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PubMed2003

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation.

The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.

Animals↗
PubMed1986

Application, limitations and research requirements of in vitro test systems in toxicology.

There is increasing application of in vitro-test systems for toxicological evaluation of chemicals, which became possible by increasing understanding of the biological endpoints present in such systems and their capability for metabolic activation and inactivation. This communication focuses on the capacities of metabolic activation and inactivation in mutagenicity test systems, using bacteria, mammalian cells in culture and isolated hepatocytes. Bacterial test procedures with S-9-fraction are specific metabolic activation systems with low inactivation capacity. Mammalian cells are either deficient in metabolic activities or contain only limited activation capacity although special cell lines derived from hepatoma cells express certain metabolic activation as well as inactivation reactions. Isolated hepatocytes contain enzymatic activities similar to those in the intact liver, which however decrease at different rates. It is the goal of present research to construct cell lines with defined and sufficient activities of these enzymes. In isolated hepatocytes chemically induced DNA repair can be readily detected when a clear discrimination between mitochondrial, semiconservative and repair synthesis is provided. In such systems genotoxicity of reactive oxygen species is decreased by physiological concentrations of alpha-keto-acids, pyruvate possessing the highest antioxidant activity. It is concluded that in vitro test systems provide a suitable tool for detecting genotoxic and toxic effects of chemicals. However, many biological parameters such as metabolic activity, degree of differentiation of the cells, cofactor requirement, and composition of the medium affect the reliability of the test system. Thus, only a detailed understanding of the biology and biochemistry of such test allow production of reliable and reproducible results.

Animals↗
PubMed1981

Systemic carnitine deficiency with peripheral nerve involvement morphological and biochemical study.

The patient, a 31-year-old woman, suffered from a weakness of the proximal muscles which subsequently worsened involving the spinal and neck muscles. During the hospitalization, the patient displayed a "burning feet" syndrome. Lab tests showed a moderate increase in CPK, Aldolase, SGPT, SGOT, lactic and pyruvic acids. Needle EMG was interpreted as "myopathic" but a marked reduction of sensitive action potentials was also detected. Muscle biopsy showed neutral lipid accumulation in muscle fibers, many small angular fibers, probably denervated ones were also evident. A liver biopsy showed fatty accumulation in hepatocytes. Free carnitine was extremely reduced in muscle and liver and just below normal level in plasma while there was a tenfold elevation of long-chain carnitine esters in plasma too. The mechanism by which carnitine deficiency can cause both neural and muscular alterations and in particular the role of carnitine in glucose metabolism is discussed.

Adult↗
PubMed1988

Experimental studies on the mechanism of ethanol formation in corpses.

Various in vitro experiments were performed for the purpose of clarifying the mechanism of ethanol production in corpses. Whereas a negligible quantity of ethanol was produced in the blood alone, which was left at room temperature, the quantity of ethanol was slightly increased by addition of glucose to the blood. When saprogens were further added, the quantity was markedly increased. Various materials were added to blood-liver homogenates as specimens, and the mixtures were stored in an incubator at 37 degrees C. As a result of the addition of an antibiotic to the mixture every day, there was hardly any production of ethanol. When alcohol dehydrogenase (ADH) and reduced nicotinamide adenine dinucleotide (NADH) were added, ethanol production was slightly increased. When acetaldehyde was added first, ethanol production was inhibited the next day, but on and after day 2, the quantity of ethanol was more than that in the control material. When pyruvic acid was added first, the results were similar to the above. Pyrazole, cyanamide, and disulfiram completely inhibited the production of ethanol. Ethanol production in corpses is believed to take place through a pathway opposite to that of ethanol metabolism in the living body, under the influence of ADH, ALDH, etc., in saprogens using carbohydrates as substrates.

Acetaldehyde↗
PubMed1991

Neutrophil function in hyperosmotic NaCl is preserved by phosphoenol pyruvate.

Hyperosmolarity in the urinary tract inhibits the host defense against bacterial infection. NaCl contributes most to osmolarity in the renal medulla and urine. Therefore, we studied the effect of hyperosmolar NaCl on neutrophil function. When osmolarity was increased by NaCl, polymorphonuclear leukocytes (PMNL) became defective in phagocytosis, intracellular killing of bacteria, chemotactic activity, and superoxide production. This coincided with a decrease in the intracellular adenosine triphosphate (ATP) content of PMNL. Both the inhibition of superoxide production and the reduction in ATP content did not occur at 4 degrees C. By increasing ATP content, phosphoenol pyruvic acid (PEP) protected against the decrease in superoxide production. These results suggest that leukocyte function is inhibited by high concentrations of NaCl due to the activation of the Na(+)-K+ pump. PEP, an ATP precursor, can protect PMNL against osmotic stress by raising the intracellular concentration of ATP.

Adenosine Triphosphate↗
PubMed1981

[Study of the valuations of serum muscular enzymes and myoglobin after maximal exercise test and during the next 24 hours (author's transl)].

Twenty-eight male subjects underwent a maximal exercise test on an ergometric bicycle, 13 untrained and 15 trained subjects. At rest and after this maximal exercise, indicated by the increase of the serum lactate and pyruvate acids, venous blood samples were taken to study the release of muscular enzymes (SGOT, SGPT, LDH, alpha HBDH, CPK, CPK MB) and myoglobin in the next 24 h (3 min, 30 min, 8 h and 24 h after the end of this test). The statistic analysis of the results shows significant increases in comparison with the values at rest, according to the enzymes and the groups of subjects, especially for CPK, SGOT, SGPT for which the results are homogeneous with a peak at the 3rd min and the 8th h in the three groups and for HBD and LDH in the groups of trained subjects. However these values remain most often within the normal limits. These results are compared with those of previous works on the same subjects.

Adult↗
PubMed1976

A constitutive mutation in a posttranscriptional regulator gene for a phenoloxidase and proteases in Podospora anserina.

A recessive mutation in the gene mod-2 results in the synthesis at low temperatures of a phenoloxidase and an arrest of growth, reversible by beta-phenyl-pyruvic acid, a protease inhibitor. Phenoloxidase synthesis is 5-fluorouracil resistant and cycloheximade sensitive. Suppression of both cold sensitivity and phenoloxidase synthesis by common factors (higher NH4+ concentrations or mutations) suggests that the protease, suspected to be responsible for cold sensitivity, also arises from preexisting mRNA molecules. Instead of being recessive and constitutive, the mod-2 mutations is suppressive and dominant when cold sensitivity and phenoloxidases synthesis are induced as the consequence of the nonallelic gene interactions C/D, C/E, or R/V responsible for protoplasmic incompatibility. Combinations of nonallelic incompatibility systems and several mod-2 mutations lead us to hypothesize that the translational control of the above proteins depends on conformational relationships between incompatibility gene products and mod-2.

Ammonia↗
PubMed1990

Biosynthesis of salsolinol, a tetrahydroisoquinoline alkaloid, in healthy subjects.

The R enantiomer of salsolinol was detected in the urine of two out of six healthy subjects, whereas 1,2-dehydrosalsolinol was present in the urine of all the subjects. (S)-salsolinol was never detected. Administration of Madopar for 7 days resulted in the presence of large amounts of (R)- and (S)-salsolinol in the urine of five out of the six subjects, the urinary excretion of 1,2-dehydrosalsolinol being generally not markedly increased. The presence of 1,2-dehydrosalsolinol in urine suggests that the biosynthesis of salsolinol in healthy volunteers should occur by condensation of dopamine with pyruvic acid, in keeping with Hahn's hypothesis. The absence of salsolinol in the urine of one subject after Madopar administration seems to indicate that the biological system(s) involved in the reduction of the C = N bond in 1,2-dehydrosalsolinol can be missing or not, or poorly, functional in some individuals, and suggests that there is no alternative pathway for the formation of salsolinol in healthy volunteers.

Adult↗
PubMed1996

Production of an extracellular polysaccharide by Agrobacterium sp DS3 NRRL B-14297 isolated from soil.

A bacterium isolated from soil and identified as Agrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.5:2.4:1, together with 3.7% (w/w) pyruvic acid. Methylation analyses showed the presence of (1-->3)-, (1-->4)- and (1-->6)-linked glucose, (1-->3)- and (1-->4, 1-->6)-linked galactose and a small portion of (1-->3)-linked mannose residues. Succinic acid was not present. The molecular weight of the polysaccharide was estimated by light scattering to be 2 x 10(6) Da. The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55 degrees C. Maximum yield of the polysaccharide, 20 g L-1, was reached in 48 h at 30 degrees C incubation.

Carbohydrate Conformation↗
PubMed1980

The radiolysis of aqueous acetonitrile: compounds of interest to chemical evolution studies.

Oxygen-free aqueous solutions of CH3CN (0.1 M, pH 6) were exposed to gamma rays from a 60Co source, the mixture of nonvolatile radiolytic products was fractionated and the fractions were analysed. Succinic, maleic, fumaric, malonic and pyruvic acids were identified. Glycol aldehyde, glucose and probably ribose were observed in the hydrolysate of fractionated material. It has been suggested that an oligomer is formed which has a fragment with the polyhydroxy structure and on hydrolysis releases the carbohydrates. Radiolytic products which release amino acids on hydrolysis were found in several fractions. The amino acid contents of the hydrolysates were up to about 2.8% of the fraction mass. The presence of several protein and nonprotein amino acids suggests that their origin should be in a peptidic structure, which is probably a fragment of an oligomer radiolytically produced. A direct analysis of the irradiated solution shows the presence of acetaldehyde, propionaldehyde, glyoxal and of biacetyl. Experimental findings are discussed and a free-radical mechanism is proposed to account for the chemical changes observed.

Acetonitriles↗
PubMed1993

Toxicokinetics of diethylene glycol (DEG) in the rat.

Oral doses of 1 and 5 ml/kg 14C-diethylene glycol (DEG) given to rats were rapidly and almost completely absorbed, the invasion constants being 2.95 h-1 and 4.24 h-1. The kinetics of invasion were determined with the method of residuals (Rowland and Tozer 1989) and by reconstruction of the invasion curves according to Kübler (1970). 14C-DEG was rapidly distributed from the blood into the organs and tissues in the order kidneys > brain > spleen > liver > muscle > fat, i.e. the same order as the blood flow. The relative volume of distribution, app. VD, was determined at 298 ml, indicating distribution over the whole body. After oral doses of 1, 5, and 10 ml 14C-DEG/kg 64, 87, and 91% of 14C activity in rat blood disappeared in 12-16 h with a half-life of 3.4 h and the remaining 9, 5, and 4% with half-lives of 39 h, 45 h, and 49 h. A total of 73-96% of 14C activity in blood was excreted with the urine and 0.7-2.2% with the faeces. From the cumulative urinary excretion kinetics half-lives of 6 h were determined for doses of 1 and 5 ml/kg and 10 h for the dose of 10 ml/kg. After doses of 5 ml/kg and 10 ml/kg 14C-DEG semi-logarithmic plots of elimination rate versus time were constant for 5 and 9 h, respectively, indicating that DEG accelerated its renal elimination by inducing osmotic diuresis. Thereafter urinary excretion followed first order kinetics with elimination half-lives of 3.6 h. After oral doses of 5 ml/kg 14C-DEG given to rats of 336 g body weight with an app. VD of 297 ml, the total clearance of 14C activity was determined at 63 ml/h, and the renal clearance of unmetabolized DEG was 66 ml/h. The ratio of ClDEG to Cl(inulin) = 0.64 indicated that DEG and its metabolite 2-hydroxyethoxyacetate (2-HEAA) were reabsorbed from the tubuli into the blood capillaries. DEG produced metabolic acidosis, which was completely balanced after doses of 1 and 5 ml/kg, but doses greater than 10 ml/kg produced non-compensated metabolic acidosis, hydropic degeneration of the tubuli, oliguria, anuria, accumulation of urea-N, and death in uraemic coma.

Administration, Oral↗
PubMed1976

Regulation of proteolytic enzymes in Podospora anserina: selection and properties of self-lysing mutant strains.

Previous results showed that cell disintegration in the fungus Podospora anserina occured through the action of two proteases, enzymes whose messengers were normally latent during the extension stage of the thallus. We selected three mutant strains in which the constitutive activity of the protease messengers was expressed by an arrest of growth early in development (10 to 30 hours after spore germination) and a reaction of cell disintegration, in the thallus, suppressible with beta-phenyl pyruvic acid, a protease inhibitor. The mutant character is recessive in one strain. In the case of the two strains in which the mutant trait is dominant, reversion studies have revealed that the deregulation resulted from the specific interaction between two genes and we have succeded in creating two non allelic incompatibility systems comparable to the non allelic gene interactions responsible for the incompatibility phenomena found between wild type races. We know, on the whole, that 11 loci are involved in the regulation of the proteases: five were revealed as incompatibility loci and six were discovered from investigations on four self-lysing mutant strains. It is suspected that all these genes act at the post-transcriptionnal level of the synthesis of specific proteolytic enzymes. We propose that the products of two genes act as "repressors" to prevent the protease messengers from being constitutively translated and that the products of the nine remaining genes exert a positive control by inducing translation, at the appropriate time, through the action of effectors resulting from specific interloci cooperation.

Alleles↗
PubMed1981

Explant culture of rat esophagus in a chemically defined medium.

Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, beta-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [3H]thymidine into DNA and [3H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [3H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30 degrees C, and in either 50 or 20 % 02. Ninety-five percent O2 was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.

Animals↗
PubMed2006

Genetic mapping of sulfur assimilation genes reveals a QTL for onion bulb pungency.

Onion exhibits wide genetic and environmental variation in bioactive organosulfur compounds that impart pungency and health benefits. A PCR-based molecular marker map that included candidate genes for sulfur assimilation was used to identify genomic regions affecting pungency in the cross 'W202A' x 'Texas Grano 438'. Linkage mapping revealed that genes encoding plastidic ferredoxin-sulfite reductase (SiR) and plastidic ATP sulfurylase (ATPS) are closely linked (1-2 cM) on chromosome 3. Inbred F(3) families derived from the F(2 )population used to construct the genetic map were grown in replicated trials in two environments and bulb pungency was evaluated as pyruvic acid or lachrymatory factor. Broad-sense heritability of pungency was estimated to be 0.78-0.80. QTL analysis revealed significant associations of both pungency and bulb soluble solids content with marker intervals on chromosomes 3 and 5, which have previously been reported to condition pleiotropic effects on bulb carbohydrate composition. Highly significant associations (LOD 3.7-8.7) were observed between ATPS and SiR Loci and bulb pungency but not with bulb solids content. This association was confirmed in two larger, independently derived F(2) families from the same cross. Single-locus models suggested that the partially dominant locus associated with these candidate genes controls 30-50% of genetic variation in pungency in these pedigrees. These markers may provide a practical means to select for lower pungency without correlated selection for lowered solids.

Base Sequence↗
PubMed2006

Overexpressing GLT1 in gpd1Delta mutant to improve the production of ethanol of Saccharomyces cerevisiae.

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.

Biomass↗