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At least 883 records · Page 49Linked to original sources
PubMed1994

Specific proton pump inhibitors E3810 and lansoprazole affect the recovery process of gastric secretion in rats differently.

After a single subcutaneous administration (30 mg/kg) of proton pump inhibitor 2-[(4-(3-methoxypropoxy)-3-methylpyridin-2-yl)-methylsulfiny l]- 1H-benzimidazole sodium salt (E3810), or lansoprazole in rats, time courses of inhibitory and recovery processes of acid secretion in vivo and pump enzyme activity in isolated microsomes were measured. The acid secretion rate which reflects H+,K(+)-ATPase activity in the secretory canalicular (apical) membrane was compared with that in the microsomal fraction which consists mostly of resting, intracellularly-pooled tubulovesicles. We found that the canalicular pump was first inhibited, followed by slow inhibition of the microsomal pump enzyme activity, with the rate of the latter process depending on the inhibitors. It took 2.5 hr for the half-maximal inhibition of the microsomal pump in E3810-treated rats, and 6 hr in lansoprazole-treated rats. The acid secretion and the microsomal enzyme activity completely recovered within 48 hr after the administration of E3810, but recovered by only 20% even 96 hr after the administration of lansoprazole. Incubation with dithiothreitol of isolated microsomes obtained from E3810-treated rats reactivated the enzyme activity, but not from rats treated with lansoprazole. These results suggest that dissociation of inhibitor from the pump and/or intracellular transport of the pump is affected differently by these inhibitors. Furthermore, it is possible that the half life of the proton pump protein is much longer (greater than 96 hr) than the previously proposed value of 30-48 hr.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1984

Enzyme immunoassay for the detection of group A streptococcal antigen.

A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 10(4) or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 10(3) CFU per test. Heterologous microorganisms tested at 10(6) CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen.

Animals↗
PubMed2003

Exploitation of astrocytes by glioma cells to facilitate invasiveness: a mechanism involving matrix metalloproteinase-2 and the urokinase-type plasminogen activator-plasmin cascade.

The presence of reactive astrocytes around glioma cells in the CNS suggests the possibility that these two cell types could be interacting. We addressed whether glioma cells use the astrocyte environment to modulate matrix metalloproteinase-2 (MMP-2), a proteolytic enzyme implicated in the invasiveness of glioma cells. We found that astrocytes in culture produce significant amounts of the pro-form of MMP-2 but undetectable levels of active MMP-2. However, after coculture with the U251N glioma line, astrocyte pro-MMP-2 was converted to the active form. The mechanism of pro-MMP-2 activation in glioma-astrocyte coculture was investigated and was found to involve the urokinase-type plasminogen activator (uPA)-plasmin cascade whereby uPA bound to uPA receptor (uPAR), leading to the conversion of plasminogen to plasmin. The latter cleaved pro-MMP-2 to generate its active form. Furthermore, key components (i.e., uPAR, uPA, and pro-MMP-2) were contributed principally by astrocytes, whereas the U251N glioma cells provided plasminogen. In correspondence with this biochemical cascade, the transmigration of U251N cells through Boyden invasion chambers coated with an extracellular matrix barrier was increased significantly in the presence of astrocytes, and this was inhibited by agents that disrupted the uPA-plasmin cascade. Finally, using resected human glioblastoma specimens, we found that tumor cells, but not astrocytes, expressed plasminogen in situ. We conclude that glioma cells exploit their astrocyte environment to activate MMP-2 and that this leads to the increased invasiveness of glioma cells.

3T3 Cells↗
PubMedDate not supplied

Alterations in free radical scavenger system profile of type I diabetic rat brain.

The activities of the enzymes related to glutathione synthesis, degradation, and functions as well as reactive oxygen scavenging enzymes were analyzed in different brain regions, such as cerebral hemisphere, cerebellum, brainstem, thalamus, and hypothalamus after 1 and 3 mo of streptozotocin-induced diabetes in rats. Parallel studies were also made in age-matched control rats and insulin-treated diabetic rats. The content of glutathione (GSH) and its synthesizing enzyme gamma-glutamylcystein synthetase and also superoxide dismutase (SOD) and catalase activities (reactive oxygen scavenging enzymes) were significantly decreased from almost all the brain regions studied. However, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GTP), and glutamine synthetase (GS) activities were increased in the diabetic rat brain. Insulin treatment to the diabetic rats resulted in partial to full recovery in these enzymes activities. The present results emphasize the potentially serious alterations of brain free radical scavenger system in uncontrolled Type I diabetes.

Animals↗
PubMed1995

Evolutionary link between glycogen phosphorylase and a DNA modifying enzyme.

We report here an unexpected similarity in three-dimensional structure between glucosyltransferases involved in very different biochemical pathways, with interesting evolutionary and functional implications. One is the DNA modifying enzyme beta-glucosyltransferase from bacteriophage T4, alias UDP-glucose:5-hydroxymethyl-cytosine beta-glucosyltransferase. The other is the metabolic enzyme glycogen phosphorylase, alias 1.4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase. Structural alignment revealed that the entire structure of beta-glucosyltransferase is topographically equivalent to the catalytic core of the much larger glycogen phosphorylase. The match includes two domains in similar relative orientation and connecting helices, with a positional root-mean-square deviation of only 3.4 A for 256 C alpha atoms. An interdomain rotation seen in the R- to T-state transition of glycogen phosphorylase is similar to that observed in beta-glucosyltransferase on substrate binding. Although not a single functional residue is identical, there are striking similarities in the spatial arrangement and in the chemical nature of the substrates. The functional analogies are (beta-glucosyltransferase-glycogen phosphorylase): ribose ring of UDP-pyridoxal ring of pyridoxal phosphate co-enzyme; phosphates of UDP-phosphate of co-enzyme and reactive orthophosphate; glucose unit transferred to DNA-terminal glucose unit extracted from glycogen. We anticipate the discovery of additional structurally conserved members of the emerging glucosyltransferase superfamily derived from a common ancient evolutionary ancestor of the two enzymes.

Amino Acid Sequence↗
PubMed1996

Interaction of organophosphorus compounds with carboxylesterases in the rat.

Carboxylesterases (CarbE) are involved in detoxication of organophosphorus compounds (OPC) through two mechanisms: hydrolysis of ester bonds in OPC which contain them and binding of OPC at the active site of CarbE which reduces the amount of OPC available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was undertaken in order to contribute to better understanding of the role of CarbE in detoxication of OPC. The results obtained have shown that inhibitory potency (I50) of dichlorvos, sarin and soman towards rat liver CarbE was 0.2 microM, 0.5 microM and 4.5 microM, respectively, for 20-min incubation at 25 degrees C. Second-order rate constants (k(a)) for liver CarbE inhibition were 2.3 x 10(5) M-1 min-1, 6.9 x 10(4) M-1 min-1 and 1.1 x 10(4) M-1 min-1 for dichlorvos, sarin and soman, respectively. The corresponding values for plasma CarbE could not be calculated because of dominant spontaneous reactivation of inhibited CarbE. CarbE inhibited with these OPC in vitro spontaneously reactivate with half-times of 18, 143 and 497 min for sarin, dichlorvos and soman in plasma and 111, 163 and 297 min for sarin, soman and dichlorvos in liver, respectively. These results were also confirmed in experiments in vivo in which rats were subcutaneously treated with 0.5 LD50 of these agents. The half-times of spontaneous reactivation of rat plasma CarbE in vivo were 1.2, 2.0 and 2.7 h for dichlorvos, sarin and soman, respectively. These findings have changed current understanding of the mechanism of interaction of CarbE with OPC and involvement of the enzymes in detoxication of OPC, suggesting an active and important role of the enzymes in metabolic conversions of OPC to their less toxic metabolites.

Animals↗
PubMed1986

The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences.

We have examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S-adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways.

Adenine↗
PubMed1979

Biphasic kinetics of metal ion reactivation of trypsin-thiol complexes.

This report describes biphasic kinetic data obtained when trypsin was inhibited by a thiol-containing inhibitor present in Ehrlich ascites tumour cells and then subjected to addition of Hg2+, Cu2+ or Ag+. This resulted in an initial re-activation of the trypsin, followed by inhibition of the enzyme with the addition of higher concentrations of these ions. The significance of these observations is 2-fold: (i) help to elucidate the mechanism of metal ion activation of latent enzymes, and (ii) also indicate that, in certain circumstances, the concentration of added metal ion determines whether the metal acts as an activator or an inhibitor of enzyme activity.

Copper↗
PubMed1979

Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition.

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.

Animals↗
PubMed1999

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase.

BACKGROUND: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B12) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt-carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an alpha2beta2gamma2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase-cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. RESULTS: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers (alphabetagamma)2. The cobalamin molecule is bound between the alpha and beta subunits in the 'base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The alpha subunit includes a (beta/alpha)8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. CONCLUSIONS: This is the first crystallographic indication of the 'base-on' mode of cobalamin binding. An unusually long cobalt-base bond seems to favor homolytic cleavage of the cobalt-carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.

Amino Acid Sequence↗
PubMed1985

The calcium dependence of insulin degradation by rat skeletal muscle.

The effect of calcium on insulin degradation by intact or homogenized skeletal muscle, by skeletal muscle cytosol, and by partially purified skeletal muscle insulin-degrading protease activity was examined. After a 15-min lag phase, intact soleus muscles degraded [125I]insulin to trichloroacetic acid-soluble products in a time-dependent fashion. Degradation was accelerated by the addition of calcium (greater than or equal to 1 mM), such that maximal stimulation (2-fold) was obtained with 10 or 25 mM calcium. Calcium stimulated insulin degradation by skeletal muscle homogenate and by cytosol in a nearly identical manner. Furthermore, after inactivation of the purified skeletal muscle, insulin-degrading protease by dialysis against EDTA, this enzyme was reactivated fully (greater than 80%) by a 100 microM concentration of free Ca2+. These observations identify a previously unrecognized but important influence of calcium on cellular insulin handling and provide further evidence for a major role of the calcium-activated enzyme, insulin protease, in cellular insulin degradation.

Animals↗
PubMed1993

Enzyme changes in remodelling epithelial cells: a histochemical study of the rat jejunum in vivo during and following exposure to deoxycholic acid.

Loops of rat jejunum were exposed in vivo to different concentrations of deoxycholic acid (DOC; 0, 2.5, 5, 10 and 20 mM). Following a 30 min exposure period, DOC was washed out of the loops and the intestines were allowed to recover for 15 or 150 min. Frozen tissue for enzyme histochemistry was collected during exposure and following the recovery periods. As shown previously, exposure to DOC caused a dose-dependent loss of epithelial cells at the villous tips and denudation of the lamina propria. Flattened epithelial cells bordering the denuded areas were, however, responsible for a rapid restoration of epithelial continuity, which was completed within 15 min. In the present study, these flattened cells showed normal reactivity for non-specific esterase and succinate dehydrogenase. In contrast, following a prolonged recovery period (150 min), a subpopulation of enterocytes at the villous tips that otherwise appeared normal showed decreased reactivity for brush border enzymes and non-specific esterase, and a positive reaction for mucin. A shutdown in the synthesis of cytoplasmic enzymes and redistribution of cell surface enzymes could be responsible for these late occurring enzyme changes, that were consistently observed after 150 min of recovery from DOC at 20 mM. Alternatively, retention of goblet cells and/or a modification in enzyme synthesis may explain the presence of mucin that was demonstrated in the epithelial cells which had low enzyme reactivity.

Alkaline Phosphatase↗
PubMed1984

Monoclonal antibodies to human terminal transferase.

Human terminal deoxynucleotidyltransferase was used to produce mouse monoclonal antibodies. Three different assays were used to screen for terminal transferase antibodies and antibody-producing hybridoma cells: an enzyme binding assay, an enzyme-linked immunosorbent assay, and a "terminal transferase-specific antibody" cytochemical procedure that allows visualization of the homogeneity of the hybridoma clones producing antibodies to terminal transferase. Of the 12 mouse hybridoma clones isolated, seven produce IgG1 and five produce IgG2a immunoglobulins. Only one of these monoclonal antibodies is inhibitory to human terminal transferase activity. Four of the monoclonal antibodies react with terminal transferase peptides after separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the remainder appear to recognize only native determinants on the human enzyme. Cross-reactivity studies with purified calf thymus enzyme show that although four of the monoclonal antibodies bind to native enzyme, none react with calf thymus terminal transferase peptides after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting structural differences within the cross-reactive determinants. A more general survey on the affinity of these monoclonal antibodies to human terminal transferase for the enzyme from other species using an enzyme-linked immunosorbent assay demonstrates that none of the monoclonal antibodies reacts with chicken thymus terminal transferase. The affinity of binding for terminal transferase from other mammals to this panel of monoclonal antibodies varies with the clones as expected, but few bind as strongly as the human enzyme. These results suggest that although antigenic determinants are conserved through evolution, fine structural changes within the determinants do occur.

Animals↗
PubMed1992

Hepatitis C virus RNA and antibody response in the clinical course of acute hepatitis C virus infection.

Hepatitis C virus RNA, anti-hepatitis C virus immune response and biochemical markers of liver injury were investigated in 17 patients with acute non-A, non-B hepatitis. At the first observation, 1 to 3 wk from the clinical onset, all patients had hepatitis C virus RNA in their serum, and most (15 of 17) were positive for second-generation anti-hepatitis C virus enzyme immunoassay. Follow-up serum samples were available for 10 patients. The rate of recombinant immunoblot assay-confirmed anti-hepatitis C virus enzyme immunoassay reactivities increased from 67% in the first 3 wk to 86% after 21 wk. Elevated ALT levels were associated with hepatitis C virus RNA positivity in most of cases, but the viral nucleic acid was also detected in sera with normal or slightly increased enzyme values. None of the single antibodies tested were related to hepatitis C virus RNA positivity or to the clinical phase of the infection. Therefore hepatitis C virus RNA determination might provide important additional information as compared with anti-hepatitis C virus markers, allowing earlier diagnosis, discrimination of active infection and, possibly, prognostic evaluation.

Acute Disease↗
PubMed1998

Cytidine monophosphate-N-acetylneuraminate hydroxylase in the starfish Asterias rubens and other echinoderms.

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesised by an NADH-dependent hydroxylase which acts on CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Although Neu5Gc is the predominant sialic acid in many echinoderms, little is known about the hydroxylase from organisms of this phylum. We show here that in contrast to the mammalian enzyme, the hydroxylase from various echinoderms is predominantly membrane-bound and exhibits optimal activity in the presence of 100 mM NaCl. A detailed characterisation of the hydroxylase from echinoderms was performed using fractionated gonads of the starfish Asterias rubens. Solubilisation using detergents led to an inactivation of the hydroxylase. However, the solubilised enzyme was reactivated by the addition of cytochrome b5 reductase together with the amphiphilic or soluble form of cytochrome b5. Although these latter proteins were only available from a mammalian source, the high affinity of the hydroxylase for cytochrome b5 suggests that, as with the mammalian enzyme, these electron carriers participate in the catalytic cycle of the hydroxylase from A. rubens in vivo. The relevance of these results to the interaction between cytochrome b5 and the hydroxylase is discussed.

Animals↗