse cerebral cortex. Pretreatment in vivo with a hypnotic dose of ethanol (but not a subhypnotic dose), or exposure to a corresponding concentration in vitro, was effective. This increase in sensitivity of gamma-aminobutyric acid receptor-operated chloride channels to the actions of DMCM was due to an increase in both the potency and efficacy of DMCM. Sensitization to DMCM was reversible and was not observed 24 hr after a single injection of ethanol. Pretreatment with ethanol (10, 50 and 100 mM) in vitro produced sensitization to DMCM in a concentration-dependent manner, similar to that produced by in vivo exposure; this increase in sensitivity did not develop if the membranes were pretreated with ethanol at 0 degrees C. Similarly, in vitro exposure to pentobarbital (200 microM) or flunitrazepam (1 microM) enhanced the actions of the inverse agonist Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]BZ-3- carboxylate). Acute ethanol exposure did not alter low-affinity gamma-aminobutyric acidA receptor binding or muscimol action, or the ability of a BZ agonist, flunitrazepam, to augment muscimol-activated chloride flux. Ethanol exposure did not alter [3H]flumazenil (Ro15-1788) binding to central BZ receptors, its displacement by DMCM or allosteric modulation of DMCM binding by muscimol (muscimol-shift).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Benzodiazepine agonist and inverse agonist actions on GABAA receptor-operated chloride channels. II. Chronic effects of ethanol.

Mice were made tolerant to and dependent on ethanol by administration of a liquid diet. Gamma-aminobutyric acid (GABA) receptor-dependent uptake of 36Cl- by mouse cortical microsacs was used to study the actions of benzodiazepine (BZ) agonists and inverse agonists. Chronic exposure to ethanol attenuated the ability of a BZ agonist, flunitrazepam, to augment muscimol-stimulated uptake of 36Cl- and enhanced the actions of BZ inverse agonists, Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,4]-benzodiazepine - 3-carboxylate) and DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), to inhibit GABAA receptor-operated chloride channels. Augmentation of chloride flux by pentobarbital was not reduced by chronic ethanol exposure. Attenuation of flunitrazepam efficacy was transient and returned to control levels within 6 to 24 hr after withdrawal from ethanol, but increased sensitivity to Ro15-4513 was observed as long as 8 days after withdrawal. Chronic exposure to ethanol did not alter [3H]SR 95531 ([2-(3'-carbethoxy-2'propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide] binding to low-affinity GABAA receptors or muscimol stimulation of chloride flux; and did not alter [3H]Ro15-4513 or [3H]flunitrazepam binding to central BZ receptors or allosteric modulation of this binding by muscimol (i.e., muscimol-shift). These results suggest that chronic exposure to ethanol reduces coupling between BZ agonist sites and the chloride channel, and may be responsible for the development of cross-tolerance between ethanol and BZ agonists. In contrast, coupling between BZ inverse agonist sites and the chloride channel is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Phosphorylation-independent effects of second messenger system modulators on gamma-aminobutyric acidA receptor complex function.

Recent studies investigating the functional significance of gamma-aminobutyric acidA (GABAA) receptor complex phosphorylation have employed membrane-permeant compounds to manipulate second messenger systems. Although these compounds affect GABAA receptor function, the dependence of these effects on phosphorylation has not been established. Here we report that several second messenger system modulations can decrease GABAA receptor function independently of their effects on protein phosphorylation. Brain membrane vesicles were lysed and resealed in the presence of EDTA to chelate internal Mg2+. Under these conditions, phosphorylation of vesicle proteins was almost completely inhibited, as determined by incorporation of 32P into phosphoproteins. In these lysed/resealed vesicles, an inhibition of muscimol-stimulated 36Cl- uptake was observed with the cAMP analogs 8-(4-chlorophenylthio)-cAMP, N6,O2'-dibutyryl-cAMP, and 8-bromo-cAMP, the protein kinase inhibitor H7, and the adenylate cyclase activator forskolin. In both intact and EDTA-treated lysed/resealed microsacs, cAMP analogs and H7 inhibited binding of the GABAA receptor ligand [3H]SR 95531 at concentrations shown to inhibit muscimol-stimulated 36Cl- uptake. Forskolin was observed to inhibit the binding of t-butylbicyclophosphoro-[35S]thionate, a ligand that binds to a site on the chloride channel. These results demonstrate that compounds commonly used to alter second messenger systems affect the receptor sites and function of the GABAA receptor chloride channel by mechanisms that do not involve protein phosphorylation. In light of these findings, results obtained with these compounds should be interpreted with caution.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Sequence of the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase in two patients with thiamine-responsive maple syrup urine disease.

Some patients with maple syrup urine disease respond to thiamine administration with a reduction in ketoaciduria and increase in activity of branched-chain alpha-ketoacid dehydrogenase. The biochemical mechanism underlying this effect is unknown but may result from decreased affinity of the mutant enzyme for thiamine or from stabilization of the abnormal enzyme by thiamine. The E1 alpha subunit of the complex participates in the thiamine-dependent decarboxylation of branched-chain alpha-ketoacids. We sequenced the E1 alpha subunit by using reverse transcription of RNA followed by enzymatic amplification of cDNA in two patients with thiamine-responsive maple syrup urine disease. The deduced amino acid sequence of this subunit in the patients was identical to that in normal controls, suggesting that in the patients the thiamine-binding site is abnormal because of a mutation in the E1 beta subunit. Other possible explanations are (a) that a mutation in the E1 beta or E2 subunits either alters thiamine binding by E1 alpha because of allosteric interactions or causes the complex to be unstable and (b) that thiamine stabilizes the complex.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

Calcium-dependent chloride currents elicited by injection of ethanol into Xenopus oocytes.

Electrophysiological techniques were used to study the response of native Xenopus laevis oocytes to intracellular injection and bath application of ethanol. Injection of ethanol produced dose-dependent transient inward currents accompanied by large current fluctuations, with estimated intracellular concentrations ranging from 10 to 300 mM. The response duration varied between 2 and 15 min, with an onset delay of 2-15 s. The inward current sometimes consisted of a fast and a slow component. Bath application of equivalent concentrations elicited similar but considerably smaller responses. The current showed a reversal potential of -20 +/- 10 mV, corresponding to an increase in chloride permeability. The response was eliminated in the presence of low chloride saline and was blocked by the chloride channel inhibitors SITS and DIDS. Ethanol responses were inhibited by the intracellular injection of the calcium chelator EGTA and were unaffected when the extracellular calcium was lowered. It is concluded that ethanol injection into Xenopus oocytes elicits a release of calcium from intracellular stores, which then activates an increased membrane permeability to chloride.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Stabilization of glycogen stores and stimulation of glycogen synthesis in hepatocytes by phenacyl imidazolium compounds.

LY177507 is representative of a series of phenacyl imidazolium compounds that cause marked lowering of blood glucose levels in animal models of noninsulin-dependent diabetes mellitus. In studies conducted with isolated rat hepatocytes, LY177507 inhibited net glucose production from a variety of substrates, inhibited glycolysis from exogenous glucose and endogenous glycogen, inhibited glycogenolysis, and stimulated glycogenesis. These effects of LY177507 appear to be the consequence of activation of glycogen synthase and inactivation of glycogen phosphorylase. In vivo studies with normal fed rats demonstrated a decrease in blood glucose, an increase in hepatic glycogen stores, and an inactivation of glycogen phosphorylase. Phenacyl imidazolium compounds appear to lower blood glucose levels and affect hepatic carbohydrate metabolism by a mechanism unlike other known hypoglycemic compounds.

Animals↗
PubMed1989

Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure.

Aldehyde Oxidoreductases↗
PubMed1989

Genetic differences in coupling of benzodiazepine receptors to chloride channels.

The affinity and density of [3H]flunitrazepam binding sites and the ability of flunitrazepam to augment GABA-activated chloride flux were determined using membrane vesicles prepared from cortex of long-sleep (LS, alcohol-sensitive), short-sleep (SS, alcohol-resistant) and individual heterogeneous stock (HS) mice. LS membranes were more sensitive than SS membranes to the effects of flunitrazepam (and ethanol) on chloride flux but did not differ in the binding of [3H]flunitrazepam. Likewise, individual HS mice displayed marked differences in enhancement of chloride flux by flunitrazepam, but little difference in binding of flunitrazepam. These results suggest that there is little genetic variation in the binding of benzodiazepines to receptors, but there is a marked diversity in the coupling of these receptors to chloride channels. This implies the existence of 'spare receptors' in LS and most HS mice, but not in SS mice. These findings also suggest that genetic differences in alcohol sensitivity in vivo may be related to coupling of benzodiazepine binding to chloride channel function.

Animals↗
PubMed1989

Cloning and sequence analysis of a cDNA for 3-hydroxyisobutyrate dehydrogenase. Evidence for its evolutionary relationship to other pyridine nucleotide-dependent dehydrogenases.

A 1.7-kilobase pair cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase has been isolated by screening a rat liver lambda gt11 library with a 17-base oligonucleotide probe which corresponds to a portion of the N-terminal amino acid sequence of rabbit liver 3-hydroxyisobutyrate dehydrogenase. The cDNA contains an open reading frame of 1038 base pairs which includes an amino acid sequence that matches the N-terminal 35 amino acid sequence of rabbit 3-hydroxyisobutyrate dehydrogenase at 33 residues. The cDNA predicts a 300-amino acid mature protein with an amino acid composition and molecular weight very similar to that of rabbit liver 3-hydroxyisobutyrate dehydrogenase. Northern blot analysis of total RNA from several rat tissues shows an mRNA of approximately 2.0 kilobase pairs in each tissue. Relative mRNA levels were: kidney greater than liver = heart greater than muscle. The amino acid sequence of 3-hydroxyisobutyrate dehydrogenase shows similarity to several other pyridine nucleotide-dependent dehydrogenases. The resemblance to malate and lactate dehydrogenases suggests that the nucleotide-binding domain is located in the N-terminal region of the protein.

Alcohol Oxidoreductases↗