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PubMed1976

A bioassay for NSILA-S in individual serum samples and its relationship to somatotropin.

A practical bioassay for the acid-ethanol soluble non-suppressible insulin-like activity (NSILA-S) of individual serum samples has been developed utilizing the incorporation of 14C-glucose into the lipid faction of isolated adipocytes. NSILA-S activity was correlated with somatotropin status. Thus, the mean potencies (+/-SD) relative to an extract of pooled normal human serum were: normal samples 1.11 +/- 0.14, acromegalic 2.91 +/- 0.72, and somatotropin deficient 0.13 +/- 0.06. This variation in NSILA-S was not due to variability in extraction recoveries. The within assay precision was 9% (coefficient of variation) and the between assay 23%. This method allows the simultaneous extraction and processing of relatively large numbers of samples, and compares favorably with other more complex methods. Because of the evidence that NSILA-S may be related to the somatomedins, the present method should provide a simpler and more reliable alternative to the cartilage bioassays used to measure somatomedin activity.

Acromegaly↗
PubMedDate not supplied

[Pathways of inclusion of isotopes 2H and 13C into exometabolites in course of glucose utilization by medusomycete].

The inclusion of 2H and 13C isotopes into the products of glucose utilization by medusomycete during its growth on deuterated media was studied by high-resolution NMR spectroscopy. Both unlabeled and 13C-labeled (in positions 1, 2, 6) glucose was used. It was shown that the glucose utilization proceeds by the classical Embden-Meyerhof-Parnas pathway. The incorporation of deuterium to the methyl group of ethanol can occur only during glucose-fructose-6-phosphate and phosphoenolpyruvate--pyruvate conversion. None of these stages by themselves is responsible for the existing distribution of deuterium atoms. The maximum inclusion of deuterium to the methyl group is no more than two atoms for the first glucose fragment (C1-C2-C3) and no more than one, for the second fragment (C4-C5-C6). The methylene group of ethanol is more accessible for deuterons because the proton surroundings of carbon atoms C2 and C5 completely changes. It was concluded that the maximum proton exchange occurs at positions C2 and C5; at positions C1, the proton exchange is lesser, and at position C6 it is the least. It was also shown that about 10% C1-C3 of triose leave the glycolysis cycle and are used in other processes.

Carbon Isotopes↗
PubMed1972

Ferredoxin- and nicotinamide adenine dinucleotide-dependent H 2 production from ethanol and formate in extracts of S organism isolated from "Methanobacillus omelianskii".

S organism ferments ethanol to acetate and H(2) but grows poorly on ethanol unless the partial pressure of H(2) is kept low, as when it is grown in combination with an H(2)-utilizing methanogenic bacterium. The present study shows that S organism contains an alcohol dehydrogenase and a formate dehydrogenase, both of which require nicotinamide adenine dinucleotide (NAD) for activity. Hydrogen is evolved from NADH generated by these activities via a ferredoxin-dependent oxidation of NADH to NAD and H(2). NADH:NADP oxido-reductase activity was also demonstrated. The relationship of these activities to the growth of S organism is discussed.

Alcohol Oxidoreductases↗
PubMed2000

Transfection of C6 glioma cells with glia maturation factor upregulates brain-derived neurotrophic factor and nerve growth factor: trophic effects and protection against ethanol toxicity in cerebellar granule cells.

Glial cells play active roles in neuronal survival, as well as neuroprotection against toxic insult. Recent studies suggest that the brain protein glia maturation factor (GMF) is involved in intracellular signaling in glia. This study investigated whether or not GMF plays a role in the survival-promoting and neuroprotective functions of glia. C6 glioma cells were transfected in vitro with GMF utilizing an adenovirus vector. The transfected cells overexpressed GMF intracellularly, but did not secrete the protein. The conditioned medium (CM) was obtained from the GMF-transfected cells (CM-GMF) and tested on primary neuronal cultures, consisting of cerebellar granule cells (CGC). The CGC cultures were utilized because these cultures have a background level of cell death, and the survival-promoting, i.e. neurotrophic effect, of the CM could be tested. In addition, since CGC cultures are ethanol-sensitive (ethanol enhances neuronal death), the neuroprotective effect of the CM against ethanol-induced cell death was tested also. We demonstrated that the CM-GMF had an enhanced neurotrophic effect as well as an increased neuroprotective effect against ethanol-induced cell death compared to control CM obtained from untransfected C6 cells (CM-Mock) or CM obtained from cells transfected with an unrelated gene (CM-LacZ). Because neurotrophins have trophic and protective effects, we investigated whether GMF-transfection upregulated the expression of neurotrophins in C6 cells. RT-PCR verified that GMF-transfected C6 cells had increased mRNA levels for BDNF and NGF. Immunoblotting corroborated the RT-PCR results and indicated that CM-GMF contained greater concentrations of BDNF and NGF protein compared to CM-Mock and CM-LacZ. A soluble TrkB-IgG fusion protein, which selectively binds BDNF and prevents its binding to the neuronal TrkB receptor, eliminated the neurotrophic effect of CM-GMF; whereas anti-NGF antibody was ineffective in preventing this effect, suggesting that the neurotrophic effect was due to BDNF. On the other hand, both the TrkB-IgG fusion protein and anti-NGF reduced neuroprotection, suggesting that BDNF and NGF both contribute to the neuroprotective effect of CM-GMF. In conclusion, GMF upregulates the expression of BDNF and NGF in C6 cells, and these factors exert neurotrophic and neuroprotective functions on primary neurons.

Adenoviridae↗
PubMed2005

Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos.

The present study examines the effects of ethanol (ETOH), dimethyl sulfoxide (DMSO), and acetone on zebrafish embryos and the implications of the observed results on the use of these solvents to zebrafish early life stage tests. The embryos were exposed to different concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 0.05, 1, 1.5, and 2.0% v/v) of the respective solvents by diluting reagent-grade solvent with reconstituted water [DIN 38415-6-Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen. Deutsches Institute für Normung e.V]. The following endpoints were investigated (mortality, hatching rate, abnormalities, heart rate, and hsp 70 induction). No effect on survival was recorded for both acetone and DMSO even up to the highest concentration. On the other hand, embryos exposed to 1.5% and 2.0% ethanol showed a significant reduction in survival rate. No developmental defects occurred with any of the solvents at the 0.1% concentration. However, starting with 1.0%, weak to very pronounced abnormalities (weak pigmentation, edema, crooked bodies, eye defect, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the solvent type and the concentration used. Ethanol has been shown to be the most embryotoxic solvent while DMSO and acetone have comparably lesser effects. Heat shock protein 70 was induced by all solvents but at different concentration ranges. DMSO has been shown to be the most potent inducer of stress proteins. Based on the study, the chemicals tested here may be used as carrier solvents in the zebrafish embryo assay at levels below 1.5, 1.5, and 1% v/v for acetone, DMSO, and ethanol, respectively. For stress protein analysis of the exposed embryos, however, the solvent levels should be below 0.1%, 0.01%, and 1.5%, respectively. Additional and separate investigations utilizing other biomarkers should be carried out to further validate the suitability of using these solvents in a typical zebrafish embryo assay.

Abnormalities, Drug-Induced↗
PubMed1976

The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes.

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.

Acholeplasma laidlawii↗
PubMed1972

Isolation of Vi antigen and a simple method for its measurement.

Vi antigen was purified from saline extract of Citrobacter strain 5396/38 by enzymatic digestion, concentration with ethanol, and precipitation with hexadecyltrimethylammonium bromide. The Vi antigen prepared by this method was 18 and 230 times more protective in mice than two other preparations isolated by an earlier method utilizing acid hydrolysis. A sensitive and specific method of electroimmunodiffusion was described for measurement of Vi antigen.

Animals↗
PubMedDate not supplied

Strategies for reducing solvent toxicity in extractive ethanol fermentation.

Extractive fermentation is a widely preferred technique in which the products of fermentation are removed from the fermentation medium by a proper solvent, in order to avoid the inhibitory effects of the products. In this work, decanol, which has a high distribution coefficient with respect to the biocompatible solvents, was used in extractive ethanol fermentation. In order to reduce decanol toxicity, Saccharomyces cerevisiae cells were immobilized in calcium alginate gel. Further, sunflower oil and Al2O3 were added to the immobilization media. Experiments were performed in 250-mL Erlenmeyer flasks that were placed in the constant-temperature bath of a constant stirring-rate shaker. Ethanol concentrations were measured to observe the effect of various parameters on ethanol production. Immobilization media included 10, 20, and 30% sunflower oil, or 5, 10, and 20% Al2O3, or Al2O3 and sunflower oil together. The ratio of the volume of aqueous phase to that of decanol phase ranged from 2:1 to 6:1. It was observed that protection depends on the oil, Al2O3, and decanol amounts. Utilization of sunflower oil (30%) and Al2O3 (5%) together yielded best results.

Aluminum Oxide↗
PubMedDate not supplied

Cerebral metabolic alterations in rats following prenatal alcohol exposure: a deoxyglucose study.

Numerous reports dealing with the gross morphological, microanatomical, and behavioral changes occurring with prenatal alcohol exposure in rats have appeared. In order to define the cerebral metabolic correlates of prenatal alcohol exposure, in vivo glucose utilization rates in brain were assayed using an autoradiographic [14C]deoxyglucose technique. Pregnant Long-Evans rats were fed liquid diets containing either 35% or 0% ethanol-derived calories during day 6 through day 20 of gestation. These liquid diets were isocaloric and a pair-feeding procedure was employed. The offspring were tested on shuttle avoidance techniques at 90 days of age; at 105 days of age all animals were subjected to the deoxyglucose procedures. Prenatal alcohol exposure resulted in a deficit in shuttle avoidance and yielded chronic effects on brain metabolism. Significant decreases in glucose utilization were seen in many structures within sensory, motor, and limbic systems. Significant increases in glucose consumption were found in the hypothalamic-neurohypophysial axis. In summary long-term behavioral and brain metabolic alterations were seen in rats exposed prenatally to alcohol.

Animals↗
PubMed2004

A systems approach to preoperative surgical patient skin preparation.

BACKGROUND: The merits of a systems approach to preoperative surgical patient skin preparation are presented. A "system" may be defined as the addition of an antiseptic bath(s) or shower(s) within 24 hours before the application of a Food and Drug Administration-compliant patient preoperative skin preparation at the time of surgery. The expected outcome from this added effort is a further reduction in the transient and normal flora that contaminate the skin surrounding the operative site, and contribute to surgical infections. The Centers for Disease Control and Prevention has recommended the addition of this step to the preoperative site preparation regimen. METHODS: A human in vivo study comparing an alcohol-based (ethanol) system to an iodine-based approach, and 2 sets of clinical observations, are presented here. RESULTS: The data confirm the utility of the systems approach. The in vivo study demonstrates that significant reductions (> or =2 log(10)) in normal and transient flora can be achieved quickly and maintained out to 72 hours after the surgery, allowing for wound closure in a prolonged state of skin antisepsis. Clinical observations support the in vivo findings. After initiation of the system, the infection rates for the monitored procedures have decreased. CONCLUSIONS: The data presented here delineate the merits of a systems approach to preoperative surgical patient skin preparation. This approach has a positive impact on infection rates and may reduce the additional costs associated with this preventable event.

Anti-Infective Agents, Local↗
PubMed1996

Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants.

Kluyveromyces lactis mutants defective in the glycolytic enzyme phosphoglucose isomerase are able to grow in glucose media and to produce ethanol, but they depend on a functional respiratory chain and do not grow in glucose-antimycin media. We postulate that this is due to the necessity of reoxidizing, in the mitochondria, the NADPH produced by the pentose phosphate pathway, which may be highly active in these mutants in order to bypass the blockade in the phosphoglucose isomerase step. This oxidation would be mediated by a cytoplasmic-side mitochondrial NAD(P)H dehydrogenase that would pass the electrons to ubiquinone. Data supporting this hypothesis are provided.

Antimycin A↗
PubMedDate not supplied

[Thiamine and biotin requirements of yeasts growing on methanol and ethanol].

The thiamine and biotin requirement of eight methanol assmilating yeast cultures belonging to four genera was studied during their growth in media containing methanol or ethanol. All the cultures, regardless of the species, required biotin during their growth in media containing either of the carbon sources. The cultures required thiamine (or its pyrimidine moiety) only when they utilized methanol. The paper discusses possible mechanisms responsible for disorders in thiamine synthesis in yeast cultures growing in media with methanol.

Biotin↗
PubMed2006

Utility of near-infrared reflectance spectroscopy to predict nutrient composition and in vitro digestibility of total mixed rations.

Total mixed ration (TMR) samples (n = 110) were analyzed for dry matter (DM), crude protein (CP), soluble CP, neutral detergent fiber (NDF), NDF CP, starch, ash, fat, total ethanol-soluble carbohydrate, and nonfiber carbohydrate (NFC). Rapidly and slowly degraded and undegraded in situ CP fractions and in vitro DM, organic matter, and NDF digestibility were determined on each TMR. The TMR were scanned using near-infrared reflectance spectroscopy (NIRS); spectra were retained with NIRS calibration and cross-validation statistics were determined using partial least squares regression methods. The CP, NDF, starch, in vitro DM, and in vitro indigestible NDF contents of TMR were predicted by NIRS with good degrees (R2 >0.85) of accuracy with proportionally low standard errors of prediction. Moderate utility of NIRS to predict the NFC (R2 = 0.83) and fat content (R2 = 0.81) of TMR was observed. Rapidly, slowly, and undegraded in situ CP fractions in TMR were not well predicted by NIRS. Similarly, soluble CP, NDF CP, total ethanol-soluble carbohydrate, and in vitro NDF digestibility (% of NDF) were not well predicted by NIRS. Ratios of nutrient range to reference laboratory method error were calculated and found to be positively related (R2 = 0.84) to NIRS predictability of a given TMR nutrient, suggesting some laboratory procedures were not precise enough to yield suitable NIRS predictions. Data suggest that NIRS has utility to predict basic nutrients such as CP, NDF, starch, NFC, and fat in TMR. However, difficulty was observed using NIRS in predicting key biological nutrients in TMR such as in situ CP fractions and in vitro NDF digestibility. Difficulty of NIRS in predicting these nutrients is related to the level of reference method error in relationship to the range of nutrient values in TMR, but other sources of prediction error may exist.

Animal Feed↗
PubMed2002

New sample preparation technique for the determination of avoparcin in pressurized hot water extracts from kidney samples.

A new approach is presented for the determination of avoparcin in tissue. Complete recovery from spiked swine kidney was achieved with hot water modified with 30% ethanol (v/v). The samples were extracted at 75 degrees C and 50 atm by accelerated solvent extraction. In situ sample clean-up was achieved by using matrix solid-phase dispersion utilizing the acrylic polymer XAD-7 HP, and by adding triethylammonium phosphate (TEAP) to the extraction solvent. The aqueous extracts were concentrated by solid-phase extraction (SPE) on the hydrophilic interaction chromatography (HILIC) material polyhydroxyethyl aspartamide. Complete analyte retention was possible during SPE when the kidney extracts were modified with 70% ethanol. A 200 A, 5 microm HILIC column with UV225 detection was used for the separation of avoparcin. The retention time was less than 15 min with 47% aqueous component in acetonitrile and 15 mM TEAP as eluent. The average recovery of avoparcin from kidney samples was 108%.

Animals↗
PubMed1976

Desulfuromonas acetoxidans gen. nov. and sp. nov., a new anaerobic, sulfur-reducing, acetate-oxidizing bacterium.

Anaerobic sea or fresh water media with acetate and elemental sulfur yielded enrichments of a new type of strictly anaerobic, rod-shaped, laterally flagellated, Gram-negative bacterium. Three pure culture-strains from different sulfide-containing sea water sources were characterized in detail and are described as a new genus and species Desulfuromonas acetoxidans. The new bacterium is unable to ferment organic substances; it obtains energy for growth by anaerobic sulfur respiration. Acetate, ethanol or propanol can serve as carbon and energy source for growth; their oxidation to CO2 is stoichiometrically linked to the reduction of elemental sulfur to sulfide. Organic disulfide compounds, malate or fumarate are the only other electron acceptors used. Butanol and pyruvate are used in the presence of malate only; no other organic compounds are utilized. Biotin is required as a growth factor. The following dry weight yields per mole of substrate are obtained: in the presence of sulfur: 4.21 g on acetate, 9.77 g on ethanol; in the presence of malate: 16.5 g on acetate, 34.2 g on ethanol and 46.2 g on pyruvate. Accumulations of cells are pink; cell suspensions exhibit absorption spectra resembling those of c-type cytochromes (abs. max. at 419, 523 and 553 nm). Malate-ethanol grown cells contain a b-type cytochrome in addition. In the presence of acetate, ethanol or propanol, Desulfuromonas strains form robust growing syntrophic mixed cultures with phototrophic green sulfur bacteria.

Acetates↗
PubMed2004

Development of stable liquid formulations for adenovirus-based vaccines.

We have evaluated the stability profiles of adenovirus type-5 (Ad5)-based vaccine formulations to identify liquid formulations that are stable during long-term storage at 4 degrees C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non-ionic surfactants and cryoprotectants as well as excipients known to inhibit free-radical oxidation. A non-ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze-thaw-induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate-80 as the non-ionic surfactant, metal-ion catalyzed free-radical oxidation is an important mechanism of Ad5 inactivation. The free-radical oxidation inhibitors ethanol and histidine, combined with the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first-order inactivation mechanism with apparent activation energies for virus inactivation of 26.5 +/- 0.9 and 28.7 +/- 0.6 kcal/mol in the absence and presence of free-radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long-term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost <0.1 logs of infectivity after 24 months of storage at 4 degrees C. The immunogenicity of a recombinant Ad5-based human immunodeficiency virus (HIV) vaccine candidate expressing HIV-1 gag (MRKAd5gag) formulated in A195, was shown to be equivalent to the same vaccine formulated in A105. Therefore, the use of EDTA, ethanol, and histidine did not significantly alter the immunogenicity of the vaccine in mice. The identification of 4 degrees C stable liquid formulations should significantly enhance the utility of Ad5 as a vector for vaccines and gene therapy.

Adenoviruses, Human↗