Pine wilt disease, which is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has caused huge damage to pine forests around the world. In this study, we analysed the PWN transcriptome to investigate the expression of genes related to the associated bacterial species Pseudomonas fluorescens and found that the gene adh-1 encoding alcohol dehydrogenase (ADH) was upregulated. The open reading frame of adh-1, which encoded a protein of 352 amino acid residues, was cloned from B. xylophilus. Recombinant ADH with a relative molecular weight of 39 kDa, was present mainly in inclusion bodies and was overexpressed in Escherichia coli BL21 (DE3) and purified after refolding. The biochemical assay revealed that recombinant ADH could catalyse the dehydrogen reaction of eight tested alcohols including ethanol in the presence of NAD+. Quantitative real-time RT-PCR analysis indicated that ethanol upregulated adh-1 expression in PWN. Results of RNA interference and inhibition of ADH treatment indicated that downregulating expression of adh-1 or inhibition of ADH could reduce ethanol tolerance and the vitality and reproduction ability of B. xylophilus, suggesting that adh-1 is involved in pathogenicity of PWN.

Previous studies have shown that the interaction between limiting vitamin A (VA) and an alcohol dehydrogenase 1 C (ADH1C) variant in beef cattle results in increased intramuscular fat in the longissimus thoracis muscle in one genotype when fed low dietary VA. Although quality grade is important for increased profitability and improving taste characteristics of beef products, limiting VA too drastically can impair animal welfare. The objectives of this study were to determine if this marker-assisted management strategy would be effective, and whether any impairment in immune function would occur in a feedlot setting. Mixed breed beef steers (n=2000) were sorted into 40 feedlot pens so that all combinations of ADH1C genotype (TT or CT), VA level (50% or 100% of recommended) and hormonal implant status (implanted (IMP) or non-implanted (NI)) were equally represented within the population. The VA×ADH1C interaction was not observed. An implant status × ADH1C interaction was observed with average daily gain (ADG; P=0.03). Steers that were IMP and CT had higher ADG than IMP TT (CT=1.69 and TT=1.62 kg/day), whereas both genotypes in the NI steers were lower (CT=1.29 and TT=1.32 kg/day). Implant status was shown to affect dry matter intake (DMI; IMP=8.55 and NI=7.87 kg; P<0.01), total days-on-feed (IMP=164.4 and NI 210.5 days; P<0.01), USDA yield grade (YIELD; IMP=2.40 and NI=2.77; P<0.01), marbling score (MARB; IMP=392 and NI=455; P<0.01), longissimus thoracis area (LTA; IMP=85.0 and NI=80.7 cm2; P=0.01) and backfat thickness (FAT; IMP=8.0 and NI 10.0 mm; P<0.01). Overall, IMP animals finished on fewer total days-on-feed with higher ADG, DMI, larger LTA, and lower YIELD, MARB and FAT. To investigate immune function parameters, crossbred steers (n=18) were selected from a prior feeding trial so that all combinations of ADH1C (TT, CT and CC) and VA (25% or 75%) were equally represented. Blood cell count analysis and peripheral blood mononuclear cell proliferation and stimulation assays were conducted. None of these immune parameters were affected by VA level. Treatment and mortality records were examined in the 2000 steer population, where no correlations with ADH1C, implant status or VA level were observed. Due to no VA × ADH1C interaction, this nutrigenetic marker-assisted management strategy is not effective at this time in commercial beef cattle feedlots, however, supplementing VA at a level as low as 25% of recommended in finishing rations would likely not result in signs of immune dysfunction.

Previously, the single nucleotide polymorphism in alcohol dehydrogenase (ADH1C c.-64T>C) was shown to have an association with intramuscular fat (IMF) in the longissimus thoracis (LT) muscle when vitamin A was limited in finishing rations of beef steers. The purpose of this study was to determine the optimum vitamin A supplementation level, in combination with ADH1C genotype, to increase IMF of the LT muscle. In total, 45 TT genotype, 45 CT and 27 CC Black Angus crossbred steers were backgrounded on a commercial ration containing 3360 IU vitamin A/kg dry matter (DM). During finishing, the steers were randomly assigned to one of three vitamin A treatments at 25%, 50% and 75% of the National Research Council recommendation of 2200 IU/kg DM. Treatments were administered via an oral bolus. Carcass quality was evaluated and a sample from the LT muscle was collected for analysis of IMF. A treatment×genotype interaction (P=0.04) was observed for IMF; TT steers on the 75% treatment had higher IMF relative to CT and CC steers on the same treatment. Western blot analysis showed that TT steers had higher (P=0.02) ADH1C protein expression in hepatic tissue. Previously, TT steers exhibited increased IMF when fed limited vitamin A. In the current study, the lack of variation in IMF between treatments and genotypes at the lower vitamin A treatment levels was likely due to the majority of the steers grading Canada AAA (USDA Choice). However, the western blot data supports that TT steers are expected to have higher IMF deposition, due to an increased production of ADH1C. The interaction between ADH1C genotype and vitamin A supplementation level has the potential for use in marker-assisted management programs to target niche markets based on increased marbling.

Recently, Macgregor et al. (2009) demonstrated significant associations of ADH polymorphisms with reactions to alcohol and alcohol consumption measures in an Australian sample. The aim of the present study was to replicate these findings in a Dutch sample. Survey data on alcohol phenotypes came from 1,754 unrelated individuals registered with the Netherlands Twin Register. SNPs in the ADH gene cluster located on chromosome 4q (n = 491) were subdivided in seven gene sets: ADH5, ADH4, ADH6, ADH1A, ADH1B, ADH1C and ADH7. Within these sets associations of SNPs with alcohol consumption measures, age at onset variables, reactions to alcohol and problem drinking liability were examined. Of the original 38 SNPs studied by Macgregor et al. (2009), six SNPs were not available in our dataset, because one of them had a minor allele frequency < .01 (rs1229984) and five could not be imputed. The remaining SNP associations with alcohol phenotypes as identified by Macgregor et al. (2009) were not replicated in the Dutch sample, after correcting for multiple genotype and phenotype testing. Significant associations were found however, for reactions to alcohol with a SNP in ADH5 (rs6827292, p = .001) and a SNP just upstream of ADH5 (rs6819724, p = .0007) that is in strong LD with rs6827292. Furthermore, an association between age at onset of regular alcohol use and a SNP just upstream of ADH7 (rs2654849, p = .003) was observed. No significant associations were found for alcohol consumption and problem drinking liability. Although these findings do not replicate the earlier findings at the SNP level, the results confirm the role of the ADH gene cluster in alcohol phenotypes.

Alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH2) are responsible for metabolizing the bulk of ethanol consumed as part of the diet and their activities contribute to the rate of ethanol elimination from the blood. They are expressed at highest levels in liver, but at lower levels in many tissues. This pathway probably evolved as a detoxification mechanism for environmental alcohols. However, with the consumption of large amounts of ethanol, the oxidation of ethanol can become a major energy source and, particularly in the liver, interferes with the metabolism of other nutrients. Polymorphic variants of the genes for these enzymes encode enzymes with altered kinetic properties. The pathophysiological effects of these variants may be mediated by accumulation of acetaldehyde; high-activity ADH variants are predicted to increase the rate of acetaldehyde generation, while the low-activity ALDH2 variant is associated with an inability to metabolize this compound. The effects of acetaldehyde may be expressed either in the cells generating it, or by delivery of acetaldehyde to various tissues by the bloodstream or even saliva. Inheritance of the high-activity ADH β2, encoded by the ADH2*2 gene, and the inactive ALDH2*2 gene product have been conclusively associated with reduced risk of alcoholism. This association is influenced by gene–environment interactions, such as religion and national origin. The variants have also been studied for association with alcoholic liver disease, cancer, fetal alcohol syndrome, CVD, gout, asthma and clearance of xenobiotics. The strongest correlations found to date have been those between the ALDH2*2 allele and cancers of the oro-pharynx and oesophagus. It will be important to replicate other interesting associations between these variants and other cancers and heart disease, and to determine the biochemical mechanisms underlying the associations.

Breast cancer is a multifactorial disease that is triggered by gene–environment interactions. Epidemiological research has identified alcohol consumption as a significant non-hormonal-related and consistent risk factor, and is thus a preventable cause of breast cancer. The reasons why alcohol drinking increase breast cancer risk are unclear, and several hypotheses exist. The perturbation of estrogen metabolism and response appears to one mechanism underlying the association. Alcohol consumption also increases breast density in postmenopausal women and affects sex steroid levels. Other plausible mechanisms include: (1) mutagenesis by acetaldehyde, which is a metabolite of ethanol; (2) by enhancing the susceptibility of the mammary gland to carcinogenesis by affecting DNA repair, stimulating cell proliferation or altering mammary gland structural development; (3) influencing the disposition and function of essential nutrients or dietary factors considered cancer protective (e.g. affecting folate and one-carbon metabolism pathways); (4) inducing genome instability and DNA damage; and (5) inducing oxidative damage via ethanol or estrogen metabolism. To better understand the etiological nature of the effect of alcohol on breast carcinogenesis, further studies at the cellular and molecular levels on the interaction between ethanol and other risk factors, estrogen, carcinogens are needed.

Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human σσ isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human β1β1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4-methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human σσ alcohol dehydrogenase (ADH) isoenzyme to 2.5 Å resolution. Comparison of this structure to the human β1β1 isoenzyme structure indicated that the side-chain position for Met141 in σσ ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in σσ ADH to Leu (σ141L) lowers the Ki for 4-methylpyrazole from 350 to 10 μM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate-binding pocket at a position close to the methyl group of 4-methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X-ray structure of the σ141L mutant enzyme was determined to 2.4 Å resolution. The three-dimensional structure of the mutant enzyme is identical to the wild-type enzyme, with the exception of the residue at position 141. Thus, the differences in 4-methylpyrazole binding between the mutant and wild-type σσ ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class-specific differences in 4-methylpyrazole binding to the human ADH isoenzymes.

Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30° in their melting temperatures. The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 Å, respectively. Comparison of these two very similar structures (RMS difference in Cα = 0.8 Å) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, “charged-neutral” hydrogen bonds, and prolines as well as improved stability of α-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.

The cyanobacterium Microcystis PCC7806, grown under continuous light, fermented endogenously stored glycogen to equimolar amounts of acetate and ethanol when incubated anaerobically in the dark. In addition, H2, CO2 and some L-lactate were produced. This fermentation pattern differed from that displayed by cells which had been grown under an alternating light/dark (16/8 h) cycle. Such cells produced much more ethanol than acetate, while no lactate was formed. These differences could not be related to the levels of key enzymes of fermentation, which were identical in the two cultures. The cultures grown under continuous light contained twice as much glycogen as the light/dark-grown cells and the former metabolized it at a rate approximately 3 times as fast as the latter culture. Fermentation in the culture grown under continuous light showed low carbon recovery (59–80%) and high oxidation/reduction balance (approximately 1·5). On the basis of calculations of ATP yield it was concluded that this culture was capable of growth driven by fermentation. The increase in structural cell material would account for the missing carbon and reduction equivalents.