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Biomedical subjects

G L Mendz

Publications and source records attributed to G L Mendz.

At least 37 records · Page 2Linked to original sources
PubMed1997

Purine metabolism and the microaerophily of Helicobacter pylori.

The requirements for purine nucleotide synthesis, the effects of purine analogues, and the metabolism of adenine in the bacterium Helicobacter pylori were investigated employing cell culture techniques and one-dimensional NMR spectroscopy. Bacterial cells grew and proliferated in media lacking preformed purines, indicating that H. pylori can synthesize purine nucleotides de novo to meet its requirements. Blocking of this pathway in the absence of sufficient preformed purines for salvage nucleotide synthesis led to cell death. Analogues of purine nucleobases and nucleosides taken up by the cells were cytotoxic, suggesting that salvage routes could be exploited for therapy. Adenine or hypoxanthine were able to substitute for catalase in supporting cell growth and proliferation, suggesting a role for these bases in maintaining the microaerophilic conditions essentially required by the bacterium.

Adenine↗
PubMed1996

The urea cycle of Helicobacter pylori.

The presence and activities of the enzymes of the urea cycle in the bacterium Helicobacter pylori were investigated employing one- and two-dimensional NMR spectroscopy and radioactive tracer analysis. Cell suspensions, lysates and membrane preparations generated L-ornithine and ammonium at high rates in incubations with L-arginine, indicating the presence of arginase activity. Anabolic ornithine transcarbamoylase (OTCase) activity was identified by the formation of heat-stable products in incubations of cell-free extracts with ornithine and radiolabelled carbamoyl phosphate. The heat-labile product that resulted from incubations of cell-free extracts with citrulline radiolabelled in the guanidino moiety revealed the presence of catabolic OTCase activity. Argininosuccinate formation and catalysis indicated the presence of argininosuccinate synthetase and argininosuccinase activities. The findings suggested that H. pylori has a urea cycle which acts as an effective mechanism to extrude excess nitrogen from cells.

Arginine↗
PubMed1995

Fumarate reductase: a target for therapeutic intervention against Helicobacter pylori.

The potential of fumarate reductase as a therapeutic target against the human pathogen Helicobacter pylori was investigated by studying the cytotoxicity of morantel, oxantel, and thiabendazole, known to inhibit the enzyme in parasitic worms. Nuclear magnetic resonance spectroscopy was employed to investigate the effects of the inhibitors on the fumarate reductase activity of laboratory-adapted and wild-type bacterial strains. Production of succinate from fumarate in H. pylori cells was inhibited by morantel, oxantel, and thiabendazole. Cell growth and viability techniques were used to examine the bacteriostatic and bactericidal effects of the three anthelmintics. Each of the antiparasites arrested growth and produced cell death in liquid cultures, although the minimal inhibitory and bactericidal concentrations of these compounds are such that they would not be of therapeutic use. The strength of the effects as measured by minimal inhibitory and bactericidal concentrations was oxantel > thiabendazole > morantel. The findings suggested that fumarate reductase is an essential component of the metabolism of H. pylori and as such constitutes a possible target for therapeutic intervention in the treatment of the bacterium.

Aconitate Hydratase↗
PubMed1995

Conformation of a tetradecapeptide epitope of myelin basic protein.

The peptide AcAla-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys-Tyr, which comprises the first 14 residues of the acetylated N-terminus of myelin basic protein, is an epitopic site for two monoclonal antibodies to the human protein. The conformations of the tetradecapeptide in aqueous solutions were investigated employing high-resolution 1H- and 13C-NMR spectroscopy. Two-dimensional techniques were used to assign the spectra observed from both nuclei. Nuclear-Overhauser-effect data, amide proton temperature coefficients, 13C spin-lattice relaxation times, distance geometry calculations and dynamic simulated annealing provided evidence that the solution conformations of the tetradecapeptide included a nascent alpha-helix in the N-terminal segment, and a loop extending from Ser7 to Ser12 that bring His10 and Tyr14 into close proximity.

Amino Acid Sequence↗
PubMed1995

Characterisation of glucose transport in Helicobacter pylori.

The nature of the glucose transport system in the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. Fast D-glucose uptake was demonstrated by using two methods of measuring glucose transport. The transport of 2-deoxy-D-glucose was inhibited competitively by D-glucose; and the efflux of 2-deoxy-D-glucose from cells also was affected by the presence of D-glucose. The transport of 2-deoxy-D-glucose was saturable with a Km of 4.8 mM and Vmax of 146.6 pmol (microliter cell water)-1 at 20 degrees C. The transport was temperature-dependent with energies of activation of 6.8 and 51.0 kJ mol-1 for 0.2 and 20 mM 2-deoxy-D-glucose, respectively. The temperature dependence and saturable nature of transport suggested the presence of one or more glucose carriers. The substrate specificity of the transport system was studied by measuring the effects of mono- and disaccharides on the rates of transport of the glucose analogue. The most significant inhibitory effects were obtained with D-galactose and L-arabinose. Lack of transport inhibition by L-glucose established the stereospecificity of the transporters for the D-isomer of glucose. Higher rates of 2-deoxy-D-glucose transport were measured in the presence of sodium ions than for other monovalent cations, and the presence of amiloride inhibited transport of the monosaccharide. No inhibition was observed with cytochalasin B, phloretin or phloridzin. The results suggested the existence of specific D-glucose transporters and that the glucose transport system of H. pylori is significantly different from other known bacterial transporters.

Amiloride↗
PubMed1995

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein.

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and 31P, 13C, and 1H nuclear magnetic resonance spectroscopy (NMR), and electron magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5-, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and 13C- and 1H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% alpha-helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of alpha-helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles.

Animals↗
PubMed1995

Aminoacid utilization by Helicobacter pylori.

Utilization of aminoacids during growth by laboratory adapted and wild type Helicobacter pylori strains was investigated employing nuclear magnetic resonance spectroscopy and aminoacid analysis. All H. pylori strains tested showed growth rates with doubling times of approx. 11.5 hr in liquid cultures with semi-defined media or with defined aminoacid broth without carbohydrates. Fast utilization of several aminoacids at rates between 80 and 250 microM/hr was observed in culture broths inoculated with approx. 10(7) cells/ml; and acetate, formate and succinate accumulated as catabolic products in the growth media. Suspensions of bacterial cells and lysates in isotonic solutions converted arginine, asparagine, aspartate, glutamine, and serine used as sole substrates at significant rates; and under these conditions the principal metabolic products observed were acetate, formate, succinate and lactate. The findings of the study indicated that H. pylori can survive employing aminoacids as the basic nutrients, and suggested some of these metabolites were utilized via fermentative pathways with common characteristics to those found in anaerobes.

Amino Acids↗
PubMed1995

Acetyl-CoA carboxylase activity in Helicobacter pylori and the requirement of increased CO2 for growth.

A biotinylated acetyl-CoA carboxylase from the microaerophilic bacterium Helicobacter pylori was partially purified and characterized. The approximate molecular mass of the native enzyme was estimated at 235 kDa by native PAGE. A single band corresponding to approximately 24 kDa was detected by SDS-PAGE, suggesting that the native enzyme is a multi-protein complex. The protein was isolated from the soluble fraction of the cell. Catalytic activity was acetyl-CoA-dependent and inhibited by avidin but unaffected by avidin pretreated with excess biotin. The end-product of the reaction was identified as malonyl-CoA and the reaction was shown to be reversible by NMR spectroscopy. The activity of the enzyme was 0.29 mumol min-1 (mg protein)-1. The Vmax for bicarbonate was calculated at 0.73 mumol min-1 (mg protein)-1, and the affinity of the enzyme for this substrate was relatively low, with an apparent Km of 16.6 mM. These data provide the first evidence of a possible physiological role for the requirement of high levels of CO2 for growth in vitro of this bacterium.

Acetyl-CoA Carboxylase↗
PubMed1994

The Entner-Doudoroff pathway in Helicobacter pylori.

Evidence for the presence of the enzymes of the Entner-Doudoroff pathway in Helicobacter pylori was obtained using 1H and 31P nuclear magnetic resonance spectroscopy. Bacterial lysates generated 6-phosphogluconate and NADH or NADPH in incubations with glucose-6-phosphate and NAD+ or NADP+, indicating the presence of glucose-6-phosphate dehydrogenase activities. Formation of pyruvate was observed in time courses of incubations of bacterial lysates with 6-phosphogluconate as the only substrate, suggesting the presence of 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase activities. The existence of these enzymes and of triose phosphate isomerase was confirmed by observing the appearance of dihydroxyacetone phosphate in time courses of bacterial lysates incubated with 6-phosphogluconate. Aldolase activity was measured by the production of pyruvate and dihydroxyacetone phosphate in lysates incubated with 2-keto-3-deoxy-6-phosphogluconate as the sole substrate. Dehydrogenase, dehydratase and aldolase activities were observed in several bacterial strains including wild types from fresh isolates. Kinetic parameters were measured for the three activities. The cellular location of the enzymes was investigated by comparing the activities measured in the pellet and supernatant fractions obtained by centrifugation of lysate suspensions. The concentration of compounds causing 50% inhibition of enzyme activity was determined from dose-response curves. The data suggested the presence of two glucose-6-phosphate dehydrogenases linked to NAD+ and NADP+ activities. Using inhibitors differences between the H. pylori and mammalian KDPG aldolases were detected. The presence of these enzyme activities in H. pylori provided evidence for the existence of the Entner-Douderoff pathway in the bacterium.

Fructose-Bisphosphate Aldolase↗
PubMed1994

Pyruvate metabolism in Helicobacter pylori.

The metabolism of pyruvate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Generation of pyruvate from L-serine in incubations with whole cell lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was formed also in cell suspensions and lysates from phosphoenol pyruvate. Metabolically competent cells incubated aerobically with pyruvate yielded alanine, lactate, acetate, formate, and succinate. The production of alanine and lactate indicated the presence of alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed-acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton into the Kreb's cycle. Addition of pyruvate to various liquid culture media did not affect bacterial growth or loss of viability. The variety of products formed using pyruvate as the sole substrate showed the important role of this metabolite in the energy metabolism of H. pylori.

Acetates↗
PubMed1994

Salvage synthesis of purine nucleotides by Helicobacter pylori.

The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.

Adenine↗
PubMed1994

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori.

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.

Aspartate Carbamoyltransferase↗
PubMed1993

Glucose phosphorylation in Helicobacter pylori.

Saccharide kinase activities in Helicobacter pylori were investigated by incubating bacterial lysates with ATP and mono- or disaccharides and monitoring directly the appearance of phosphorylated products using 13C or 31P nuclear magnetic resonance spectroscopy. The monosaccharides employed included two trioses, two tetroaldoses, one tetroketose, five aldopentoses, two ketopentoses, five aldohexoses, three ketohexoses, and gluconic and glucuronic acids; the disaccharides studied were maltose, trehalose, cellobiose, sucrose, lactose, gentobiose, and melibiose. D-Glucose was the only sugar phosphorylated among all the carbohydrates examined. The kinase activity of lysates was studied by measuring the rates of formation of glucose 6-phosphate. The substrate specificity, the relatively high KM, and the absence of substrate inhibition suggested that the enzyme is a glucokinase rather than a hexokinase. Most of the glucose kinase activity was observed with the pellet fraction obtained by centrifugation, suggesting an association of the enzyme with the bacterial cell envelope.

Adenosine Triphosphate↗
PubMed1993

Fluorinated probes to measure carboxylesterase activities using 19F NMR spectroscopy: application to erythrocytes and Helicobacter pylori.

Eight fluorinated compounds were tested as putative probes to measure carboxylesterase activity employing 19F nuclear magnetic resonance spectroscopy. The method takes advantage of the sensitivity of fluorine resonances to the changes in the chemical bonding in the covalent structures where they are located. Determination of the kinetic parameters of ethyl fluoroacetate and diethyl fluoromalonate in hemolysates showed that these probes were well suited to study carboxylesterase activities in complex systems the potential of these probes for noninvasive applications was demonstrated in measurements of carboxylesterase kinetic parameters in intact erythrocytes. The presence of carboxylesterases was established in several strains of the bacterium Helicobacter pylori employing 19F nuclear magnetic resonance spectroscopy, and the kinetic parameters of these enzymes for ethyl fluoroacetate and diethyl fluoromalonate were measured.

Animals↗
PubMed1993

Glucose utilization and lactate production by Helicobacter pylori.

The transport and incorporation of D-glucose into the human pathogen Helicobacter pylori was investigated employing radioactive tracer analysis and 1H and 13C nuclear magnetic resonance spectroscopy. The bacterium was found to utilize D-glucose contrary to the accepted view that it cannot catabolize carbohydrates. Under the experimental conditions employed, the rate of transport of [14C]glucose was 3.24 mmol min-1 (g protein)-1, and the rate of incorporation into the cellular mass was 1.06 mumol h-1 (g protein)-1. The utilization of [13C]glucose showed biphasic characteristics with a slower initial period followed by a phase with a rate of utilization at least an order of magnitude faster. The apparent rates of decline of glucose levels during both phases varied between strains and depended on the growth conditions of the bacteria prior to harvesting. The main product of glucose catabolism was identified as lactate. These findings provide new perspectives into the physiology of H. pylori and have implications for the active search to develop appropriate therapies for the micro-organism.

Biological Transport, Active↗
PubMed1993

Fumarate catabolism in Helicobacter pylori.

The metabolism of fumarate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Metabolically competent cells generated malate and succinate from fumarate as the sole substrate indicating the presence of fumarase and fumarate reductase activities in the bacterium. In incubations of fumarate with cell lysates accumulation of lactate, acetate, formate and alanine was observed after the initial production of malate and succinate. The results indicate the existence of active fumarate catabolism in H. pylori and suggest the possibility of an ATP generating mechanism which may play an important role in the bioenergetics of the bacterium.

Acetates↗
PubMed1992

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.

Animals↗