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PubMed2006

Production of ethanol from starch by free and immobilized Candida tropicalis in the presence of alpha-amylase.

Candida tropicalis is a potentially useful organism for the commercial production of ethanol as it is capable of fermenting starch at a low rate. To enhance this carbon source utilization and increase the rate of alcohol production, we pretreated corn soluble starch with alpha-amylase. Starch liquefaction was sufficient to drive the fermentation and to convert 96% substrate to ethanol. Indeed, in the presence of exogenous alpha-amylase, 9% (w/v) soluble starch was converted to 43.1g ethanol/l in 65 h with a productivity of 0.65 g/l h. Thus, bio-ethanol production using free and calcium alginate-immobilized C. tropicalis does not require the saccharification step. Furthermore, fed-batch fermentation by free C. tropicalis cells increased the final concentration to 56 g ethanol/l, reaching published values for Saccharomyces cerevisiae recombinant strains expressing both alpha-amylase and glucoamylase.

Candida tropicalis↗
PubMed2001

Uroplakin as a marker for typing metastatic transitional cell carcinoma on fine-needle aspiration specimens.

BACKGROUND: Immunohistological markers specific for a single type of epithelium are rare. Recently, urothelium tissue-specific genes were cloned. The genes encoded a family of transmembrane proteins, uroplakins, that are expressed only in urothelial mucosa. Using uroplakin antibodies on paraffin-embedded tissue, a previous study demonstrated positive staining in 66% of metastatic transitional cell carcinoma (TCC) cases and negative staining in all other tumors (including breast, ovarian, lung, and gastrointestinal carcinomas) tested. The current study addresses the diagnostic value of uroplakins in conventional fine-needle aspiration (FNA) material in establishing a diagnosis of metastatic TCC. METHODS: Representative slides from 27 FNA cases of metastatic TCC and 52 non-TCC carcinomas were collected. The avidin-biotin-peroxidase method was utilized, using polyclonal antiuroplakin as the primary antibody on 95% ethanol-fixed, Papanicoloau-stained direct smears. RESULTS: Twenty-five of 27 metastatic TCC cases (93%) were found to stain positively for uroplakin with a superficial membrane/microluminal staining pattern. A few cells with diffuse membranous staining also were noted in 48% of the positive metastatic TCC cases. The superficial membrane/microluminal staining pattern was not observed in any of the non-TCC carcinomas. However, approximately 6% of these cases (3 of 52 cases) did show rare tumor cells with diffuse membranous staining. CONCLUSIONS: The application of uroplakin antibodies to 95% ethanol-fixed FNA direct smears has improved the sensitivity of the antibody for metastatic TCC while maintaining a specificity comparable to that of paraffin-embedded tissue. The authors believe that these antibodies have diagnostic potential in cytopathology in the evaluation of metastatic TCC.

Adult↗
PubMed1982

Metronidazole phosphate--a water-soluble prodrug for parenteral solutions of metronidazole.

To develop a parenteral solution of relatively water-insoluble metronidazole (2-methyl-5-nitro-1H-imidazole-1-ethanol), its phosphate ester was synthesized via two routes. One route utilized 2-cyanoethyl phosphate and the other utilized pyrophosphoryl tetrachloride. The first method used dicyclohexylcarbodiimide as a coupling agent and the cyanoethyl group was removed under mild alkaline conditions. The second method was a one-step procedure in which free acid of metronidazole phosphate was isolated as a crystalline solid. The solubility of metronidazole in various solvents was determined at 25 degrees. From the pH-dependence of its aqueous solubility, the pKa of the conjugate acid of metronidazole was estimated to be 2.62, which agreed well with the pKa values of other nitroimidazoles. Metronidazole phosphate behaved as a zwitterionic compound in an acidic medium with a minimum solubility at pH 2.0. At pH 7, its solubility was approximately 50 times that of metronidazole. The phosphate ester was so soluble at pH higher than 7 that it was difficult to measure the solubility accurately. In human serum, the hydrolysis of metronidazole phosphate followed zero-order kinetics at an initial concentration of 0.25 mg/ml or higher, presumably due to enzyme saturation (0.035 mg/ml/hr at 37 degrees). A reversed-phase HPLC procedure was adopted to monitor the appearance of metronidazole and the disappearance of metronidazole phosphate. Subcutaneous administration of metronidazole phosphate to rats produced a blood level of bioactivity comparable to that observed after administration of metronidazole.

Animals↗
PubMed1980

Energetics of growth of Microbacterium thermosphactum at low temperatures.

Microbacterium thermosphactum was grown at 5 degrees C in glucose-limited continuous cultures. The end products of glucose metabolism were L-lactate and ethanol, and these compounds accounted for 86--92% of the glucose utilized. With input glucose concentrations less than 3 mV, YgluMax was found to be 40--43, YATPMax 20--21 and ms 0.1--0.2. These values are almost identical to those found previously for cultures at 25 degrees C and show that this psychrotroph grows with a very high energetic efficiency over a wide range of temperatures. With a higher (but still limiting) input glucose concentration of 5.6 mM at 9 degrees C, cellular efficiency declined as there was a marked reduction in Yglu. This decrease was accounted for in mathematical terms by an increase in ms to 0.7, whilst YgluMax and gh energetic efficiency over a wide range of temperatures. With a higher (but still limiting) input glucose concentration of 5.6 mM at 9 degrees C, cellular efficiency declined as there was a marked reduction in Yglu. This decrease was accounted for in mathematical terms by an increase in ms to 0.7, whilst YgluMax and gh energetic efficiency over a wide range of temperatures. With a higher (but still limiting) input glucose concentration of 5.6 mM at 9 degrees C, cellular efficiency declined as there was a marked reduction in Yglu. This decrease was accounted for in mathematical terms by an increase in ms to 0.7, whilst YgluMax and YATPMax remained high at 38 and 19 respectively.

Actinomycetales↗
PubMed1986

Assay of plasma leupeptin using the reversible binding of leupeptin to bovine pancreatic trypsin.

A competitive binding radioassay for leupeptin has been developed utilizing the reversible binding of leupeptin to bovine pancreatic trypsin. An ethanol precipitation step was introduced to separate trypsin-bound leupeptin from its free form. Advantages of this method are simplicity of the procedure and avoidance of the preparation of antiserum. The possible metabolites of leupeptin exhibit no significant inhibitory effect on leupeptin-trypsin binding in this system. This method was applied to the determination of plasma leupeptin levels in dogs after oral administration of the peptide.

Animals↗
PubMed1981

Determination of acetaldehyde in human blood by a gas chromatographic method with negligible artefactual acetaldehyde formation.

A method is described for determination of acetaldehyde in blood by head space gas chromatography. The method utilizes sodium nitrite-sulfosalicylic acid as an inhibitor of the ethanol oxidizing systems by means of which the interference of ethanol is reduced considerably. The detection limit was 0.4 mumol/l, the recovery 101.5 +/- 5.2% and the coefficient of variation was 7.8% (1.5 mumol/1 acetaldehyde). There was no disappearance of acetaldehyde if the head space vials were kept at -20 degree C for 24 h. In the comparison study with the semicarbazide method our results were 0.7-4.1 mumol/l lower. The values for acetaldehyde in blood after ethanol ingestion (0.5 g/kg) by volunteers were 0.5-1.3 mumol/l.

Acetaldehyde↗
PubMed1984

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum.

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.

Animals↗
PubMed2003

In vivo evaluation of riboflavin-containing microballoons for floating controlled drug delivery system in healthy human volunteers.

Hollow microspheres (microballoons) floatable in JPXIII no. 1 solution were developed as a floating controlled drug delivery system. Microballoons (MB) were prepared by the emulsion solvent diffusion method utilizing enteric acrylic polymers dissolved in a mixture of dichloromethane and ethanol. To assess the usefulness of the intragastric floating properties of MB in sustained pharmacological action, nonfloating microspheres (NF) possessing riboflavin release profiles equivalent to those of MB were prepared. Riboflavin powder, riboflavin-containing MB, and riboflavin-containing NF were administrated orally to each of three healthy volunteers. Riboflavin pharmacokinetics was investigated via analysis of urinary excretion of riboflavin. As a result, although urinary excretion of riboflavin following administration of MB was not sustained in the fasted state, urinary excretion of MB was significantly sustained in comparison to riboflavin powder and NF in the fed condition. Additionally, the excretion half-life time (t(1/2)) of MB was prolonged significantly by feeding. Furthermore, MB provided significantly high total urinary excretion (%) of riboflavin compared to NF in the fasted and the fed conditions. In view of the finding regarding similar riboflavin release profiles of MB and NF in JP XIII no. 1 solution (pH 1.2) and no. 2 solution (pH 6.8), the intragastric floating properties of MB appeared to be beneficial with respect to sustained pharmacological action.

Administration, Oral↗
PubMed1999

Characterization of a new xylanolytic bacterium, Clostridium xylanovorans sp. nov.

A new xylanolytic bacterium designated strain HESP1T (T = type strain) was isolated from a methanogenic digester. Strain HESP1T was a motile, rod shaped, spore-forming bacterium, which possessed a Gram-positive type cell wall. Glucose, fructose, lactose, trehalose, maltose, raffinose, sucrose, xylan, mannitol, cellobiose, galactose, mannose, melibiose, ribose were fermented to produce, acetate, butyrate, H2, CO2, formate, isobutyrate, and ethanol. Fumarate was fermented to acetate. Glycerol and methanol were also utilized. Sulfate, thiosulfate, nitrate, sulfur and fumarate were not used as electron acceptors. Strain HESP1T had a G + C content of 40 mol% and grew optimally at 37 degrees C and pH 7 on a fructose containing medium. Phylogenetically, strain HESP1T was most related to Clostridium aminovalericum (similarity of 94%) than to C. populeti, C. herbivorans and Eubacterium xylanophilum (average similarity of 92%), all members of subcluster XIVa of the low G + C containing Gram-positive branch. However, strain HESP1T shared little phenotypic and genotypic traits with C. aminovalericum and on the basis of this and phylogenetic evidence, we propose to tentatively designate strain HESP1T as a new species of the genus Clostridium, Clostridium xylanovorans sp. nov. The type strain is HESP1T (= DSM 12503).

Base Sequence↗
PubMed2004

In situ bioreduction of technetium and uranium in a nitrate-contaminated aquifer.

The potential to stimulate an indigenous microbial community to reduce a mixture of U(VI) and Tc(VII) in the presence of high (120 mM) initial NO3- co-contamination was evaluated in a shallow unconfined aquifer using a series of single-well, push-pull tests. In the absence of added electron donor, NO3-, Tc(VII), and U(VI) reduction was not detectable. However, in the presence of added ethanol, glucose, or acetate to serve as electron donor, rapid NO3- utilization was observed. The accumulation of NO2-, the absence of detectable NH4+ accumulation, and the production of N2O during in situ acetylene-block experiments suggest that NO3- was being consumed via denitrification. Tc(VII) reduction occurred concurrently with NO3- reduction, but U(VI) reduction was not observed until two or more donor additions resulted in iron-reducing conditions, as detected by the production of Fe(II). Reoxidation/remobilization of U(IV) was also observed in tests conducted with high (approximately 120 mM) but not low (approximately 1 mM) initial NO3- concentrations and not during acetylene-block experiments conducted with high initial NO3-. These results suggest that NO3(-)-dependent microbial U(IV) oxidation may inhibit or reverse U(VI) reduction and decrease the stability of U(IV) in this environment. Changes in viable biomass, community composition, metabolic status, and respiratory state of organisms harvested from down-well microbial samplers deployed during these tests were consistent with the conclusions that electron donor additions resulted in microbial growth, the creation of anaerobic conditions, and an increase in activity of metal-reducing organisms (e.g., Geobacter). The results demonstrate that it is possible to stimulate the simultaneous bioreduction of U(VI) and Tc(VII) mixtures commonly found with NO3- co-contamination at radioactive waste sites.

Biodegradation, Environmental↗
PubMed2004

Evolving protein functional diversity in new genes of Drosophila.

The mechanism by which protein functional diversity expands is an important evolutionary issue. Studies of recently evolved chimeric genes permit direct investigation of the origin of new protein functions before they become obscured by subsequent evolution. Found in several African Drosophila species, jingwei (jgw), a recently evolved gene with a domain derived from the still extant short-chain alcohol dehydrogenase (ADH) through retroposition, provides an opportunity to examine this previously undescribed process directly. We expressed JGW proteins in a microbial expression system and, after purification, investigated their enzymatic properties. We found that, unexpectedly, positive Darwinian selection for amino acid replacements outside the active site of JGW produced a novel dehydrogenase with altered substrate specificity compared with the ancestral ADH. Instead of detoxifying and assimilating ethanol like its Adh parental gene, we observe that JGW efficiently utilizes long-chain primary alcohols found in hormone and pheromone metabolism. These data suggest that protein functional diversity can expand rapidly under the joint forces of exon shuffling, gene duplication, and natural selection.

Alcohol Dehydrogenase↗
PubMed2006

Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes.

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.

Acetates↗
PubMed1976

Comparison of the delayed actions of growth hormone and somatomedin on adipose tissue metabolism.

Although acute insulin-like effects of growth hormone (GH) on adipose tissue are readily demonstrable in vitro, it has not been possible to reproduce in vitro the delayed (3 h) inhibition of glucose utilization seen after the administration of GH in vivo. To examine the possibility that somatomedin (Sm), the postulated mediator of GH action, might, like GH, have biphasic effects on adipose tissue metabolism and mediate the delayed inhibition of glucose utilization, Sm was prepared by gel filtration from an acid-ethanol extract of normal rat plasma. The partially purified material increased 35SO4 incorporation into costal cartilage of hypophysectomized rats and produced in adipose tissue an acute stimulation of glucose oxidation that was not suppressed by insulin anti-serum. Unlike GH, whose insulin-like effects disappear in 3--4 hours, Sm-stimulated glucose oxidation remained linear for the entire 4 hour incubation period. Although acute stimulation of tissues with GH rendered them refractory to renewed insulin-like stimulation by GH, no such refractoriness to the action of Sm was seen. Furthermore, acute stimulation with Sm failed to render tissues refractory either to itself or to GH. Finally, Sm failed to reproduce the delayed lipolytic effects produced by GH in conjunction with glucocorticoids. These results make it highly unlikely that Sm alone accounts for the delayed metabolic effects of GH.

Adipose Tissue↗
PubMed1983

Graft pretreatment with cyclosporin A. Long-term study including four trials in one and a half years.

Four trials were conducted to establish whether Cyclosporin A (Cy A) could be used as a graft pretreatment to prolong the graft function and animal survival of kidneys transplanted into bilaterally nephrectomized mongrel dogs. The first nonrandomized trial analyzed various parameters such as concentration, flush temperatures, effect of reflushing, and the need for subsequent minimal immunosuppression. By analyzing data from the ten groups in this trial, we established that 50 mg/l Cy A could be flushed successfully at either 4 C or 25 C without nephrotoxic effects. This concentration was found to prolong graft function and animal survival in some animals greater than 100 days. Reflushing the kidney after primary graft pretreatment with Cy A did not entirely remove the immunosuppressive effect of the drug as significant prolongation of survival also was observed in this group. In addition, minimal immunosuppression with azathioprine was necessary to observe a delay in the onset of rejection and prolongation of animal survival when Cy A was used for graft pretreatment. Trials 2 and 4 compared the efficacy of the original batch that was obtained later. The effects observed in Trial 1 and reproduced in Trial 3, which were randomized and double blinded using the original batch, were not seen in either Trials 2 or 4. This may have been related to the amount of time that the Cy A was dissolved in ethanol prior to its use or the batch of Cy A utilized. These preliminary results indicate that Cy A, when used for graft pretreatment under certain conditions, can delay the onset of rejection and prolong animal survival of transplanted renal allograft recipients.

Animals↗
PubMed1993

Prediction of alcohol-related harm by laboratory test results.

We examined the value of laboratory markers of excessive alcohol (ethanol) intake as predictors of mortality, morbidity, and health-care utilization in a cohort of 330 patients attending an acute ambulatory care service. Among men, all four markers examined--gamma-glutamyltransferase (GGT) and aspartate aminotransferase (AST) activities, high-density lipoprotein cholesterol (HDL-C), and mean corpuscular volume (MCV)--were predictive of medical sequelae and health-care utilization over a 3-year period. In contrast, social problems were more closely related to the amount of alcohol consumption at initial assessment than to any biological marker. Serum GGT and AST activities and MCV were predictive of medical sequelae in women. The predictive value of GGT was an independent risk factor and did not merely reflect recent alcohol intake or the presence of chronic liver disease. We conclude that these readily available laboratory tests provide important prognostic information and should be an integral part of the assessment of persons with hazardous alcohol consumption.

Alcoholism↗
PubMed1996

Interim and archival preservation of plant specimens in alcohols for DNA studies.

Three plant species, spinach, juniper and broccoli, were stored in different alcohol solutions, and the DNAs were examined to determine changes in the quality and quantity of DNA over time. The smallest changes in the genomic DNA were found in the samples stored in the lower mol wt alcohols (100% methanol and 100% and 95% ethanol) and when proteinase (Pronase E) was used in the extraction procedure. After 11 months of storage in ethanol, excellent yields of high mol wt DNA were obtained, but only when the procedure utilized Pronase E. The use of proteinase was found to be essential to obtain DNA from preserved specimens of spinach and broccoli. This appears to explain the previous reports of failures to obtain DNA from alcohol-preserved specimens. Vacuum infiltration of ethanol resulted in better DNA preservation than passive infiltration of ethanol.

Alcohols↗
PubMed1998

Influence of chronic alcohol abuse on body weight and energy metabolism: is excess ethanol consumption a risk factor for obesity or malnutrition?

OBJECTIVES: To evaluate the influence of chronic alcohol abuse on body composition and energy metabolism in patients affected by chronic alcoholism (group A) compared with a group of healthy social drinkers (group B). SETTING: A university hospital clinic in Italy. SUBJECTS: A total of 32 alcoholics without clinical or laboratory signs of liver cirrhosis and malabsorption. MEASUREMENTS: Body composition was assessed by anthropometric measurements. Resting energy expenditure (REE) and substrate oxidation rate was measured by indirect calorimetry. Daily caloric intake was computed on the basis of a food diary compiled over 7 days. RESULTS: Alcoholics showed a significantly lower body weight (P < 0.05) and a significant lower fat mass (P < 0.05) compared with controls. A higher waist-to-hip ratio was found in group A than in group B, both as a whole group (P < 0.01) or separated by gender (females, P < 0.01) and males, P < 0.001), indicating a prevalence of fat distribution in the abdominal region in alcoholics. REE was significantly higher in group A than in group B (P < 0.05). The non-protein respiratory quotient was significantly lower in group A than in group B (P < 0.001) with a consequent higher utilization of lipids (P < 0.01) and a lower carbohydrate oxidation (P < 0.05) in group A. The energy intake provided only by food ingestion was found to be significantly higher in group B (P < 0.01), whilst the total caloric intake, computed as food intake plus alcohol intake, was higher in group A (P < 0.01). CONCLUSIONS: Alcoholics, as compared with social drinkers, showed a lower body weight due essentially to a fat mass reduction, a higher REE value normalized by fat-free mass, and a preferential utilization of lipids as energy substrate. These findings might suggest that chronic ethanol abuse is able to determine an impairment of nutritional status due, at least in part, to an alteration of the substrate oxidation.

Abdomen↗
PubMed1990

Acute alcohol ingestion reduces fatty acid extraction of the heart, liver, and small intestine.

Ethanol may have profound effects on both the distribution of perfusion and substrate utilization by the liver and heart due to its vasodilating properties and the generation of high levels of circulating acetate and lactate. Since fatty acids are highly extracted by the heart and liver under normal circumstances, changes in the relationship of perfusion/fatty acid uptake may be a sensitive indicator of both altered perfusion and changes in metabolic substrate availability. To test this hypothesis, studies were performed in rats fed 3.1, 6.2, and 9.3 g/kg doses of ethanol. Fatty acid uptake was estimated with a 3-methyl substituted reagent with a chain length equivalent to 17 carbons. The methyl group in the three position prevented beta oxidation and prolonged the residence of fatty acids in the tissue. Eighteen hours after acute alcohol administration, fatty acid uptake was reduced in the heart and the small intestine; in the liver uptake was increased or unchanged. Acute ethanol administration also resulted in increased perfusion, as indicated by enhanced uptake of 201thallium by the heart, liver, and small intestine. The fatty acid extraction of the heart, liver, and small intestine, defined as the concentration of fatty acid divided by the concentration of 201thallium, was markedly decreased by alcohol ingestion. These alcohol effects were dose-dependent and temporally related. The data suggest that ethanol ingestion could potentially alter heart function during exercise or following a prolonged fast, when the heart relies primarily upon fatty acids extracted from the circulation to generated adenosine triphosphate (ATP).

Acetates↗