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Biomedical subjects

A Kotyk

Publications and source records attributed to A Kotyk.

At least 19 recordsLinked to original sources
PubMed2006

Positive effect of oral supplementation with glycosaminoglycans and antioxidants on the regeneration of osteochondral defects in the knee joint.

The effect of oral supplementation with glycosaminoglycans (GAG) and radical scavengers (vitamin E/selenium) on the regeneration of osteochondral defects was investigated in rabbits. After introduction of defined osteochondral defects in the knee joint, groups of ten animals were given a GAG/vitamin E/selenium mixture or a placebo (milk sugar) for 6 weeks. Following sacrifice, histological and histochemical analysis was performed. The amount of synovial fluid was increased in the placebo group, while the viscosity of the synovial fluid was significantly enhanced in the GAG group. The amount of sulfated GAG in the osteochondral regenerates (8.8 +/- 3.6 % vs. 6.0 +/- 5.6 %; p <0.03) was significantly higher in the GAG group. In both groups, the GAG amount in the cartilage of the operated knee was significantly higher than in the non-involved knee (p <0.05). Histological analysis of the regenerates in the GAG group was superior in comparison with the placebo group. For the first time, a biological effect following oral supplementation with GAG was demonstrated in healing of osteochondral defects in vivo. These findings support the known positive clinical results.

Administration, Oral↗
PubMed2006

Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs.

Lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) electrophoretic tissue patterns of two different orders of Elasmobranchii: Carchariniformes (Galeus melanostomus and Prionace glauca) and Squaliformes (Etmopterus spinax and Scymnorinus licha) were studied. The number of loci expressed for these enzymes was the same of other elasmobranch species. Differences in tissue distribution were noted in LDH from G. melanostomus due to the presence of an additional heterotetramer in the eye tissue. There were also differences in MDH. In fact, all the tissues of E. spinax and G. melanostomus showed two mitochondrial bands. Major differences were noted in the number of isozymes detected in the four compared elasmobranchs. The highest polymorphism was observed in E. spinax and G. melanostomus, two species that live in changeable environmental conditions. The resistance of isozymes after urea treatment was examined; the resulting patterns showed a quite good resistance of the enzymes, higher for LDH than MDH, also at urea concentration much greater than physiological one. These results indicated that the total isozyme resistance can be considered higher in urea accumulators (such as elasmobranchs) than in the non-accumulators (such as teleosts).

Adaptation, Physiological↗
PubMed2004

Role of polyols in thermal inactivation of shark ornithine transcarbamoylase.

The ability of activity modulators of ornithine transcarbamoylase (OCT) from the liver of the thresher shark Alopias vulpinus to stabilize the enzyme against thermal denaturation was investigated in the tri-buffer at pH 7.8, at temperatures ranging from 60 to 70 (o)C, in the presence of polyhydroxylic molecules such as glycerol and sugars. The study indicated that in the presence of 0.5 M sugars and 1.6 M glycerol in the preincubation medium the OCT activity increases. When trehalose is introduced directly in the reaction mixture in a range of concentration of 0.25-0.5 M, the activity is lower than that with maltose, glycerol and buffer alone. Kinetic data for carbamoyl phosphate and ornithine with and without maltose and glycerol are similar, whereas trehalose increases the kinetic values. Arrhenius plots show an increase of activation energy due to trehalose, whereas values obtained with maltose and glycerol are similar to the control.

Animals↗
PubMed2002

Purification and properties of ornithine carbamoyltransferase from loggerhead turtle liver.

Ornithine carbamoyltransferase has been purified from the liver of the loggerhead turtle Caretta caretta by a single-step procedure using chromatography on an affinity column to which the transition-state analogue, delta-N-(phosphonoacetyl)-L-ornithine (delta-PALO), was covalently bound. The procedure employed yielded an enzyme which was purified 373-fold and was judged to be homogeneous by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed a specific activity of 224. The molar mass of the C. caretta enzyme was approximately 112 kDa, the single band obtained by SDS-PAGE indicated a subunit molar mass of 39.5 kDa; hence, the enzyme is a trimer of identical subunits. It catalyzes an ordered sequential mechanism in which carbamoyl phosphate binds first, followed by L-ornithine. The Michaelis constants were 0.858 mM for L-ornithine and 0.22 mM for carbamoyl phosphate, the dissociation constant of the enzyme-carbamoyl phosphate complex was 0.50 mM.

Animals↗
PubMed2001

Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H(+)-ATPase in activated and basal-level enzyme preparations.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolizing cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Using two-dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non-active, i.e. non-phosphorylated, and the active, i.e. phosphorylated, forms of the enzyme. Previous deliberations suggesting that the B-ATPase displays some activity which is lower than that of A-ATPase were apparently wrong. It seems that, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms. On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations.

Cell Membrane↗
PubMed2001

Subcellular shifts of trimeric G-proteins following activation of baker's yeast by glucose.

Addition of glucose to a resting cell suspension of the yeast Saccharomyces cerevisiae was accompanied by marked shifts of the G alpha-protein subunits from the plasma membrane to the cell interior. This process was rapid with half-times between < 10 and 20 s. The decrease of the plasma membrane pool of the Gi alpha/Go alpha- and Gq alpha/Gl 1 alpha-protein subunits correlated with an increase in acid-sensitive forms of these proteins which was recovered in the mitochondrial and/or lysosomal membrane fraction. In contrast to cells from higher organisms glucose-stimulated yeast exhibits an extremely rapid type of the redistribution (internalization). The question remains open as to the functional significance of the internalized forms of the G-proteins as these remain sequestered from the plasma membrane well after glucose has been consumed.

GTP-Binding Proteins↗
PubMed2001

Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization.

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

Alleles↗
PubMed2000

Effects of a ferrate-containing preparation on diverse metabolic processes in yeast.

A plant-sap-derived preparation containing bi- and tervalent ferrate anions was tested on growth, respiration on glucose, and membrane transport of 6-deoxy-D-glucose (6-dGlc) and 2-aminoisobutyric acid (Aib) in several yeast species, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Lodderomyces elongisporus, Rhodotorula gracilis, and Dipodascus magnusii. Growth was enhanced by as much as 65%, respiration was not affected significantly except for a decrease in R. gracilis, transport of 6-dGlc was not affected while that of Aib was increased by up to 45% in R. gracilis and up to 27% in L. elongisporus.

Aminoisobutyric Acids↗
PubMed2000

Effects of the Fenton reagent on transport in yeast.

In the facultatively anaerobic yeast Saccharomyces cerevisiae the uptake rate and the accumulation ratio of 2-aminoisobutyric acid was decreased by some 30% by Fenton's reagent (FR), a powerful source of OH. radicals. Likewise, the uptake of glutamic acid, leucine and arginine was diminished. The mediated diffusion of 6-deoxy-D-glucose was not affected. The H+ symport of maltose and trehalose was inhibited by some 40% both in the initial rate and in the accumulation ratio. FR had a dramatic inhibitory effect when present during preincubation with 50 mmol/L glucose. In the obligately aerobic Lodderomyces elongisporus the uptake of all amino acids tested was decreased by 15-30%, that of 6-deoxy-D-glucose by about 10%. The initial rates of uptake of maltose and trehalose were depressed by FR by 40% and the acceleration of uptake observed after 8 min of incubation, was abolished by FR completely. Acidification rate of the external medium by S. cerevisiae in the presence of glucose or galactose was enhanced three-fold, that after subsequently added K+ was substantially decreased. FR appears to have a dual effect on sugar and amino acid transport processes in yeast: (1) it blocks carrier protein synthesis; (2) it inhibits the source of energy for transport. It does not appreciably affect the carrier proteins themselves.

Amino Acids↗
PubMed2000

Two forms of yeast plasma membrane H(+)-ATPase: comparison of yield and effects of inhibitors.

Classical isolation procedure for plasma membrane H(+)-ATPase of Saccharomyces cerevisiae based on fractional centrifugation yielded always a roughly two-fold greater amount of membranes when starting from glucitol-preincubated than from glucose-preincubated yeast. This difference persisted all the way to the purified plasma membranes and to the purified H(+)-ATPase. The ATP-hydrolyzing activity by plasma membranes was roughly twice greater in glucose-preincubated cells than in the D-glucitol-preincubated ones while the purified enzyme was 7 times more active after glucose than after glucitol. Effects of diethylstilbestrol, suloctidil, erythrosin B, vanadate and dicarbanonaboranuide were very similar on plasma membrane-localized and purified ATPases of both forms, suggesting that both preparations contain the two ATPase forms, the glucose-preincubated one being richer in the activated form while the glucitol-preincubated one contains less of it.

Adenosine Triphosphate↗
PubMed1999

Glucose- and K(+)-induced acidification in different yeast species.

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced in Saccharomyces cerevisiae and Schizosaccharomyces pombe but is very much lower in Lodderomyces elongisporus, Dipodascus magnusii and Rhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H(+)-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains of S. cerevisiae and in S. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K+ is observed, the effect of K+ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H(+)-ATPase-caused acidification.

Acids↗
PubMed1999

2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy.

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.

Argon↗
PubMed1998

Erythrosin 5'-isothiocyanate labels Cys549 as part of the low-affinity ATP binding site of Na+/K+-ATPase.

The high-affinity E1ATP site of Na+/K+-ATPase labeled with fluorescein 5'-isothiocyanate and its E2ATP site labeled with erythrosin 5'-isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813-28821], reside on separate and adjacent catalytic alpha subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549 residue in the tryptic fragment with the sequence Val545-Leu-Gly-Phe-Cys549-His550. Hence, Cys549 is part of or close to the low-affinity E2ATP binding site of Na+/K+-ATPase.

Adenosine Triphosphate↗
PubMed1998

Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolising cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Structure of the two enzyme forms and the effects of beta, gamma-imidoadenosine 5'-triphosphate (AMP-PNP) and of diethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. IR spectra revealed the presence of two populations of alpha-helices with different exposure to the solvent in both the A-ATPase and B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-helix populations. Thermal denaturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while DES destabilised both enzyme forms. IR spectra showed that 60% of the amide hydrogens were exchanged for deuterium in both forms at 20 degrees C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55 degrees C for both enzyme forms, while in the presence of DES it was observed at lower temperatures. The data do not contradict the possibility that the activation of H(+)-ATPase is due to the C-terminus of the enzyme dissociating from the ATP-binding site which is covered by it in the less active form.

Adenylyl Imidodiphosphate↗
PubMed1998

Extracellular acidification by Saccharomyces cerevisiae in normal and in heavy water.

Titratable acidity of the extracellular medium was compared with that calculated from pH changes in a suspension of Saccharomyces cerevisiae. After addition of cells to normal water the ratio of titratable acidity to the computed one was about 25, after addition of 50 mmol/L D-glucose it was about 13, after subsequent addition of K+ ions it was only 2. In heavy water the respective values were 30, 9, and 1. Apparently, the principal buffer-generating processes have to do with glucose metabolism but little with the K+/H+ exchange observed after addition of K+. D2O appears to block processes producing the buffering capacity of the medium, among them possibly extrusion of organic acids.

Acids↗
PubMed1998

Univalent cation fluxes in yeast.

Transport of H+, K+, Rb+ and Tl+ ions was studied in a wild-type strain of Saccharomyces cerevisiae and in its mutants defective in the high-affinity K+ transport system TRK1 and in the double mutant with an additional deletion in the TRK2 gene. In the absence of glucose K+, Rb+ and Tl+ elicited a more or less stoichiometric exchange outflow of H+, in the mutants K+ moved out of cells even in the presence of 10 mM KCl or KNO3. In the presence of glucose in the wild type, K+, Rb+ and Tl+ brought about a massive outflow of H+ while being transported inward against high concentration gradients. In the trk1 delta mutant the exchange fluxes were reduced by 65-85%, in the double mutant those of K+, Rb+ and Tl+ practically cease but outflow of H+ caused by Tl+ remained at the level of the trk1 delta mutant. It appears that, in addition to the H+ export by the PMA1-coded plasma membrane H(+)-ATPase, at least three different univalent-cation involving activities are present: the high-affinity transport system for K+ (TRK1), another system (possibly TRK2) with different responses to K+ and Rb+, vs. Tl+, and an active system for K+ export. The first two are apparently active exchange systems for K+, Rb+, and Tl+ against H+. The source of energy for these highly active transports (acting against gradients of 1000:1 and 5000:1, respectively) is unclear.

Carrier Proteins↗