[The utilization of alcohol by the organism].
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Ethanol↗
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Characterization of enzymes involved in formation of ethyl esters of long-chain fatty acids in humans.
Elevated fatty acid ethyl ester (FAEE) concentrations have been detected in postmortem organs from alcoholics and patients acutely intoxicated by alcohol, and FAEE have been implicated as mediators of ethanol-induced organ damage. The formation of FAEE is catalyzed by acyl-coenzyme A:ethanol O-acyltransferase (AEAT) and by FAEE synthase, which utilize acyl-CoA and free fatty acids, respectively, as substrates. Because little is known about the capacity of various human tissues to synthesize and hydrolyze FAEE, we investigated formation of FAEE by AEAT and FAEE synthase in tissue homogenates from human gastric ventricular and duodenal mucosa, pancreas, liver, heart, lung, and adipose tissue, gallbladder mucosa, and in serum. Liver, duodenal mucosa, and pancreas were found to have the highest capacities to synthesize FAEE, mainly due to AEAT. FAEE hydrolyzing activity was highest in liver and pancreas, but hardly detectable in adipose tissue or heart. Because fatty acids and alcohol are absorbed by the intestinal mucosa, intestine may be a major site of FAEE synthesis, and FAEE may be delivered via the circulation to other organs and taken up by lipoprotein receptor-mediated uptake. A very low rate of FAEE hydrolysis was detected in heart and adipose tissue, which probably accounts for the previously observed accumulation of FAEE in these organs.
Acyltransferases↗
Factors contributing to the loss of ethanologenicity of Escherichia coli B recombinants pL0I297 and KO11.
To be economic and to be compatible with modern continuous bioconversion systems, it is imperative that the process organism exhibits an extremely high degree of stability. In the case of ethanol production from lignocellulosic biomass, functional stability of the potential process biocatalyst can be assessed in terms of the capacity to sustain high-performance fermentation during the continuous fermentation of biomass-derived sugars. This investigation employed glucose- or xylose-limited chemostat culture to examine the functional stability of two patented, genetically engineered E. coli-namely E. coli B (ATCC 11303) carrying the Zymomonas genes for pyruvate decarboxylase and alcohol dehydrogenase II on a multicopy plasmid pLOI297 and a chromosomal pet integrant of strain 11303, designated as strain KO11. Both recombinants carry markers for antibiotic resistance and have been reported to exhibit genetic stability in the absence of antibiotic selection. Chemostats were fed with Luria broth (LB) (with 25 g/L sugar) at a dilution rate of 0.14 and 0.07/h when the feed medium was glucose-LB and xylose-LB, respectively. They pH was controlled at 6.3. With glucose, both recombinants exhibited a rapid loss of ethanologenicity even when selection pressure was imposed by the inclusion of antibiotics in the feed medium. With strain KO11, increasing the concentration of chloramphenicol from 40 to 300 mg/L, resulted in a retardation in the rate of loss of ethanologenicity, but it did not prevent it. Under xylose limitation, the plasmid-bearing recombinant appeared to be stabilized by antibiotics, but this did not reflect genetic stability, since the slower-growing revertant was washed out at a dilution rate of 0.07/h. With both recombinants, interpretation of functional stability with xylose was complicated by the inherent ethanologenicity associated with the host culture. Based on an average cost for large bulk quantities of antibiotics at $55/kg and an amendment level of 40 g/m3, the estimated economic impact regarding the potential requirement for operational stabilization by antibiotics in a plant operating in batch mode varied from a maximum of 29 cents/gal of E95 ethanol for antibiotic amendment of all fermentation media to a minimum of 0.45 cents/gal where antibiotics were used exclusively for the preparation of the inocula for every fourth batch fermentation cycle. The high degree of instability observed in these continuous fermentations does not auger well for the proposed potential industrial utility of these patented, genetically engineered constructs for the production of fuel ethanol from biomass and wastes.
Anti-Bacterial Agents↗
Derepression of galactose metabolism in melibiase producing bakers' and distillers' yeast.
Beet molasses is widely used as a growth substrate for bakers' and distillers' yeast in the production of biomass and ethanol. Most commercial yeasts do not fully utilise the carbohydrates in molasses since they are incapable of hydrolysing the disaccharide melibiose to glucose and galactose. Also, expression of genes encoding enzymes for the utilisation of carbon sources that are alternatives to glucose is tightly regulated, sometimes rates of yeast growth and/or ethanol production. The GAL genes are regulated by specific induction by galactose and repression during growth on glucose. In an industrial distillers' yeast, two genes interacting synergistically in glucose repression of galactose utilization, MIG1 and GAL80, have been disrupted with MEL1, encoding melibiase. The physiology of the wild-type strain and the recombinant strains was investigated on mixtures of glucose and galactose and on molasses. The recombinant strain started to ferment galactose when 9.7 g 1(-1) glucose was still present during a batch fermentation, whereas the wild-type strain did not consume any galactose in the presence of glucose. The ethanol yield in the recombinant strain was 0.50 g ethanol g sugar (-1) in an ethanol fermentation on molasses, compared with 0.48 g ethanol g sugar (-1) for the wild-type strain. The increased ethanol yield was due to utilization of melibiose in the molasses.
Culture Media↗
Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production.
Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol(-1), mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.
Ammonia↗
Measurement of serum T4 concentration by high performance liquid chromatography.
Although RIA techniques for the measurement of serum T4 have been extremely useful, this methodology has several disadvantages, including the requirement for the use of radioisotopes, various levels of thyronine cross-reactivity, and the ability to measure only a single iodothyronine in one assay. We have developed a high performance liquid chromatography (HPLC) method for quantitating serum T4 that utilizes the detection of dansyl-T4 compounds and obviates the problems described for RIA techniques. Serum samples were extracted with ethanol and then chloroform, reacted with dansyl chloride, and, after n-heptane extraction, placed directly on column. Utilizing this technique, dansyl-T4 was easily separated and identified. The sensitivity of detection of the dansylated-T4 in serum was 1 microgram/dl, and linearity was observed when increasing standard T4 concentrations were employed. Sensitivity to 10 ng/dl (100 fmol on column) was achieved when T4 was added to buffer. The coefficients of variation were 4.8% and 2.1% for normal and high serum samples, respectively. When 39 random serum samples were anayzed both by HPLC and RIA, there was concordance of these techniques, since the derived correlation coefficient was 0.94. In summary, the present study demonstrates that serum T4 concentrations can be measured by HPLC and that these measurements agree remarkably well with those obtained by RIA. Because of the inherent advantages of HPLC methodology over that of RIA, this technique of measurement of T4 may have wide applicability to the measurement of iodothyronines.
Chromatography, High Pressure Liquid↗
Noncompetitive ELISA for human serum insulin-like growth factor-I.
Measurement of serum insulin-like growth factor I (IGF-I) is important in assessing the growth hormone/IGF axis. Competitive immunoassay methods for IGF-I are complicated by substantial interference from IGF-binding proteins (IGFBPs) due to the relatively low ratio of specific antibody to IGFBPs in the sample, even after standard acid-ethanol sample extraction. We report of development of a noncompetitive ELISA for human IGF-I that avoids this problem by utilizing excess amounts of capture and detection antibodies. Serum samples were prepared by using an abbreviated acid-ethanol extraction method. Neutralized supernatant was added to microwells coated with IGF-I capture antibody; horseradish peroxidase-labeled detection antibody was then added, incubated for 2 h, and then developed. Compared with the "gold standard" method of acid-column chromatography, the simplified acid-ethanol extraction yields a mean +/- SD recovery of 103% +/- 5.5% despite the presence of residual IGFBPs in the extracted sample. Comparisons with a centrifugal filtration sample extraction method are also shown. The ELISA is specific for IGF-I with an absolute sensitivity of 0.03 microgram/L and inter- and intraassay CVs of 3.9-8.8% and 2.6-6.7%, respectively. The availability of a rapid IGF-I ELISA combined with a simple and reliable sample preparation procedure should facilitate clinical and research studies of this important growth factor.
Centrifugation↗
Limited access to ethanol in genetic drinking rats is suppressed while feeding is enhanced by the mixed 5-HT1A agonist/5-HT2A antagonist FG5938.
Serotonin (5-HT) receptor agonists, antagonists, and mixed agonist/antagonists have been implicated in the volitional intake of ethanol in the rat and other species. The present experiments were undertaken to determine whether FG5938 (1-[4-(p-fluorophenyl)butyl]-4-(6-methyl-2-pyridinyl)-piperazine fumarate) would alter ethanol drinking in: genetic ethanol preferring (P) rats; and a new strain of high ethanol preferring (HEP) male and female rats derived from crossbreeding of P and a variant strain of Sprague-Dawley animals. After a preference test for solutions of 3 to 30% ethanol vs. water, each rat was given limited access to its maximally preferred concentration daily between 1600 and 1800 h; fluid intakes were recorded every 0.25 h. Once fluid consumption had stabilized over 4 days, saline vehicle, 2.5 mg/kg or 5.0 mg/kg FG5938 was injected subcutaneously 0.5 h prior to ethanol access on each of 3 consecutive days; thereafter, preference testing for ethanol continued for 4 additional days. Whereas the saline vehicle was without effect, FG5938 caused a fivefold decrease in total intake of ethanol from 1.7 to 0.3 g/kg and in proportion of ethanol to total fluid consumed from 0.42 to 0.03. The onset of the significant decline in ethanol drinking occurred during the latter 1.75-h interval. Further, both doses of FG5938, but not saline, increased the intake of food significantly. The decline in ethanol drinking was virtually identical in both P and HEP males and in female HEP rats. These results demonstrate that FG5938 affects ethanol drinking only after 0.5 h of its administration. Finally, it is envisaged that the ingestion of ethanol in genetic high drinking rats is mediated, in part, by central synapses utilizing both 5-HT1A and 5-HT2A receptors.
Alcohol Drinking↗
Topiramate attenuates withdrawal signs after chronic intermittent ethanol in rats.
Topiramate, a novel anticonvulsant drug, has CNS depressant activity including enhancement of GABAergic inhibitory synaptic transmission. Drugs of this pharmacological spectrum might have utility in assuaging drug addiction. This study analyzes the ability of TPM to reduce withdrawal signs in the kindling model of ethanol dependence: chronic intermittent ethanol (CIE) rats. After CIE, persistent withdrawal signs are shown by an increased seizure susceptibility to the convulsant drug pentylenetetrazol and increased anxiety measured on the elevated plus-maze. Topiramate increased significantly the PTZ seizure threshold in CIE but not in control rats. On the elevated plus-maze, Topiramate was markedly more effective in CIE rats than in controls. Topiramate may have a therapeutic efficacy in treating alcohol withdrawal symptoms.
Animals↗
Metabolic properties of Eubacterium pyruvativorans, a ruminal 'hyper-ammonia-producing' anaerobe with metabolic properties analogous to those of Clostridium kluyveri.
Eubacterium pyruvativorans I-6(T) is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C(2) units derived from other substrates to dispose of reducing equivalents.
Adenosine Triphosphate↗
Physiological ecology of Clostridium glycolicum RD-1, an aerotolerant acetogen isolated from sea grass roots.
An anaerobic, H(2)-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H(2)-CO(2), formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O(2) in the headspace of static, horizontally incubated culture tubes; the concentration of O(2) decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O(2). In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H(2) became significant end products when RD-1 was grown on glucose in the presence of O(2). Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H(2) were produced. When the concentration of O(2) in the headspace exceeded 1% (vol/vol), supplemental H(2) was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288(T), an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288(T) demonstrated that it had negligible H(2)- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C. glycolicum DSM 1288(T). A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C. glycolicum DSM 1288(T) confirmed that RD-1 was a strain of C. glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O(2), (iii) oxic conditions favor the production of ethanol, lactate, and H(2) by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O(2) might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O(2).
Acetates↗
Long-term incomplete xylose fermentation, after glucose exhaustion, with Candida shehatae co-immobilized with Saccharomyces cerevisiae.
Saccharomyces cerevisiae and Candida shehatae were co-immobilized in an agar sheet which was introduced in an original two-chambered bioreactor asymmetrically fed in a batch mode with a mixture of glucose and xylose in a ratio of 35:15. The two sugars were consumed simultaneously. All glucose was fermented but only 20% of xylose. After incubation, yeast cells recovered from different areas of the agar sheet (close to, called Hi, and distant from, called Ho, the substrate chamber) were cultured as suspended cells in fresh culture medium provided with xylose or the sugar mixture. Xylose utilization by gel released Hi yeasts was significantly delayed compared to the Ho culture. Ethanol consumption by Hi yeasts in the two-substrate medium occurred after glucose exhaustion despite the presence of xylose. The waste medium resulting from incubation of the immobilized-cell structure inhibited xylose utilization by C. shehatae. Our results suggested that batch fermentation most probably favoured this incomplete xylose fermentation.
Bioreactors↗
Central nervous system effects of acetate: contribution to the central effects of ethanol.
Acetate, resulting from ethanol metabolism in the liver, is released into the circulation and is utilized in a number of tissues, including the brain. In its metabolism, acetate leads to the production of adenosine, a powerful physiological mediator. We have investigated the effect of acetate on central nervous system (CNS) function in rodents. Sodium acetate in doses resulting in blood concentrations comparable to those attained after the administration of 1 to 2 g/kg ethanol, had significant CNS effects. Both ethanol and acetate produced a dose-dependent impairment of motor coordination. This effect of acetate was fully blocked by the adenosine receptor blocker 8-phenyltheophylline (8PT), whereas the dose-response relationship for ethanol was shifted to the right by about 30%. The inspired concentration of sevoflurane to achieve anesthesia was significantly reduced by both these agents. General anesthesia was potentiated in a dose-dependent fashion by ethanol and by acetate. The effect of acetate on anesthetic requirements was fully blocked by 8PT. The effect of ethanol on sevoflurane anesthetic requirements was inhibited by 22 to 35% by 8PT. Locomotor activity in mice was reduced by acetate in a dose-dependent fashion, an effect that was also fully blocked by 8PT. On the other hand, ethanol at a dose of 1 to 2 g/kg increased locomotor activity. This likely results from a direct stimulatory effect of ethanol, opposed by an inhibitor effect of acetate. The administration of 8PT enhanced the stimulation of locomotor activity induced by ethanol. In conclusion, acetate, a product of ethanol metabolism has significant CNS effects that can either potentiate or antagonize the effects of the ethanol molecule per se.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetates↗
Effect of epinephrine and alprenolol on ethanol metabolism, liver cell respiration and mitochondrial function.
Chronic administration of epinephrine to adult male rats resulted in a significant increase in the rate of ethanol elimination, when given alone or together with the beta-adrenergic blocker alprenolol. This effect was observed concomitantly with an increased hepatic oxygen utilization and no changes in mitochondrial respiratory functions. Epinephrine given acutely did not modify the rate of ethanol metabolism. Blood glucose levels were enhanced in these conditions, but were unaffected in rats treated with epinephrine plus alprenolol. These results suggest that chronic epinephrine treatment induces an increased oxidative capacity in the liver characterized by enhanced rates of oxygen uptake and ethanol metabolism, which is not related to its beta-adrenergic actions.
Alprenolol↗
Comparison of ethanol production by different zymomonas strains.
A comparison of the rates of growth and ethanol production by 11 different strains of Zymomonas revealed a wide range of characteristics, with some strains being more tolerant of high sugar or ethanol concentrations and high incubation temperatures than others. Some strains were unable to utilize sucrose; others produced large amounts of levan, and one strain grew well but produced no levan. One strain, CP4, was considerably better in all respects than most of the other strains and was chosen as a starting strain for genetic improvement of ethanol production.
Journal Article↗
Simulation of batch and continuous reactors with co-immobilized yeast and beta-galactosidase.
A reaction-diffusion model was used to simulate a co-immobilized system utilizing the numerical method of orthogonal collocation. The production of ethanol from deproteinized whey using beta-galactosidase co-immobilized with Saccharomyces cerevisiae in calcium alginate gel beads was chosen as a model system. Calculated concentrations of lactose, glucose, galactose and ethanol were compared with experimental data for a batch reactor and a continuous horizontal packed-bed reactor. The mathematical model has been used to analyse the influence of internal and external mass transfer for the continuous reactor. The external mass transfer was shown to be of minor importance. The introduction of baffles decreased the backmixing in the horizontal packed-bed reactor. Internal mass transfer was found to be the main cause of the reduction in the apparent reaction rate. Thus, much of the expected increase in reaction rate is diminished by mass transfer hindrance when the cell concentration is increased.
Alginates↗
Synthesis of a secondary N-desmethyl and a tertiary N-cyclopropylmethyl bridged hexahydroaporphine as precursors to bicyclic opioid ligands.
In an attempt to generate a bicyclic 5,8-ethano derivative of N-methylmorphinan, an isomeric bicyclic hexahydroaporphine 2 was synthesized. The phenolic analogue of 2 has demonstrated affinity for mu opioid receptors in vitro and, along with 2, provided weak, primarily nonopioid analgesic action when injected intracerebroventricularly in mice. It was of interest to assess the potential opioid antagonist action of bicyclic hexahydroaporphine analogues containing cyclopropylmethyl and allyl nitrogen substituents. As the first steps in the generation of these potential opioid antagonists, the secondary bicyclic hexahydroaporphine 3 and its N-cyclopropylmethyl congener 4 were synthesized. N-Demethylation of 2 was initially attempted via the von Braun reaction, but acid-catalyzed hydrolysis of the crude N-cyano intermediate resulted in product decomposition. A successful approach to 3 involved the hydrolysis of the N-formyl precursor 1 in ethanolic potassium hydroxide. Direct alkylation of the secondary amine 3 utilizing cyclopropylmethyl bromide and sodium bicarbonate successfully generated the alkylated derivative 4. Both products were purified in hydrochloride salt form and characterized by standard analytical and spectroscopic methods. The free base form of 3 was highly sensitive to photooxidation. Opioids are known to oxidize to 10-keto structures, and secondary amines can oxidize to hydroxylamines. Infrared analysis of the decomposition product indicated the presence of both hydroxy and carbonyl groups which were absent in the spectrum of the salt. Structures of potential oxidation products are proposed.
Aporphines↗
Enzymatic synthesis of aliphatic beta-lactosides as mimic units of glycosphingolipids by use of Trichoderma reesei cellulase.
Aliphatic beta-lactosides were directly synthesized by beta-lactosyl transfer reaction from p-nitrophenyl beta-lactoside (Lac beta-pNP) to various 1-alkanols (n = 2-12), utilizing commercially available cellulase preparation of Trichoderma reesei C1. With ethanol acceptor, the enzyme induced ethyl beta-lactoside (1) in 18% yield based on the donor added in aqueous buffer system. When 1-octanol and dodecanol were acceptors, octyl beta-lactoside (2) and dodecyl beta-lactoside (3) were also obtained as transfer products, respectively. In both cases, the addition of sodium cholate as detergent to the reaction system ensured a sufficient solubility of these acceptors and resulted in a remarkable increase of the desired compounds (5-13% yields based on the donor added). Furthermore, the enzyme catalyzed the N-acetyllactosaminyl transfer reaction from p-nitrophenyl beta-N-acetyllactosaminide (LacNAc beta-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides, Gal beta1-4GlcNAc beta1-4Man (4) and Gal beta1-4GlcNAc beta1-4Glc (5), respectively. The enzyme activities transferring lactosyl and N-acetyllactosaminyl groups were not separated by chromatographies using DEAE-Sepharose Fast Flow and Sephadex 75 pg columns, indicating that the two reactions were catalyzed by a single enzyme. It was specified that a single enzyme works both transglycosylations, based on the substrate competition assay on hydrolysis.
Amino Sugars↗