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D W Crabb

Publications and source records attributed to D W Crabb.

At least 37 records · Page 2Linked to original sources
PubMed1996

The role of nuclear factor NF-Y/CP1 in the transcriptional regulation of the human aldehyde dehydrogenase 2-encoding gene.

Mitochondrial aldehyde dehydrogenase (ALDH2) activity is produced at low levels in many tissues, with highest production in liver. Transfection assays using the first 600 bp of upstream DNA provided evidence for both positive and negative regulatory elements in the proximal promoter. A region from -79 to -116 bp was protected in DNase I footprinting assays and bound in electrophoretic mobility shift assays (EMSA) by a nuclear factor found in all cell lines and tissues tested. This region, denoted FP160, contained the consensus recognition sites for Sp1 and AP2, and a CCAAT box. The CCAAT box was specifically protected by a nuclear factor in methylation interference assays. Mutagenesis of specific bp within the CCAAT box eliminated protein binding in vitro and decreased transcriptional activity from the ALDH2 promoter approximately 50% in reporter gene assays. Competition experiments showed that the nuclear factor binding to the FP160 oligodeoxyribonucleotide (oligo) was competed by oligos corresponding to an NY-Y/CP1-binding site to a greater extent than by those containing sites for CTF/NF1, C/EPB or CP2. The heat stability, resistance to proteinase K digestion, sensitivity to inhibition of DNA binding by o-phenanthroline, and immunological properties of the liver factor binding to FP160 were very similar to the corresponding properties of NF-Y/CP1. Thus, the proximal ALDH2 promoter was bound by NF-Y/CP1 and this transcription factor may be responsible for the basal expression of the gene observed in most tissues. The NFY-CP1 present in rat liver has similar properties to that previously characterized in M12 B-lymphoma cells and LMTK mouse fibroblasts.

Aldehyde Dehydrogenase↗
PubMed1996

Structural and mechanistic similarities of 6-phosphogluconate and 3-hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases.

Rat 3-hydroxyisobutyrate dehydrogenase exhibits significant amino acid sequence homology with 6-phosphogluconate dehydrogenase, D-phenylserine dehydrogenase from Pseudomonas syringae, and a number of hypothetical proteins encoded by genes of microbial origin. Key residues previously proposed to have roles in substrate binding and catalysis in sheep 6-phosphogluconate dehydrogenase are highly conserved in this entire family of enzymes. Site-directed mutagenesis, chemical modification, and substrate specificity studies were used to compare possible mechanistic similarities of 3-hydroxyisobutyrate dehydrogenase with 6-phosphogluconate dehydrogenase. The data suggest that 3-hydroxyisobutyrate and 6-phosphogluconate dehydrogenases may comprise, in part, a previously unrecognized family of 3-hydroxyacid dehydrogenases.

Alcohol Oxidoreductases↗
PubMed1996

Catabolism of isobutyrate by colonocytes.

Isolated colonocytes have more capacity for the oxidation of isobutyrate and alpha-ketoisovalerate than isolated enterocytes. Both enterocytes and colonocytes express high levels of 3-hydroxyisobutyryl-CoA hydrolase, an enzyme activity important in maintaining low intracellular concentrations of methacrylyl-CoA, a common, potentially toxic intermediate in the catabolic pathways of these compounds. In spite of comparable 3-hydroxyisobutyryl-CoA hydrolase activities in both cell types, and much greater amounts of 3-hydroxyisobutyrate dehydrogenase in colonocytes than in enterocytes, only the colonocytes produced 3-hydroxyisobutyrate as an endproduct of alpha-ketoisovalerate and isobutyrate catabolism. Butyrate very effectively inhibits isobutyrate catabolism by colonocytes, most likely by competitively inhibiting activation of isobutyrate to its CoA ester. Oleate also inhibits isobutyrate catabolism, but at a site more distal than butyrate. Starvation of rats for 72 h decreased the capacity of colonocytes for butyrate but not isobutyrate catabolism. We conclude that isobutyrate could function as a carbon source for energy and anapleurosis in colonocytes under conditions of defective butyrate oxidation or low butyrate availability.

Animals↗
PubMed1996

Dexamethasone represses phorbol ester-, forskolin-, and calcium-stimulated expression of a preproenkephalin A promoter-chloramphenicol acetyltransferase gene via a receptor-mediated mechanism.

CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50%, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10(-8)-10(-6) M dexamethasone and was blocked by the presence of the glucocorticoid antagonist RU486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.

Calcimycin↗
PubMed1996

Distribution of messenger RNAs for aldehyde dehydrogenase 1, aldehyde dehydrogenase 2, and aldehyde dehydrogenase 5 in human tissues.

BACKGROUND: The distribution of aldehyde dehydrogenases in human tissues is incompletely understood, in part because of technical limitations of gel electrophoretic and other enzyme assay methods used previously and because of the instability of the enzymes. Since these enzymes participate in detoxification of endogenous and exogenous compounds, including ethanol, their tissue distribution may be relevant to the toxicology of a number of substances and to the medical consequences of alcoholism. METHODS: The abundance of mRNA for aldehyde dehydrogenase 1 (ALDH1), aldehyde dehydrogenase 2 (ALDH2), and aldehyde dehydrogenase 5 (ALDH5) was determined by Northern blotting using poly A+ RNA from 16 adult human tissues and 5 fetal tissues. RESULTS: The highest levels of ALDH1 mRNA were found in liver, kidney, muscle, and pancreas. ALDH2 and ALDH5 were expressed in a larger number of tissues than ALDH1, with highest levels in liver, kidney, muscle, and heart. Fetal heart, brain, liver, lung, and kidney expressed ALDH2 and ALDH5, while ALDH1 was present mainly in fetal liver, kidney, and lung. CONCLUSIONS: The results are in general agreement with the distribution of enzymes studied in a limited number of tissues in the past, with the exception that the ALDH1 activity reported to exist in heart and brain may, in fact, be ALDH5. The only mRNA detected in placenta was that for ALDH5. This study extends the knowledge of the expression of these enzymes to several tissues not previously studied and establishes the tissue distribution of the new enzyme ALDH5.

Adult↗
PubMed1995

The novel aldehyde dehydrogenase gene, ALDH5, encodes an active aldehyde dehydrogenase enzyme.

The mRNA for the novel aldehyde dehydrogenase 5 (ALDH5) gene was detected in HuH7 hepatoma cells. The cells also expressed cytosolic aldehyde dehydrogenase (ALDH1) mRNA, but no mitochondrial aldehyde dehydrogenase (ALDH2) mRNA. Extracts of the hepatoma cells contained an enzymatic activity with an isoelectric point similar to that of ALDH1. This enzyme activity was insensitive to inhibition by disulfiram, a potent inhibitor of ALDH1. The enzyme was active with short chain aldehydes (acetaldehyde and propionaldehyde) and NAD+, but not with NADP+, and the activity was higher in the mitochondrial pellet than other cell fractions. These studies demonstrate the expression of ALDH5 mRNA in a human hepatoma and suggest that the gene product is enzymatically active and probably resides in the mitochondria.

Aldehyde Dehydrogenase↗
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

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

Hormonal and chemical influences on the expression of class 2 aldehyde dehydrogenases in rat H4IIEC3 and human HuH7 hepatoma cells.

We studied the effect a variety of hormones and chemical stimuli on the activity of low Km aldehyde dehydrogenase (ALDH) in rat H4IIEC3 hepatoma cells and ALDH activity in human HuH7 hepatoma cells. The low Km enzyme in H4IIEC3 cells reflects ALDH2 activity, and the ALDH activity in HuH7 likely represents ALDH5. Of the steroid hormone family, thyroid hormone, progesterone, and dihydrotestosterone increased low Km ALDH activity approximately 50%, whereas dexamethasone and estradiol had little effect. Insulin decreased the activity of low Km ALDH. None of these hormones affected the activity of ALDH in HuH7 cells. Among second messengers, 8-bromo-cAMP and A23187 increased low Km ALDH activity; HuH7 ALDH activity again was unchanged. Exposure of the cells to 22 mM ethanol reduced low Km activity by approximately 20%, whereas hydrogen peroxide, tumor necrosis factor-alpha, and interleukin-1 beta had little effect. Ultraviolet light increased the HuH7 ALDH activity. Retinaldehyde or retinolc acid reduced the HuH7 ALDH activity, but had no effect on low Km ALDH activity. These data suggest that low Km ALDH2 can be regulated by hormones and may not be constitutive as previously thought, and that the HuH7 ALDH is regulated differently.

Alcoholism↗
PubMed1995

The aldehyde dehydrogenase ALDH2*2 allele exhibits dominance over ALDH2*1 in transduced HeLa cells.

Individuals heterozygous or homozygous for the variant aldehyde dehydrogenase (ALDH2) allele (ALDH2*2), which encodes a protein differing only at residue 487 from the normal protein, have decreased ALDH2 activity in liver extracts and experience cutaneous flushing when they drink alcohol. The mechanisms by which this allele exerts its dominant effect is unknown. To study this effect, the human ALDH2*1 cDNA was cloned and the ALDH2*2 allele was generated by site-directed mutagenesis. These cDNAs were transduced using retroviral vectors into HeLa and CV1 cells, which do not express ALDH2. The normal allele directed synthesis of immunoreactive ALDH2 protein (ALDH2E) with the expected isoelectric point. Extracts of these cells contained increased aldehyde dehydrogenase activity with low Km for the aldehyde substrate. The ALDH2*2 allele directed synthesis of mRNA and immunoreactive protein (ALDH2K), but the protein lacked enzymatic activity. When ALDH2*1-expressing cells were transduced with ALDH2*2 vectors, both mRNAs were expressed and immunoreactive proteins with isoelectric points ranging between those of ALDH2E and ALDH2K were present, indicating that the subunits formed heteromers. ALDH2 activity in these cells was reduced below that of the parental ALDH2*1-expressing cells. Thus, the ALDH2*2 allele is sufficient to cause ALDH2 deficiency in vitro.

Aldehyde Dehydrogenase↗
PubMed1995

Ethanol oxidizing enzymes: roles in alcohol metabolism and alcoholic liver disease.

The elucidation of nucleotide and amino acid sequences of the genes and enzymes involved in the metabolism of ethanol has led to the ability to genotype individuals simply and rapidly. Although the different isozymes were once thought to be a likely explanation for between individual differences in alcohol elimination rates, this has not been found to be the case. Other explanations that remain to be investigated include potential regulatory variants in the genes that alter the level of expression of the enzymes, and genetic influences on activity of the malateaspartate shuttle and rates of mitochondrial NADH reoxidation. However, the isozymes encoded by ADH2*2 and ALDH2*2 have been found to influence alcohol drinking behavior or alcoholism substantially. This supports the original premise that the metabolic disposition of ethanol affects individuals' responses to it. The results suggest that any additional variants might also contribute to the spectrum of individual drinking preferences. Among heavy drinkers, the influence of the isozymes on risk of alcoholic liver disease has not been found to be great, suggesting that many other factors, perhaps interacting with the enzyme polymorphisms, are involved in determining susceptibility.

Alcohol Dehydrogenase↗
PubMed1994

Seven transmembrane domain receptor subtypes identified in NG108-15 cells by reverse transcription-polymerase chain reaction.

NG108-15 neuroblastoma x glioma cells are widely used for the study of neurotransmitter receptors. We utilized reverse transcription-polymerase chain reaction to amplify members of the seven transmembrane domain class of G-protein linked receptors using RNA isolated from NG108-15 cells. Two complementary DNAs representing receptors were obtained; based upon comparison with the sequence database, they probably represent the murine dopamine D1A receptor and a receptor closely related to the serotonin 5HT1D receptor subtype. The finding of the 5HT receptor subtype is of interest, as only the 5HT3 subtype was previously identified in NG108-15 cells by pharmacological means. Certain responses of NG108-15 cells to serotonin have been described that do not appear to be mediated by known 5HT receptor subtypes. The cDNA we cloned may therefore represent an additional 5HT1D subclass.

Amino Acid Sequence↗
PubMed1994

Characterization of the 5'-flanking sequence of rat class I alcohol dehydrogenase gene.

Expression of the rat class I alcohol dehydrogenase (ADH) gene is highest in the liver and regions of the intestine. We characterized over 3 kilobases of the gene's 5'-flanking region by sequencing and transient transfection. Alignment of the flanking sequence of the rat gene with those of the mouse and human class I genes revealed a cis-acting element, known to be a functional glucocorticoid response element in the human gene and conserved in the mouse, is interrupted in the rat promoter by a 490-base pair processed retropseudogene of the ribosomal protein S25. Southern analysis indicated that this inserted element is present in the class I ADH promoters of multiple strains of rat. Transfection analysis of the rat and mouse promoters showed that the mouse, but not the rat promoter, is inducible by dexamethasone. Electrophoretic mobility shift assays using nuclear extracts from dexamethasone-treated cells confirmed that the mouse's element interacts with the glucocorticoid receptor. Transient transfection of the 5'-flanking region of the rat gene linked to a human growth hormone reporter demonstrated the liver and intestinal specificity of the rat promoter. Two positive elements, one from nucleotides -1,327 to -977 and the other from -241 to -12, were shown to support high levels of reporter activity. In addition, a suppressive element was localized between nucleotides -403 and -241, a region of DNA situated within the domain of the S25 ribosomal protein pseudogene.

Alcohol Dehydrogenase↗
PubMed1994

Site-directed mutagenesis of phosphorylation sites of the branched chain alpha-ketoacid dehydrogenase complex.

Regulation of the branched chain alpha-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1 alpha and E1 beta under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased Km for the alpha-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1994

Genetic aspects and risk factors in alcoholism and alcoholic liver disease.

There is a great deal of evidence for genetic predisposition to alcoholism; considerably less is known regarding predisposition to alcoholic liver disease. The specific genes involved in either disorder are not well understood, although the enzymes of alcohol metabolism appear to play some role. It will be interesting to determine whether genetic factors that alter the expression of these enzymes, in addition to altering the kinetics of the enzymes, could modify responses to drinking. Work in the next few years will include determination of which responses to alcohol are indeed genetically influenced in twin studies, testing additional candidate genes for alcohol-related traits in populations and families, as well as the application of genomic mapping methodologies to alcoholic pedigrees. The latter strategy will be integrated into the larger number of studies that will grow from the Human Genome Project. Animal studies with selectively bred lines of rodents that differ in voluntary alcohol consumption will lead the way to define the neuronal and behavioral substrates responsible for differences in alcohol-drinking behavior. The use of the quantitative trait locus (QTL) mapping approach in F2 intercross between two inbred strains of rodents with opposite alcohol-response characteristics and in recombinant inbred strains derived from F2 intercross already has and will continue to help identify chromosomal locations of genes relevant to voluntary alcohol consumption. Perhaps in the future selective breeding of rodents and QTL mapping strategies can also be used to determine the biology and genetics of alcohol-induced liver injury.

Adenylyl Cyclases↗