Structure and expression of the rat class I alcohol dehydrogenase gene.

Clones containing the rat class I alcohol dehydrogenase (ADH) gene were isolated from a Charon 4A genomic library. The gene spans approximately 13 kb and comprises nine exons and eight introns. The upstream 436 bp contain canonical TATA and CCAAT sequences, an inverted CACCC box, a TG3 box found in mouse and human ADH promoters, and regions of homology to glucocorticoid response elements.

The heat-shock response in cultured cells exposed to ethanol and its metabolites.

Many stresses, including elevated temperature and exposure to toxins or heavy metals, activate a stereotyped response of cultured cells known as the heat-shock response. The products of several highly conserved heat-shock genes (heat-shock proteins) protect the cells against subsequent stresses. The intracellular signal for the response is unknown, but may include the presence of damaged and abnormal proteins in the cell.

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.

Evidence for both a regulatory mutation and a structural mutation in a family with maple syrup urine disease.

Maple syrup urine disease (MSUD) results from a deficiency of branched chain alpha-ketoacid dehydrogenase (BCKDH). We have studied the etiology of MSUD by determining the enzyme activity, protein, and mRNA levels of BCKDH in fibroblasts from a classic MSUD patient and his parents. By enzymatic amplification of the patient's mRNA followed by cloning and DNA sequencing, we have identified a T to A transversion that alters a tyrosine to an asparagine at residue 394 of the E1 alpha subunit.

Localization of the human gene for the El alpha subunit of branched chain keto acid dehydrogenase (BCKDHA) to chromosome 19q13.1----q13.2.

The gene encoding the El alpha subunit of branched chain keto acid dehydrogenase (BCKDHA) was mapped to human chromosome region 19q13.1----q13.2 using 3H-labeled cDNA hybridized in situ to human chromosomes.

Assignment of the gene for the E1 alpha subunit of branched chain alpha-ketoacid dehydrogenase to chromosome 19.

The gene encoding the E1 alpha subunit of branched chain alpha-ketoacid dehydrogenase was mapped to human chromosome 19. 32P-labeled human E1 alpha cDNA was hybridized with DNA derived from flow-sorted human chromosomes; it hybridized exclusively with that from chromosome 19.

Assaying the reporter gene chloramphenicol acetyltransferase.

These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase.

cDNA cloning of the E1 alpha subunit of the branched-chain alpha-keto acid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease.

We have cloned cDNAs encoding human and rat liver BCKDH E1 alpha subunits and deduced the primary structure of the mature protein. The sequences of the cDNA and protein are highly conserved between the two species. Significant sequence similarity has also been found between human BCKDH and PDH E1 alpha subunits.

Genotypes for aldehyde dehydrogenase deficiency and alcohol sensitivity. The inactive ALDH2(2) allele is dominant.

Many Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH2) activity responsible for the oxidation of acetaldehyde produced during ethanol metabolism. These individuals suffer the alcohol-flush reaction when they drink alcoholic beverages. The alcohol-flush reaction is the result of excessive acetaldehyde accumulation, and the unpleasant symptoms tend to reduce alcohol consumption.

Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase.

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit.

Oral medications for upper gastrointestinal endoscopy using a small diameter endoscope.

We carried out a double-blinded, randomized prospective study to compare patient tolerance of small diameter endoscopes using limited potency oral premedication to complement topical anesthesia. Patients randomly received either oral placebo, diphenhydramine (100 mg), acetaminophen (1000 mg), or both drugs 30 to 60 min prior to endoscopy. All patients received topical Cetacaine and underwent upper endoscopy with the Olympus XP10 7.

Induction of alcohol dehydrogenase activity and mRNA in hepatoma cells by dexamethasone.

Liver alcohol dehydrogenase activity was present in rat H4IIE hepatoma cells. Dexamethasone increased the enzyme activity two- to fourfold in these cells, but not in HepG2 cells. Enzyme induction was observed at dexamethasone concentrations as low as 10(-9) M, and the induction was maximal at 3 days.

Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver.

3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase, EC 1.1.1.

Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase.

We have isolated a cDNA encoding the branched chain alpha-ketoacid dehydrogenase E1 alpha subunit. A rat liver lambda gt11 expression library was screened with antibody reactive with the 2-oxoisovalerate dehydrogenase (lipoamide) component. A positive clone, lambda BZ304, contains a 1.

A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells.

Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay.

Loss of growth hormone-dependent characteristics of rat hepatocytes in culture.

The liver of rodents is sexually differentiated, i.e. the female liver differs from the male liver.

Loss of sexual differentiation of rat hepatocytes in short-term culture.

We investigated the maintenance of alcohol dehydrogenase (ADH) and estrogen receptors in primary cultures of rat hepatocytes. Female hepatocytes partially lose ADH activity and completely lose estrogen receptors during 4 days in culture. Growth hormone, which induces both the enzyme and receptor in vivo, did not induce either in vitro.

Cloning and sequencing of a cDNA encoding rat liver alcohol dehydrogenase.

We report the cloning and sequencing of a cDNA encoding rat liver alcohol dehydrogenase. The enzyme is very similar to mouse alcohol dehydrogenase but contains an amino acid insertion.

Ethanol metabolism.

Alcohol is metabolized by two pathways in humans: the ADH pathway which accounts for the bulk of the metabolism, and the MEOS pathway which contributes to the increased rate of ethanol elimination at high blood alcohol levels. The increased rate of elimination which results from chronic alcohol consumption is due to an increase in MEOS activity. The activities of these pathways are influenced by environmental factors such as smoking, diet, and endocrine factors.

Studies on the hormonal regulation of rat liver alcohol dehydrogenase.

These observations have extended and clarified the earlier reports on the effects of thyroid hormone and androgens on liver ADH activity. Thyroid hormone exerts a direct effect on ADH activity, but is not required for the expression of the enzyme. At present, it has not been possible to demonstrate a physiological role for thyroid hormone in determining the developmental and sexual variation in ADH activity.