The mutation in the mitochondrial aldehyde dehydrogenase (ALDH2) gene responsible for alcohol-induced flushing increases turnover of the enzyme tetramers in a dominant fashion.

Deficiency in mitochondrial aldehyde dehydrogenase (ALDH2), a tetrameric enzyme, results from inheriting one or two ALDH2*2 alleles. This allele encodes a protein subunit with a lysine for glutamate substitution at position 487 and is dominant over the wild-type allele, ALDH2*1. The ALDH2*2-encoded subunit (ALDH2K) reduces the activity of ALDH2 enzyme in cell lines expressing the wild-type subunit (ALDH2E). In addition to this effect on the enzyme activity, we now report that ALDH2*2 heterozygotes had lower levels of ALDH2 immunoreactive protein in autopsy liver samples. The half-lives of ALDH2 protein in HeLa cell lines expressing ALDH2*1, ALDH2*2, or both were determined by the rate of loss of immunoreactive protein after inhibition of protein synthesis with puromycin and by pulse-chase experiments. By either measure, ALDH2E enzyme was very stable, with a half-life of at least 22 h. ALDH2K enzyme had an enzyme half-life of only 14 h. In cells expressing both subunits, most of the subunits assemble as heterotetramers, and these enzymes had a half-life of 13 h. Thus, the effect of ALDH2K on enzyme turnover is dominant. These studies indicate that the ALDH2*2 allele exerts its dominant effect both by interfering with the catalytic activity of the enzyme and by increasing its turnover. This represents the first example of a dominantly acting allele with this effect on a mitochondrial enzyme's turnover.

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.

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. The subunit of this homotetrameric enzyme was sequenced and the abnormality in the inactive enzyme shown to be a substitution of lysine for glutamate at position 487. We have used the polymerase chain reaction to determine the genotypes of 24 livers from Japanese individuals. Correlating genotype with phenotype leads to the conclusion that the allele (ALDH2(2)) encoding the abnormal subunit is dominant.

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. Amplification of both mRNA and genomic DNA, in combination with allele-specific oligonucleotide hybridization, demonstrated that the father was heterozygous for this mutant allele. The mother was homozygous for the allele encoding the normal Tyr394, but expressed only about half of the normal level of mRNA and protein. The patient was genetically heterozygous for this altered allele, although only the abnormal allele was expressed as mRNA. We conclude that the patient was a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele from the mother containing a cis-acting defect in regulation which abolished the expression of one of the E1 alpha alleles. Our results revealed for the first time that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit.

Protein expression changes in the nucleus accumbens and amygdala of inbred alcohol-preferring rats given either continuous or scheduled access to ethanol.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

Binding and activation of the human aldehyde dehydrogenase 2 promoter by hepatocyte nuclear factor 4.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expressed in a tissue-specific fashion with high levels in liver, heart, kidney, and muscle, and low levels in most other tissues. The ALDH2 promoter was found to bind nuclear proteins at a pair of adjacent sites approximately 300 bp upstream from the translation start site, each of which was contacted at motifs containing the hexamer A/GGGTCA. The 3' site was shown to bind in vitro translated HNF-4. It was also shown by electrophoretic mobility shift assay utilizing antibodies against nuclear factors and rat liver nuclear extracts to be bound by hepatocyte nuclear factor 4 (HNF-4), chicken ovalbumin upstream promoter transcription factor I and II, and retinoid X receptors. A reporter construct containing four copies of this promoter element was activated by co-transfection of an HNF-4 expression plasmid in COS-1 and hepatoma cell lines. These results suggest that the tissue specificity of ALDH2 expression is in part determined by its activation by HNF-4.

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. In addition, individuals inherit different types of ADH isoenzymes which have different kinetic properties. Individuals with different phenotypic variants, e.g. the beta 1 vs beta 2 isoenzymes, appear to have different rates of ethanol elimination. The cloning of the ADH genes and the availability of molecular hybridization methods now make it possible to genotype individuals and to correlate the genotype with both alcohol elimination rates and with the risk of developing medical complications of alcoholism or even of developing alcoholism itself.

Complete amino acid sequence of rat liver alcohol dehydrogenase deduced from the cDNA sequence.

Alcohol dehydrogenase (ADH) catalyzes the rate-determining reaction in the metabolism of ethanol. We report here the complete nucleotide sequence of a cDNA encoding rat liver ADH, and the deduced amino acid (aa) sequence of the protein. The rat enzyme contains a cluster of aa substitutions and an aa insertion in the region between aa residues 111 and 118, which is near the intron-exon junction reported for the human ADH gene. It also contains an additional cysteine in the highly variable region from aa residues 108-125 which may account for the unusual lability of rat ADH compared with ADH from other species.

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. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.

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.

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.

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. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product. After these studies had been performed, we found that others have also recommended heat treatment of the cell extract prior to CAT assay. We concur with this recommendation. We suggest that EDTA plus heat treatment of the cell extract should be incorporated into all CAT assay protocols, unless it has been previously determined that extracts of the cells used do not interfere. Furthermore, the heat treatment step should be used whenever the activity of promoter-CAT constructs is compared among different cell lines, as is often done to define tissue-specific expression.

Training resident physicians in fiberoptic sigmoidoscopy. How many supervised examinations are required to achieve competence?

Twenty-five resident physicians performed 495 fiberoptic sigmoidoscopic examinations that were graded for overall skill according to a six-point competence scale. In general, 24 to 30 examinations were required to become competent at fiberoptic sigmoidoscopy. Trainees with prior rigid sigmoidoscopy experience achieved competence more quickly than those with no prior rigid sigmoidoscopy experience. As experience increased, unassisted insertion distance and luminal visualization increased, insertion time and assisted time decreased, and management scores and percent correct diagnoses improved. Trainees detected 93 to 100 percent of polyps and cancers viewed by the experienced sigmoidoscopist once competence was achieved. These data indicate that programs for training primary care physicians in fiberoptic sigmoidoscopy are feasible, help define the number of examinations required to become competent, and indicate that such trainees should be effective in cancer screening.

Rate-determining factors for ethanol metabolism in fasted and castrated male rats.

The effects of castration and fasting upon the alcohol elimination rate, liver alcohol dehydrogenase (LADH) maximum activity (Vmax), and hepatic concentrations of ethanol, acetaldehyde, and free NADH during ethanol oxidation were examined in male Wistar rats. Castration increased the Vmax of LADH and, to a lesser extent, the alcohol elimination rate in vivo. On the other hand, fasting reduced the Vmax of LADH and the alcohol elimination rate in sham-operated and castrated rats but it did not nullify the effect of castration. Castration produced small but significant changes in the hepatic concentrations of ethanol, acetaldehyde and free NADH in fed rats during ethanol oxidation. Fasting also caused significant increases in the concentration of free NADH during alcohol oxidation in both the sham-operated and castrated groups. The ratio of the steady-state velocities of LADH in situ to the maximum velocities of LADH (v/Vmax) under the different experimental conditions was calculated by using the steady-state rate equation for the enzyme mechanism of rat LADH and its kinetic constants. The calculated v/Vmax ratios were 50-62%, indicating that LADH activity was limited to about the same extent by its substrates and products under these conditions and that the changes in alcohol elimination rates produced by fasting and castration mainly reflected changes in the Vmax of LADH. The calculated steady-state velocities in situ (v) were 14-28% lower than the measured rates of alcohol elimination in vivo. The extent of agreement is probably acceptable in view of the assumptions needed to determine the free NADH concentration in liver and the existence of non-LADH-related processes for alcohol elimination in vivo.

Role of the pituitary and neonatal androgenic imprinting in the hormonal regulation of liver alcohol dehydrogenase activity.

Liver alcohol dehydrogenase activity is increased by thyroidectomy, orchidectomy, or hypophysectomy. We investigated the mechanisms of these hormonal effects by examining the effects of testosterone, dexamethasone and thyroid hormone on liver alcohol dehydrogenase activity in hypophysectomized rats and in cultured hepatocytes, and the effect of administration of androgens to neonatal female rats. Testosterone did not lower alcohol dehydrogenase activity in hypophysectomized rats, whereas dexamethasone and thyroxine produced moderate decreases in activity. Triiodothyronine reduced alcohol dehydrogenase activity of cultured hepatocytes from male and hypothyroid female rats in a dose-dependent fashion, confirming that thyroid hormone had pituitary-independent effects on the enzyme activity. Dexamethasone was required for the expression of alcohol dehydrogenase activity in cultured cells, and it increased the enzyme activity when present at supraphysiologic concentrations. Treatment of neonatal female rats with testosterone reduced the activity of the enzyme in adulthood. The difference in alcohol dehydrogenase activity in adult male and female rats appears to be determined in part by neonatal imprinting by androgens and in part by an effect of testosterone that is either mediated by or dependent upon the pituitary. Thyroid hormone reduces alcohol dehydrogenase activity by a direct effect on the liver.

Pathogenesis of alcoholic liver disease: newer mechanisms of injury.

The understanding of how alcohol damages the liver has expanded substantially over the last decade. In particular, the genetics of alcoholism, the genesis of fatty liver, the role of oxidant stress, interactions between endotoxin and the Kupffer cell, and the factors that control activation of the hepatic stellate cell (HSC) have been the focus of a great deal of research. Genetic mechanisms for increasing the risk of alcoholism include alterations in alcohol metabolizing enzymes as well as neurobiological differences between individuals. The development of fatty liver may involve both redox forces, oxidative stress, and alterations in peroxisome proliferator activated receptor function. Oxidative stress is now known to involve both microsomal and mitochondrial systems. Recent studies implicate stimulation of Kupffer cells by portal vein endotoxin as a cause of release of cytokines and chemokines, hepatocyte hyper-metabolism, and activation of HSC. These actions appear to be in part gender-dependent and may explain the susceptibility of women to alcoholic liver disease. Activation of HSC underlies liver fibrosis and cirrhosis of all types; control of this activation might permit control of the progression of fibrosis. These advances suggest a number of new approaches as therapy for alcoholic liver injury.

A direct repeat (DR-1) element in the first exon modulates transcription of the preproenkephalin A gene.

The preproenkephalin A gene is regulated by upstream cis-acting elements which respond to various signals, such as cAMP, calcium, and phorbol esters. An additional regulatory element was detected downstream of the transcription start site of the human preproenkephalin A gene in transfection experiments. The element was localized by DNAse I footprinting and methylation interference assays to a direct repeat (DR-1) element in the first (untranslated) exon. Deletion or mutation of this site reduced transcriptional activity of promoter-reporter constructs by over 50%. Antibodies against COUP-TF beta/ARP-1 and RXR transcription factors altered the pattern seen on electrophoretic mobility shift assays using double-stranded oligonucleotide containing the exon 1 protein binding site. This suggests that the factors that bind this site and modulate transcription of the PPE gene include members of the COUP-TF and retinoid X receptor families.

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.

Sensitivity of virally-driven luciferase reporter plasmids to members of the steroid/thyroid/retinoid family of nuclear receptors.

During a series of transfection experiments, the pRSV-luc plasmid used as an internal control was found to be sensitive to cotransfection with expression vectors for several members of the steroid/thyroid/retinoid superfamily of nuclear receptors. Therefore, a survey of the effect of these expression vectors on the activity of four reporter plasmids was conducted. In CV-1 cells, the activity of pRSV-luc, which contains the P. pyralis luciferase gene, was repressed by co-transfection of PPARalpha and ARP-1 and was activated by COUP-TFI. Expression of pSV40-luc, containing the same luciferase gene, was repressed by PPARalpha and HNF-4 and activated by both COUP-TFI and ARP-1. All four of these expression vectors reduced the expression of the pRL-TK plasmid, which contains the luciferase gene from Renilla reniformis. RXR expression vectors had no effect on luciferase activity in CV-1 cells but induced luciferase activity in H4IIEC3 hepatoma cells. This activation was blocked by the addition of ligand, 9-cis retinoic acid. pSV2-CAT, which contains the chloramphenicol acetyltransferase gene, was insensitive to all receptor expression vectors tested. Both the P. pyralis and R. reniformis luciferase genes appear to contain sequences that render them responsive to steroid/thyroid/retinoid nuclear receptors.

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. We have studied the molecular basis of MSUD by determining the enzyme activity and levels of BCKDH protein and mRNA, and by enzymatic amplification and sequencing of BCKDH E1 alpha-specific mRNA, from an MSUD patient and his parents. Different mutant alleles were identified in the two parents. The patient was a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father and an allele containing a defect in regulation from the mother. Our results demonstrate that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit.