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PubMed2006

Mode of action and properties of the beta-xylosidases from Talaromyces emersonii and Trichoderma reesei.

Enzymatic hydrolysis of arabinoxylan is an important prerequisite for the utilization of hemicellulose for ethanol fermentation or for making the low calorie sweetener xylitol by catalytic hydrogenation of the generated xylose. This study focus on cloning and characterization of two industrial relevant beta-xylosidases (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from Talaromyces emersonii (betaXTE) and Trichoderma reesei (betaXTR) and a comparison of these in relation to hemicellulose hydrolysis using an industrial relevant substrate. Both beta-xylosidases were expressed in A. oryzae and subsequently purified. During the enzymatic hydrolysis of xylobiose, the reaction product of both enzymes was found to be beta-D-xylose proving that the hydrolysis is proceeding via a retaining reaction mechanism. Based on sequence similarities and glycosyl hydrolases family membership, the active site residues of betaXTE and betaXTR are predicted to be Asp 242 and Glu 441, and Asp 264 and Glu 464, respectively. The involvement in catalysis of these carboxyls was examined by modification using the carbodiimide-nucleophile procedure resulting in a complete inactivation of both enzymes. The degree of xylose release from vinasse, an ethanol fermentation by-product, by betaXTE and betaXTR was 12.1% and 7.7%, respectively. Using the beta-xylosidases in combination with the multicomponent enzyme product Ultraflo L, resulted in 41.9% and 40.8% release of xylose, respectively indicating a strong synergistic effect between the exo-acting beta-xylosidases and the endo-1,4-beta-xylanases and alpha-L-arabinofuranosidase in Ultraflo L. There seems to be no measurable differences between the two beta-xylosidases when used in this specific application despite the differences in specific activity and kinetic properties.

Amino Acid Sequence↗
PubMed1992

Studies on plasmid stability, cell metabolism and superoxide dismutase production by Pgk- strains of Saccharomyces cerevisiae.

A double mutant sod1/pgk1 strain of Saccharomyces cerevisiae has been constructed in order to investigate the effects of different environmental conditions on yeast physiology, plasmid stability, and superoxide dismutase (SOD) production. Strains were transformed with yeast episomal plasmids (YEp) containing both PGK1 and SOD1 genes and were grown on fermentable carbon sources and under vigorous aeration. Under these conditions, the presence of the PGK1 gene was made essential for growth and both genes were efficiently expressed. However, plasmid-borne PGK1 was found not to increase the stability of YEp vectors in batch cultures of Pgk- cells. Paradoxically, plasmid stability increased during the respiratory phase of growth. An investigation of the metabolism of Pgk- cells demonstrated that these glycolytic pathway mutants do not appreciably metabolize glycerol. Thus Pgk+, plasmid-containing, cells have a selective advantage during the respiratory phase of batch growth since they can utilize both glycerol and ethanol.

Clone Cells↗
PubMed2004

Determination of catechins by means of extraction with pressurized liquids.

The technique of extraction with pressurized liquids is applied for extraction of catechin and epicatechin in tea leaves and in grape seeds. The extracts obtained are then analyzed by HPLC. A comparison has been made of the recoveries obtained employing extraction by magnetic stirring, ultrasound-assisted extraction, and extraction with pressurized liquids. In the three extraction systems, four different pure solvents were utilized, namely water, methanol, ethanol, and ethyl acetate. Methanol produced the best results. For this comparison, an initial step was to check the stability of catechins during the process of extraction using pressurized liquids at high temperature (100-200 degrees C). It has been confirmed that recoveries of these two compounds begin to fall, to below 95%, at 130 degrees C and above. Pressurized liquid extraction using methanol as solvent, produces results, in terms of recovery of catechin and of epicatechin, notably higher than any of the other conditions of extraction tested. The duration of the extraction cycle was also evaluated and re-extractions were performed to ensure the full recovery from the samples. It was found that, forcatechin, the R.S.D. of the method is 3.21%, and for epicatechin the R.S.D. was 2.96% (n = 5). The final optimized pressurized liquid extraction method allows for the determination of catechin and epicatechin in diverse types of samples with a rapid (10 min) and reproducible method.

Catechin↗
PubMed2006

Structure-guided engineering of xylitol dehydrogenase cosubstrate specificity.

Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 angstroms resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2' hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.

Carrier Proteins↗
PubMed1980

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.

Acetates↗
PubMedDate not supplied

Determination of annatto in high-fat dairy products, margarine and hard candy by solvent extraction followed by high-performance liquid chromatography.

Utilizing solvents such as ethanolic aqueous ammonia, petroleum ether, hexane and chloroform, annatto components alpha- and beta-norbixin and alpha- and beta-bixin were extracted from cheese, butter, margarine and hard candy. After transferring the extract into a solution of aqueous acetic acid in methanol, bixin and norbixin were determined quantitatively using high-performance liquid chromatography (HPLC) and an absorbance detector set at 500 nm. Recovery of norbixin from spiked cheese samples averaged 92.6% over a range of 1 to 110 micrograms/g. Commercial cheese samples were found to contain 1.1-68.8 microgram/g total norbixin, and two samples also contained 5.1-5.6 micrograms/g total bixin. Samples of uncoloured butter were spiked with bixin and recovery averaged 93.2% over a range of 0.1 to 445 micrograms/g. Levels of 0.2 microgram/g total bixin and 0.91 microgram/g total norbixin were found in one commercial butter sample; the others contained trace levels of both compounds. Hard candies were prepared in the laboratory and recovery studies conducted. Recovery of norbixin averaged 88%.

Bixaceae↗
PubMed1996

Energy value of moderate alcohol consumption by humans.

We investigated the effects of an equal-energetic substitution of ethanol for dietary carbohydrate in high-and low-fat diets on energy expenditure and body composition. During the controlled feeding study, subjects maintained their weights and consumed only food and drink provided by the US Department of Agriculture Beltsville Human Nutrition Research Center's Diet Study Facility. Subjects (16 men and 32 women) were divided equally into two groups and consumed either a high-or low-fat diet for 16 wk. The feeding period was divided into two 8-wk periods during which either ethanol or carbohydrate was added to the diet (5% of total daily energy intake) in a crossover design. The metabolizable energy content of the diets (with supplements) was determined for all subjects through measurement of total food intake and fecal and urinary losses for 7 d during both 8-wk periods. Energy expenditure, measured for 24 h in a room calorimeter at the end of each 8-wk period, was the same for both periods. Metabolizable energy intake and changes in total-body energy content were used to calculate the total amount of energy expended by each subject for 7 wk during each 8-wk period. Total energy expenditure for 7 wk was the same when subjects consumed either ethanol or carbohydrate. These data clearly show that on an energy basis ethanol and carbohydrate are utilized in the diet with the same efficiency. These data are consistent with the efficiency of use of alcohol for maintenance of metabolizable energy being the same as that for carbohydrate.

Adult↗
PubMedDate not supplied

Brain nitric oxide synthase levels increase in response to antenatal ethanol exposure.

AIMS: Our previous in vitro data have indicated that ethanol can increase nitric oxide synthase (NOS) expression. Thus, the Aims of this study were to determine whether ethanol produces the same effect in vivo. METHODS: To accomplish this, we utilized the well-established prenatal ethanol (EtOH) exposure model in the guinea pig to examine the effect on brain NOS expression and activity. RESULTS: Brain homogenates isolated from offspring of guinea pigs fed EtOH exhibited an increase in NOS protein expression and NOS activity compared to controls. Increased expression of neuronal NOS was observed only in soluble fractions of brain homogenates (P < 0.05 vs. control). Increased expression of a approximately 60 kDa band was detected in the soluble fraction that was immunoreactive against an antiserum raised against inducible NOS. In addition, an immunoreactive band of the correct predicted molecular weight for iNOS was found in the particulate fraction although the expression was unchanged between control and EtOH-treated animals. Endothelial NOS protein expression could not be detected in either soluble or particulate fractions from control or EtOH-treated animals. CONCLUSIONS: These results suggest that EtOH may exert its toxic effects antenatally via a mechanism of altered nitric oxide availability from NOS.

Animals↗
PubMed2000

The application of the atomic force microscope to studies of medically important protozoan parasites.

Both living and fixed specimens of the medically-important parasitic protozoa, Trypanosoma cruzi, Toxoplasma gondii, Giardia lamblia, Entamoeba histolytica, and Acanthamoeba spp. were studied by atomic force microscopy (AFM). The preparation of fixed specimens was similar to methods used for either scanning or transmission electron microscopy. AFM scanning was performed using both contact and tapping modes. A classical fixation procedure utilizing glutaraldehyde followed by ethanol dehydration was not suitable for all parasite species. AFM images could not be obtained from fixed samples of T. cruzi, T. gondii or E. histolytica. However, excellent topographic images could be obtained from specimens of G. lamblia and Acanthamoeba under identical conditions. Critical point drying permitted AFM imaging of both trypomastigote and epimastigote stages of T. cruzi. Phase imaging of T. cruzi elucidated unique surface details at a level of resolution not visible using any other imaging modalities. AFM elasticity map imaging of T. cruzi-infected and T. gondii-infected cells demonstrated that both parasites were markedly firmer than the surrounding host cell cytoplasm. The parasitophorous vacuole surrounding replicating T. gondii tachyzoites was also visualized by elasticity map imaging. These data suggest that although much remains to be learned about preparing parasitic protozoa for AFM imaging, the technique has the potential of providing unique and important insights into these disease causing organisms.

Amoebida↗
PubMed2001

Desulfomusa hansenii gen. nov., sp. nov., a novel marine propionate-degrading, sulfate-reducing bacterium isolated from Zostera marina roots.

The physiology and phylogeny of a novel sulfate-reducing bacterium, isolated from surface-sterilized roots of the marine macrophyte Zostera marina, are presented. The strain, designated P1T, was enriched and isolated in defined oxygen-free, bicarbonate-buffered, iron-reduced seawater medium with propionate as sole carbon source and electron donor and sulfate as electron acceptor. Strain P1T had a rod-shaped, slightly curved cell morphology and was motile by means of a single polar flagellum. Cells generally aggregated in clumps throughout the growth phase. High CaCl2 (10 mM) and MgCl2 (50 mM) concentrations were required for optimum growth. In addition to propionate, strain P1T utilized fumarate, succinate, pyruvate, ethanol, butanol and alanine. Oxidation of propionate was incomplete and acetate was formed in stoichiometric amounts. Strain P1T thus resembles members of the sulfate-reducing genera Desulfobulbus and Desulforhopalus, which both oxidize propionate incompletely and form acetate in addition to CO2. However, sequence analysis of the small-subunit rDNA and the dissimilatory sulfite reductase gene revealed that strain P1T was unrelated to the incomplete oxidizers Desulfobulbus and Desulforhopalus and that it constitutes a novel lineage affiliated with the genera Desulfococcus, Desulfosarcina, Desulfonema and 'Desulfobotulus'. Members of this branch, with the exception of 'Desulfobotulus sapovorans', oxidize a variety of substrates completely to CO2. Strain P1T (= DSM 12642T = ATCC 700811T) is therefore proposed as Desulfomusa hansenii gen. nov., sp. nov. Strain p1T thus illustrates the difficulty of extrapolating rRNA similarities to physiology and/or ecological function.

Biodegradation, Environmental↗
PubMed2002

Methanocalculus taiwanensis sp. nov., isolated from an estuarine environment.

Two novel hydrogenotrophic methanogens, designated strains P2F9704aT and P2F9705, were isolated from an estuary in Eriln Shi, Taiwan. The cells of strain P2F9704aT were non-motile, irregular cocci 0.9-1.4 microm in diameter. They stained gram-negative. The cells catabolized formate and H2+CO2 to produce methane, but did not utilize acetate, methanol, trimethylamine, ethanol or secondary alcohols as methanogenic substrates. The optimal growth parameters for strain P2F9704aT were pH 6.7, 37 degrees C and 0.5% NaCl. Acetate was required for cell growth even though it was not a substrate for methanogenesis. The trace element tungsten was not required but slightly stimulated the growth of strain P2F9704aT. However, tungsten extended the growth ranges relating to temperature, pH and salt. The sequences of the 16S rRNA genes of strains P2F9704aT and P2F9705 were nearly identical and possessed 99.1 and 98.5% similarity to the genes of Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, strain P2F9704aT possessed 14 and 12% DNA relatedness with respect to Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, the optimal salt concentrations, the cellular protein profiles and the molecular masses of surface-layer protein subunits of strain P2F9704aT were different from those of the other two known Methanocalculus species. On the basis of these observations, it is proposed that these two organisms should be placed in a new species, namely Methanocalculus taiwanensis. The type strain is P2F9704aT (= OCM 671T = CCRC 16182T = DSM 14663T).

Archaeal Proteins↗
PubMed1976

Acetyl-CoA production and utilization during growth of the facultative methylotroph Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate.

In Pseudomonas AM1, conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase, an acetoacetate: succinate coenzyme A transferase (specific for succinyl-CoA) and an inducible beta-ketothiolase. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase has been partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant (ICT54) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant (ICT51), lacking 'malate synthase' activity has been isolated and its characteristics indicate that this activity is normally essential for growth, of Pseudomonas AM1 on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C1 compounds. The growth properties of a revertant (ICT51R) and of a mutant lacking malyl-CoA lyase (PCT57) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by way of acetyl-CoA.

Acetate-CoA Ligase↗
PubMed2003

Desulfovibrio hydrothermalis sp. nov., a novel sulfate-reducing bacterium isolated from hydrothermal vents.

Mesophilic, hydrogenotrophic, sulfate-reducing bacteria were isolated from a deep-sea hydrothermal chimney sample collected at 13 degrees N on the East-Pacific Rise at a depth of 2,600 m. Two strains (BL5 and H9) were found to be phylogenetically similar to Desulfovibrio profundus (similarity >99%), whereas two other strains (H1 and AM13T) were found to be phylogenetically distinct (similarity 96.4%) from Desulfovibrio zosterae, their closest relative. Strain AM13T was characterized further. It was a barophilic, Gram-negative, non-sporulating, motile, vibrio-shaped or sigmoid bacterium possessing desulfoviridin. It grew at temperatures ranging from 20 to 40 degrees C, with an optimum at 35 degrees C in the presence of 2.5% NaCl. The pH range for growth was 6.7-8.2 with an optimum around 7.8. Strain AM13T utilized H2/CO2, lactate, formate, ethanol, choline and glycerol as electron donors. Electron acceptors were sulfate, sulfite and thiosulfate, but not elemental sulfur or nitrate. The G + C content of DNA was 47 mol%. Strain AM13T (= DSM 14728T = CIP107303T) differed from D. zosterae not only phylogenetically, but also genomically (DNA-DNA reassociation value between the two bacteria was 23.8%) and phenotypically. This isolate is therefore proposed as the type strain of a novel species of the genus Desulfovibrio, Desulfovibrio hydrothermalis sp. nov.

DNA, Bacterial↗
PubMed1995

Validated fluorimetric HPLC analysis of acetaldehyde in hemoglobin fractions separated by cation exchange chromatography: three new peaks associated with acetaldehyde.

Stable hemoglobin-acetaldehyde adducts present in hemoglobin fractions separated by polyaspartic acid cation exchange chromatography were quantified by fluorimetric HPLC. The fluorescent species eluted from the HPLC was confirmed by mass spectrometry to be consistent with the expected product from reaction of acetaldehyde, 1,3-cyclohexanedione (CHD), and ammonium ion. Hemolysate (2.2 mM hemoglobin) was incubated in equivalent volumes of either phosphate-buffered saline or 5 mM acetaldehyde at 37 degrees C for 30 min and washed three times with H2O to remove free acetaldehyde and labile adducts before the injection of 14.7 mg hemoglobin onto the cation exchange column. Fluorimetric HPLC analysis of hemolysate samples either with or without in vitro reaction with acetaldehyde revealed that most acetaldehyde resides in the hemoglobin A0 fraction. The reaction with acetaldehyde in vitro resulted in a significant increase in fast-eluting minor hemoglobin species on cation exchange chromatography concomitant with increased acetaldehyde in the HbA1a+b, HbA1c, and HbA1-AcH fractions. We report three new cation exchange chromatographic peaks after reaction with acetaldehyde: HbA1-AcH-3, HbA1c-1, and HbA0-1. Each new peak was found to associate with a significant quantity of CHD-reactive acetaldehyde. These experiments provide additional evidence that stable adducts form between acetaldehyde and hemoglobin and that these adducts occur in multiple hemoglobin species separated by cation exchange chromatography. Further characterization and structural assignment of these species are warranted in view of their potential utility as markers for ethanol intake.

Acetaldehyde↗
PubMed1973

Bubble stability test compared with lecithin assay in prediction of respiratory distress syndrome.

A simple test for surfactant, utilizing bubble stability in ethanol, was performed in 106 samples of amniotic fluid obtained from 94 patients. Of these patients 80 delivered within 48 hours of the collection of the sample. The results were compared with the lecithin concentration in the same amniotic fluid samples and with the quality of respiration in the neonate. The test was "positive," indicating fetal pulmonary maturity, in 37 cases and none of these infants developed respiratory distress syndrome (R.D.S.). In only one of these cases, however, was gestation less than 37 weeks. The test was "intermediate" or "negative" in 43 cases but in 35 of these infants respiration at birth was perfectly normal.Performed by the method described by its originators, this simple test gives too many false negative results to be of value in routine clinical practice, although a positive result is helpful. The concept of the test is ingenious, however, and further developments may be expected.

Amniotic Fluid↗
PubMed1980

Properties of a phosphoprotein phosphatase from rat epididymal fat pads: deactivation of hormone-sensitive triglyceride lipase and activation of glycogen synthase in adipose tissue.

A phosphoprotein phosphatase has been partially purified from rat epididymal fat pads by a procedure utilizing ammonium sulfate and ethanol precipitations and chromatography on DEAE-Sephadex A-50. The phosphatase was eluted from Sephadex G-75 columns with an apparent molecular weight of 28 000. The phosphoprotein phosphatase catalyzed the reversible deactivation of protein kinase activated chicken adipose tissue hormone-sensitive triglyceride lipase. Phosphatase activity measured with activated triglyceride lipase as substrate was completely dependent upon the presence of metal ions (Mg2+, Ca2+, or Mn2+) and was inhibited by inorganic phosphate and adenine nucleotides. The fat pad phosphatase increased the rate of activation of glycogen synthase in rat adipose tissue infranatant fractions from fed and 24-h fasted rats but had little or no effect on synthase activity in infranatant fractions from rats fasted for 48 h. Fasting had no effect on rat fat pad phosphatase activity measured with triglyceride lipase as substrate, but phosphatase activity was decreased in preparations from diabetic rats.

Animals↗
PubMed2001

Misoprostol therapeutics revisited.

Misoprostol, a prostaglandin E1 analog, is a racemate of four stereoisomers. On administration it rapidly de-esterifies to its active form, misoprostolic acid. Misoprostolic acid is 85% albumin bound and has a half-life of approximately 30 minutes. It is excreted in urine as inactive metabolites. No significant drug interactions have been reported. Besides its gastrointestinal protective and uterotonic activities, misoprostol regulates various immunologic cascades. It inhibits platelet-activating factor and leukocyte adherence, and modulates adhesion molecule expression. It protects against gut irradiation injury, experimental gastric cancer, enteropathy, and constipation. It improves nutrient absorption in cystic fibrosis. Misoprostol has utility in acetaminophen and ethanol hepatotoxicity, hepatitis, and fibrosis. It is effective in asthmatics and aspirin-sensitive asthmatic and allergic patients. It lowers cholesterol and severity of peripheral vascular diseases, prolongs survival of cardiac and kidney transplantation, synergizes cyclosporine, and protects against cyclosporine-induced renal damage. It works against drug-induced renal damage, interstitial cystitis, lupus nephritis, and hepatorenal syndrome. It is useful in periodontal disease and dental repair. Misoprostol enhances glycosoaminoglycan synthesis in cartilage after injury. It prevents ultraviolet-induced cataracts and reduces intraocular pressure in glaucoma and ocular hypertension. It synergizes antiinflammatory and analgesic effects of diclofenac or colchicine and has been administered to treat trigeminal neuralgic pain. It reduces chemotherapy-induced hair loss and recovery time from burn injury, and is effective in treating sepsis, multiple sclerosis, and pancreatitis.

Animals↗
PubMed1980

Swedish company doctors' attitudes to benzodiazepines and neuroleptics used as minor tranquillizers.

A survey was carried out among 150 Swedish company doctors to assess their attitudes towards 2 benzodiazepines and 4 neuroleptics widely used as minor tranquillizers. The participants rated their opinions on 6 drug characteristics considered important when prescribing anxiolytics. The profile for the two benzodiazepines, diazepam and oxazepam, showed that whilst these drugs were regarded as having a greater anxiolytic effect than the neuroleptics they were also considered as having a higher amount of negative effects compared to the neuroleptics as a hole. The neuroleptics had a more varied profile than the benzodiazepines. Two of the neuroleptics, flupenthixol and especially fluphenazine, had a more favourable profile than the other two, dixyrazine and thioridazine, being regarded as having a greater anxiolytic effect without a greater amount of negative effects. The more favourable profiles for flupenthixol and fluphenazine are probably an indication that these two drugs are used with satisfactory results in special clinical situations when it is more important to avoid possible side-effects.

Anti-Anxiety Agents↗