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Whole brain methionine-enkephalin of ethanol-avoiding and ethanol-preferring c57BL mice.

These experiments systematically investigated ethanol preference in both the C57Bl/6N and C57Bl/6J mice utilizing three-choice 2-bottle preference test. In addition, these sublines were evaluated for whole brain methionine-enkephalin levels, which were significantly lower in C57Bl/6J mice (alcohol preferring) compared to C57Bl/6N mice (alcohol non-preferring). This finding supports the involvement of the peptidyl opiates in ethanol seeking behavior.

Alcohol Drinking↗

Interaction of amphiphilic molecules with biological membranes. A model for nonspecific and specific drug effects with membranes.

The nonspecific interactions of propranolol, timolol, and ethanol with model and sarcoplasmic reticulum membranes were determined utilizing radioisotopic association differential scanning calorimetry, and neutron diffraction. Differential scanning calorimetry performed on mixtures of these amphiphilic compounds and model membrane bilayers composed of dimyristoyllecithin showed that propranolol was approximately 25 times more lipid-soluble than timolol and at least 100 times more lipid-soluble than ethanol. Neutron diffraction showed that the solvation of propranolol was within the fatty acyl chain region of the lipid bilayer. This solvation correlated with the effect of propranolol to inhibit ATP-dependent calcium transport in isolated rabbit skeletal muscle sarcoplasmic reticulum, a membrane that lacks beta-adrenergic receptors. In contrast, the major site of interaction of ethanol was within the aqueous compartment hydrating the sarcoplasmic reticulum membrane. A model for nonspecific drug interaction with the sarcoplasmic reticulum membrane based on the site of interaction of these amphiphiles and their relative potencies to inhibit calcium transport by these membranes is proposed. In principle, this model could be extended to specific drug interactions with membranes.

Animals↗

Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum.

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.

Cellobiose↗

Genetic and environmental factors in ethanol self-administration.

Findings presented in this paper from pharmacogenetic studies of oral ethanol self-administration suggest a correlation between ethanol preference and self-administration and indicate that there are important genetic as well as environmental determinants of ethanol reinforced behavior. AA (alcohol accepting) and ANA (alcohol nonaccepting) rats, animals bred selectively for differential ethanol preference, showed large differences in operant responding for ethanol. AA rats drank significantly more ethanol than water, and their intake varied as a function of ethanol concentration. Intake of water and ethanol solutions did not differ in the ANA rats. In two inbred strains of rats, F344 and LEWIS, ethanol maintained higher response rates and was consumed in larger volumes than the water vehicle. In a third series of studies, C57BL/6J mice, which exhibit high ethanol preference and low sensitivity, readily self-administered ethanol in an operant situation. Conversely, BALB/cJ mice, which exhibit low preference and high sensitivity, were not positively reinforced by ethanol. The results demonstrate the experimental control possible by the utilization of genetically defined animals, even when complex learned behavioral sequences are being measured, and indicate that genotype and environment interact in a complex but definable way to determine the degree to which ethanol comes to function as a positive reinforcer.

Animals↗

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol.

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.

Biomass↗

Effect of ethanol on thromboxane and prostacyclin production in the human placenta.

Fetal alcohol syndrome (FAS) is frequently associated with intrauterine growth retardation (IUGR). One cause of ethanol-induced IUGR is thought to be related to increased pressor activity in the human placenta, resulting in decreased oxygenation and nutrient transport to the fetus. Thus, we have investigated the effect of ethanol on paracrine substances, such as thromboxane and prostacyclin, that act as vasoregulators within the intrauterine tissues. In these studies we have utilized the perfused single human cotyledon system to study the effect of ethanol on placental prostanoid production. We assessed the effect of longer (240 min) and more acute (60 min) exposure to ethanol on release of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) at the maternal and fetal sides of the placenta. Thromboxane was increased by both longer and shorter ethanol exposure, especially on the fetal side of the placenta. Prostacyclin was essentially unchanged with exposure to ethanol. The thromboxane:prostacyclin ratio also tended to increase with both 60- and 240-min ethanol exposure, but a statistically significant increase was seen only at a few time points. In the 60-min ethanol exposure, an increase in thromboxane was observed both during and following exposure to ethanol. The increase in the thromboxane milieu observed with ethanol exposure may lead, at least in part, to the IUGR which is frequently associated with FAS. Prevention of this effect of ethanol on thromboxane production might be a beneficial intervention for FAS.

6-Ketoprostaglandin F1 alpha↗

Ethanol production using concentrated oak wood hydrolysates and methods to detoxify.

Ethanol production from concentrated oak wood hydrolysate was carried out to obtain a high ethanol concentration and a high ethanol yield. The effect of added inhibitory compounds, which are typically produced in the pretreatment step of steam-explosion on ethanol fermentation, was also examined. p-Hydroxybenzoic aldehyde, a lignin-degradation product, was the most inhibitory compound tested in this study. Compounds with additional methyl groups had reduced toxicity and the aromatic acids were less toxic than the corresponding aldehydes. The lignin-degradation products were more inhibitory than the sugar-derived products, such as furfural and 5-hydroxymethylfurfural (HMF). Adaptation of yeast cells to the wood hydrolysate and detoxification methods, such as using charcoal and overlime, had some beneficial effects on ethanol production using the concentrated wood hydrolysate. After treatment with charcoal and low-temperature sterilization, the yeast cells could utilize the concentrated wood hydrolysate with 170 as well as 140 g/L glucose, and produce 69.9 and 74.2 g/L ethanol, respectively, with a yield of 0.46-0.48 g ethanol/g glucose. In contrast, the cells could not completely utilize untreated wood hydrolysate with 100 g/L glucose. Low-temperature sterilization, with or without charcoal treatment, was very effective for ethanol production when highly concentrated wood hydrolysates were used. Low-temperature sterilization has advantages over traditional detoxification methods, such as using overlime, ion exchange, and charcoal, because of the reduction in the total cost of ethanol production.

Journal Article↗

Large-scale gene profiling of the liver in a mouse model of chronic, intragastric ethanol infusion.

BACKGROUND/AIMS: The mechanisms underlying alcohol-induced liver injury are not fully elucidated. An approach in this direction would consist of an all-inclusive assessment of gene expression in the liver. The purpose of this study was to perform a comprehensive analysis of gene expression in the livers of mice treated with ethanol by means of intragastric infusion. METHODS: An ethanol- or glucose-enriched liquid diet was fed to animals for 4 weeks via a long-term gastrostomy catheter. The animals were killed and plasma alanine:2-oxoglutarate aminotransferase (ALT) assay, liver histology and total RNA analysis by microarray gene technology were performed. RESULTS: Alcohol increased ALT, induced steatosis, necrosis and inflammation. A total of 12,423 genes were analyzed for expression out of which 4867 were expressed by the liver. Alcohol repressed expression of 11 genes, induced expression of 13 genes, and up- or down-regulated expression of 44 and 42 genes >2-fold, respectively. Gene expression analysis identified several genes that have not previously been tested for alcohol effects. CONCLUSIONS: This study: (i) expands the knowledge of mechanism(s) of action of ethanol; (ii) indicates novel pathways of ethanol action on the liver, and (iii) illustrates the utility of microarray gene analysis in hepatology research.

Alanine Transaminase↗

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains.

BACKGROUND: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. RESULTS: We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. CONCLUSION: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.

Journal Article↗

Cellodextrin utilization and beta-glucosidase production by Bacteroides polypragmatus.

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a beta-glucosidase with a pI value of 4.2 and a molecular weight of approximately 100,000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The Km values obtained with p-nitrophenyl-beta-D-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55 degrees C. It was also stable during long-term storage at either 4 degrees C or -20 degrees C, provided that 10% (v/v) glycerol was added to preparations maintained at -20 degrees C.

Bacteroides↗

Metabolism of ethanol and associated hepatotoxicity.

Over the last three decades, direct hepatotoxic effects of ethanol were established, some of which were linked to redox changes produced by NADH generated via the alcohol dehydrogenase (ADH) pathway and shown to affect the metabolism of lipids, carbohydrates, proteins, and purines. It was also determined that ethanol can be oxidized by a microsomal ethanol oxidizing system (MEOS) involving a specific cytochrome P-450; this newly discovered ethanol-inducible cytochrome P-450 (P-450 IIEi) contributes to ethanol metabolism, tolerance, energy wastage (with associated weight loss), and the selective hepatic perivenular toxicity of various xenobiotics. Their activation by P-450IIEi now provides an understanding of the increased susceptibility of the heavy drinker to the toxicity of industrial solvents, anaesthetic agents, commonly prescribed drugs, over-the-counter analgesics, and chemical carcinogens. P-450 induction also explains depletion (and toxicity) of nutritional factors such as vitamin A. As a consequence, treatment with vitamin A and other nutritional factors is beneficial, but must take into account a narrowed therapeutic window in alcoholics who have increased needs for nutrients and also display an enhanced susceptibility to some of their adverse effects. Acetaldehyde (the metabolite produced from ethanol by either ADH or MEOS) impairs hepatic oxygen utilization and forms protein adducts, resulting in antibody production, enzyme inactivation, and decreased DNA repair. It also stimulates collagen production by the vitamin A storing cells (lipocytes) and myofibroblasts, and causes glutathione depletion. Supplementation with S-adenosyl-L-methionine partly corrects the depletion and associated mitochondrial injury, whereas administration of polyunsaturated lecithin opposes the fibrosis. Thus, at the cellular level, the classic dichotomy between the nutritional and toxic effects of ethanol has now been bridged. The understanding of how the ensuing injury eventually results in irreversible scarring or cirrhosis may provide us with improved modalities for treatment and prevention.

Journal Article↗

Microbial production of 1,3-propanediol.

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO2, H2, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH2, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a "green" polyester is a most cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism.

Adenosine Triphosphate↗

Xylose transport by the anaerobic thermophile thermoanaerobacter ethanolicus and the characterization of a D-xylose-binding protein

Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-beta-glucoside. Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein. Metabolic labeling with 14C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose. The apparent Kd of the protein for xylose was approximately 1.5 &mgr;M, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations. Uptake experiments with cells also suggested the presence of a separate low-affinity system. Binding activity varied less than 20% over a pH range of 4-8, and the level of activity was virtually unaffected when temperature was varied between 40 degreesC and 80 degreesC. This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism.

Journal Article↗

Characterization of the psychrotolerant acetogen strain SyrA5 and the emended description of the species Acetobacterium carbinolicum.

A psychrotolerant, obligate anaerobic, acetogenic bacterium designated strain SyrA5 was isolated from black anoxic sediment of a brackish fjord. Cells were Gram-positive, non-sporeforming rods. The isolate utilized H(2)/CO(2), CO, fructose, glucose, ethanol, ethylene glycol, glycerol, pyruvate, lactate, betaine and the methyl-groups of several methoxylated benzoic derivatives such as syringate, trimethoxybenzoate and vallinate. The optimum temperature for growth was 29 degrees C, whilst slow growth occurred at 2 degrees C. The strain grew optimally with NaCl concentrations below 2.7% (w/v), but growth occurred up to 4.3% (w/v) NaCl. Growth was observed in the range from pH 5.9 to 8.5, optimum at pH 8. The G+C content was 44.1 mol%. Based upon 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, the organism was classified in the genus Acetobacterium. Strain SyrA5 shared a 16S rRNA sequence similarity with A. carbinolicum of 100%, a fthfs gene (which codes for the N5,N10 tetrahydrofolate synthetase) sequence identity of 98.5-98.7% (amino acid sequence similarities were 99.4-100%) and a RNA-DNA hybridization homology of 64-68%. Despite a number of phenotypic differences between strain SyrA5 and A. carbinolicum we propose including strain SyrA5 as a subspecies of A. carbinolicum for which we propose the name Acetobacterium carbinolicum subspecies kysingense. The type strain is SyrA5 (=DSM 16427(T), ATCC BAA-990).

Acetobacterium↗

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization.

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.

Aldehyde Oxidoreductases↗

Percutaneous absorption, dermatopharmacokinetics and related bio-transformation studies of carbaryl, lindane, malathion, and parathion in isolated perfused porcine skin.

The percutaneous absorption of topically applied pesticides is a primary route for systemic exposure and potential toxicity. The isolated perfused porcine skin flap (IPPSF) is an in vitro model for studying percutaneous absorption of xenobiotics as well as cutaneous metabolism and toxicity in an anatomically intact viable skin preparation. In the present studies, percutaneous absorption of four different pesticides, carbaryl (C), lindane (L), malathion (M), and parathion (P), was assessed topically in an ethanol vehicle. A 4-compartment pharmacokinetic model was utilized to model their absorption profile. The order of absorption was C > P > L > M for the 8-h experimental period, but C > L > P > M for a model-extrapolated 6-day prediction. Metabolism of C and P was also assessed by high performance liquid chromatography (HPLC). The HPLC results indicate a significant first-pass effect for both pesticides after topical application, with parathion being metabolized to paraoxon and para-nitrophenol and carbaryl to naphthol. In addition, comparison of the metabolic data of P with previous results underscores the difference between non-recirculating and recirculating IPPSF systems in xenobiotic metabolism studies.

Animals↗

Acid-base balance during hypoxic hypometabolism: selected vertebrate strategies.

An important functional advantage of hypoxic hypometabolism is that it blunts the acid-base consequences of hypoxia. Hypoxia can lead to anaerobiosis and metabolic acidosis and, in animals that are apneic, to respiratory acidosis. A fall in blood and tissue pH is a major limiting factor in hypoxic tolerance and a variety of strategies occur in vertebrates, in concert with hypometabolism, to respond to this acid-base challenge. These include sequestering of lactic acid away from the circulating blood during the hypoxic exposure, either in underperfused tissues or in mineralized tissues, supplementing extracellular buffering by releasing bone mineral into the circulation, and utilizing alternative metabolic pathways for anaerobiosis to produce ethanol rather than lactate as the principal end-product. For submerged air-breathing ectotherms, effective cutaneous O2 and CO2 exchange can also allow an animal to avoid or minimize both anaerobiosis and respiratory acidosis. These responses serve to maintain a viable acid-base state in the body and to extend the time that the hypoxic stress can be endured.

Acclimatization↗

Members of the genus Arthrobacter grow anaerobically using nitrate ammonification and fermentative processes: anaerobic adaptation of aerobic bacteria abundant in soil.

Members of the genus Arthrobacter are usually regarded as obligate aerobic bacteria. The anaerobic growth and energy metabolism of two Arthrobacter species were investigated. Arthrobacter globiformis utilized both nitrate ammonification and lactate, acetate and ethanol producing fermentation processes for anaerobic growth. Only nitrate supported anaerobic growth of Arthrobacter nicotianae. Anaerobically induced respiratory nitrate reductase activity was detected in both strains. Neither of the tested strains used the alternative electron acceptors fumarate, dimethylsulfoxide or trimethylamine-N-oxide.

Aerobiosis↗