Biomedical subjects

B Mayer

Publications and source records attributed to B Mayer.

At least 145 records · Page 8Linked to original sources

PubMed1997

Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction.

The oxygenase domain of inducible NO synthase (residues 1-498, iNOSox) is the enzyme's catalytic center. Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and binds l-arginine [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801]. To help identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli. The iNOSox was 80% dimeric but contained a low-spin heme iron that bound DTT as a sixth ligand. The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket. The H4biopterin-replete iNOSox could catalyze conversion of Nomega-hydroxyarginine to citrulline and NO in a H2O2-supported reaction. Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single site in its N-terminal region (K117). H4biopterin protected against the cleavage whereas l-arginine did not. The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically inactive, showed no capacity to bind H4biopterin or l-arginine, and did not dimerize when provided with these molecules, indicating that residues 1-117 were important for iNOSox dimerization and H4biopterin/l-arginine interaction. A deletion mutant missing residues 1-114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to l-arginine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wild-type iNOSox, narrowing the region of importance to amino acids 66-114. Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, L-arginine binding, or catalytic function. These results suggest that residues 66-114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization. H4biopterin binding appears to stabilize the protein structure in this region, and through doing so activates iNOS for NO synthesis.

Amino Acid Sequence↗

PubMed1997

Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin.

The characteristics of tetrahydrobiopterin (H4biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7, 8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H4biopterin), a novel pterin-based inhibitor of nitric oxide synthase. The 4-amino compound completely inhibited enzyme stimulation by 10 microM H4biopterin with a half-maximally active concentration of 7.2 +/- 0.39 microM, whereas H2biopterin and sepiapterin were much less potent. Binding studies using [3H]H4biopterin at 4 degrees C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min-1, each accounting for approximately 50% of total binding. Dissociation of [3H]H4biopterin occurred with rate constants of 0.005 and 0.0028 min-1 in the absence and presence of l-arginine, respectively. Specific binding of 10 nM [3H]H4biopterin was antagonized by unlabeled H4biopterin and its 4-amino analog with half-maximal effects at 84 +/- 6 and 34 +/- 3.2 nM, respectively. Binding of H4biopterin and 4-amino-H4biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by l-arginine. Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H4biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis.

Animals↗

PubMed1997

Thiols and neuronal nitric oxide synthase: complex formation, competitive inhibition, and enzyme stabilization.

To elucidate how thiols affect neuronal nitric oxide synthase (nNOS) we studied the binding of thiols to tetrahydrobiopterin (BH4)-free nNOS. Dithiothreitol (DTT), 2-mercaptoethanol, and L- and D-cysteine all bound to the heme with Kd values varying from 0.16 mM for DTT to 41 mM for L-cysteine. DTT, 2-mercaptoethanol, and L-cysteine yielded absorbance spectra with maxima at about 378 and 456 nm, indicative of bisthiolate complexes; the maximum at 426 nm with D-cysteine suggests binding of the neutral thiol. From the results with 2-mercaptoethanol we deduced that in 2-mercaptoethanol-free, BH4-free nNOS the sixth heme ligand is not a thiolate. DTT binding to nNOS containing one BH4 per dimer was biphasic. Apparently, the BH4-free subunit bound DTT with the same affinity as the BH4-free enzyme, whereas the BH4-containing subunit exhibited a > 100-fold lower affinity, indicative of competition between DTT and BH4 binding. Binding of DTT to the BH4-containing subunit was suppressed by L-arginine, whereas high-affinity binding was not affected, suggesting that L-arginine binds only to the BH4-containing subunit. DTT competitively inhibited L-citrulline production by nNOS containing one BH4 per dimer (Ki approximately 11 mM). Comparison of DTT binding and inhibition suggests that the heme of the BH4-free subunit is not involved in catalysis. Thermostability of nNOS was studied by preincubating the enzyme at various temperatures prior to activity determination. At nanomolar concentrations, nNOS was stable at 20 degrees C but rapidly deactivated at higher temperatures (t1/2 approximately 6 min at 37 degrees C). At micromolar concentrations, inactivation was 10 times slower. Absorbance and fluorescence measurements demonstrate that inactivation was not accompanied by major structural changes. The stabilization of nNOS by thiols was illustrated by the fact that omission of 2-mercaptoethanol during preincubation for 10 min at 30 degrees C led to an activity decrease of up to 90%.

Animals↗

PubMed1997

Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization.

The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147+/-24.1 nM and 52+/-9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6+/-0.5 to 0. 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8+/-0.04 nm, corresponding to a hydrodynamic volume of 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55x10(-24) m3, suggesting that haem availability regulates eNOS dimerization.

Animals↗

PubMed1997

Metabolic fate of peroxynitrite in aqueous solution. Reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry.

Peroxynitrite, the reaction product of nitric oxide (NO) and superoxide (O-2) is assumed to decompose upon protonation in a first order process via intramolecular rearrangement to NO3-. The present study was carried out to elucidate the origin of NO2- found in decomposed peroxynitrite solutions. As revealed by stopped-flow spectroscopy, the decay of peroxynitrite followed first-order kinetics and exhibited a pKa of 6.8 +/- 0.1. The reaction of peroxynitrite with NO was considered as one possible source of NO2-, but the calculated second order rate constant of 9.1 x 10(4) M-1 s-1 is probably too small to explain NO2- formation under physiological conditions. Moreover, pure peroxynitrite decomposed to NO2- without apparent release of NO. Determination of NO2- and NO3- in solutions of decomposed peroxynitrite showed that the relative amount of NO2- increased with increasing pH, with NO2- accounting for about 30% of decomposition products at pH 7.5 and NO3- being the sole metabolite at pH 3.0. Formation of NO2- was accompanied by release of stoichiometric amounts of O2 (0.495 mol/mol of NO2-). The two reactions yielding NO2- and NO3- showed distinct temperature dependences from which a difference in Eact of 26.2 +/- 0.9 kJ mol-1 was calculated. The present results demonstrate that peroxynitrite decomposes with significant rates to NO2- plus O2 at physiological pH. Through formation of biologically active intermediates, this novel pathway of peroxynitrite decomposition may contribute to the physiology and/or cytotoxicity of NO and superoxide.

Hydrogen-Ion Concentration↗

PubMed1997

Patterns of mobilization of copper and iron following myocardial ischemia: possible predictive criteria for tissue injury.

Direct evidence for substantial iron and copper mobilization into the coronary flow immediately following prolonged, but not short, cardiac ischemia is presented. When small volumes of coronary flow fractions (CFFs) were serially collected upon reperfusion, after 25-60 min of ischemia, the copper and iron levels in the first CFF were 50-fold and 12- to 15-fold higher, respectively, than corresponding pre-ischemic values. The copper and iron levels after shorter periods (15-21 min) of ischemia were only about five-fold higher than the pre-ischemic values. This demonstrates that the resumption of coronary flow is accompanied by a burst of both metal ions. The levels of Cu/Fe in the CFFs correlated well with the loss of cardiac function following global ischemia of varying duration. After 18 min of ischemia, the residual cardiac function was less than 50%, and the damage was essentially reversible. After 25 min of ischemia, it exceeded 50% and was only partially reversible, while after 35 min, the damage exceeded 80%, and was mostly irreversible. The results are in accord with the hypothesis that copper and iron play causative roles in myocardial injury through mediation of hydroxyl radical production. Thus, the pattern of Cu/Fe mobilization from the tissue into the CFF can be used for the prediction of the severity of myocardial damage following ischemia, and could be developed into useful modalities for intervention in tissue injury.

Animals↗

PubMed1997

A synthetic peptide corresponding to the putative dihydrofolate reductase domain of nitric oxide synthase inhibits uncoupled NADPH oxidation.

A stretch of about 150 amino acids located between the heme and the calmodulin recognition sequence of nitric oxide synthase (NOS) has been strongly conserved within isoforms and was proposed to participate in pteridine binding because of sequence similarities to the folate binding site of dihydrofolate reductase (DHFR). In the present study we tested four synthetic peptides corresponding to sequences located within the putative DHFR domain of rat neuronal NOS for their effects on catalytic and binding activities of the recombinant enzyme purified from baculovirus-infected insect cells. Three of the selected peptides had no effects at concentrations of up to 0.1 mM, but one peptide, corresponding to amino acid residues 564-582 of neuronal NOS, led to a concentration-dependent inhibition of L-citrulline formation. The potency of the peptide decreased with increasing assay concentrations of NOS, pointing to a competitive interaction with a specific structure of the enzyme. The peptide was not competitive with L-arginine and H4biopterin, did not antagonize binding of radiolabeled NG-nitro-L-arginine or H4biopterin, and had no effect on Ca2+/calmodulin-dependent reduction of cytochrome c. However, the presence of the peptide led to a pronounced inhibition of NADPH oxidation in the absence of L-arginine and prevented stimulation of this reaction by the amino acid substrate. These results indicate that sequence 564-582 of neuronal NOS does not contribute to L-arginine or H4biopterin binding but is critically involved in the electron transfer from the reductase domain to the heme.

Amino Acid Sequence↗

PubMed1997

Release of nitric oxide from donors with known half-life: a mathematical model for calculating nitric oxide concentrations in aerobic solutions.

The NO concentrations released from donor compounds are difficult to predict as they are determined by formation and inactivation reactions. To calculate the concentrations of NO over time, we have developed a mathematical model which is based on a system of two differential equations describing the first order decomposition of the NO donor in association with the third order reaction of NO with oxygen. Although there is no closed formula for the solution, it can be easily computed by any standard numerical differential solver or simulation software with the following input parameters: initial concentration and decomposition rate constant of the NO donor, O2 concentration, and rate constant for NO autoxidation. The model was validated by monitoring NO release from 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) with a Clark-type NO-sensitive electrode at two different temperatures (25 and 37 degrees C) and DEA/NO concentrations ranging from 1 to 10 microM. Under all conditions, there was an excellent agreement between experimental and calculated data. In addition to the computer modeling, we present graphical plots which allow a rough but very easy estimation of the actual NO concentrations if appropriate computer software should not be available.

Kinetics↗

PubMed1997

Spatial relationships of enteric nerve fibers to vagal motor terminals and the sarcolemma in motor endplates of the rat esophagus: a confocal laser scanning and electron-microscopic study.

Enteric co-innervation of motor endplates in the rat esophagus was studied with confocal laser scanning and electron microscopy. Enteric fibers were demonstrated with immunocytochemistry for nitric oxide synthase, vasoactive intestinal peptide or NADPH-diaphorase histochemistry. Vagal motor terminals were identified with calcitonin gene-related peptide (CGRP) immunocytochemistry. Teloglia was stained with immuno- cytochemistry for S100, and TRITC-tagged alpha-bungarotoxin was used to delineate endplate areas in immmunofluorescence preparations. Both confocal imaging and electron microscopy revealed intimate relationships between enteric and vagal terminals on the one hand, and enteric terminals and the sarcolemma on the other. In addition, electron microscopy could point out direct apposition of a significant proportion of enteric varicosities to vagal motor terminals without intervening teloglial processes. These morphological data are compatible with pre- and postsynaptic modulatory effects of enteric neurons on vagal neuromuscular transmission in striated esophageal muscle.

Animals↗

PubMed1997

Nitric oxide/cGMP pathway components in the Leydig cells of the human testis.

In this study we sought to determine whether the main components of the nitric oxide (NO) pathway are localized within the Leydig cells of the human testis and whether the soluble guanylyl cyclase (sGC), the enzyme that accounts for NO effects, is functionally active in these cells. Using an amplified immunocytochemical technique, immunoreactivity for nitric oxide synthase (NOS-I), sGC and cyclic guanosine monophosphate (cGMP) was detected within the cytoplasm of human Leydig cells. Distinct differences in staining intensity were found between individual Leydig cells, between cell groups and between Leydig cells of different patients. By means of a specific cGMP-RIA, a concentration-dependent increase in the quantity of cGMP was measured in primary cultures of human Leydig cells following exposure to the NO donor sodium nitroprusside. In addition, NOS-I immunoreactivity was seen in Sertoli cells, whereas cGMP and sGC immunoreactivity was found in Sertoli cells, some apically situated spermatids and residual bodies of seminiferous tubules. Dual-labelling studies and the staining of consecutive sections showed that there are several populations of Leydig cells in the human testis. Most cells were immunoreactive for NOS-I, sGC and cGMP, but smaller numbers of cells were unlabelled by any of the antibodies used, or labelled for NOS-I or cGMP alone, for sGC and cGMP, or for NOS-I and sGC. These results show that the Leydig cells possess both the enzyme by which NO is produced and the active enzyme which mediates the NO effects. There are different Leydig cell populations that probably reflect variations in their functional (steroidogenic) activity.

Adult↗

PubMed1997

Occurrence of enzymes of free radical metabolism suggests the possible cytotoxic capacity of the transitional epithelium of the human ureter.

Three enzymes, viz., tartrate-resistant acid phosphatase (TRAP), nitric oxide synthase I (NOS-I), and superoxide dismutase (SOD), involved in the production and metabolism of free radicals or radical equivalents, were demonstrated by immunocytochemistry in the urothelium of the ureters of six patients of various ages. Two of these enzymes (TRAP and NOS-I) were colocalized in the most apical and lateral border of the superficial cells of the urothelium. In contrast, SOD showed a patchy or granular distribution within the supranuclear region of these cells. Intra- and subepithelial macrophages exhibited a weak TRAP, but no NOS-I or SOD, immune reaction. On the basis of the immunocytochemical findings, arguments in favor of a cytotoxic function of the superficial cells of the human urothelium are presented.

Acid Phosphatase↗

PubMed1997

Neurochemical differentiation of rat enteric neurons during pre- and postnatal life.

The presence of nitric oxide synthase (the enzyme which synthesizes nitric oxide), vasoactive intestinal peptide (VIP) and pituitary adenylyl-cyclase-activating peptide (PACAP) in the rat enteric nervous system between term (E18) and 90 days postpartum (P90) was investigated by immunohistochemistry. Neuronal maturity was ascertained by emanining for the presence of two novel neuron intermediate filament proteins: alpha-internexin, which is transiently expressed in developing neurons, and peripherin, which is expressed in differentiating as well as in mature neurons. The alimentary canal of the foetus at E18, unfed newborn, suckling, weaning and adult rats was studied. Throughout the rat gut the myenteric neurons were recognized by the time the foetal stage was reached and the submucous neurons by the suckling period. alpha-Internexin was present at E18; its levels increased during ageing and were markedly reduced at P30. Peripherin was first detected at birth; its levels increased with ageing and practically almost all of the neurons and nerve fibres were labelled from P30. Nitric oxide synthase was present at E18 and the number of labelled neurons gradually increased with ageing, while both VIP and PACAP could be detected by the end of the suckling period in an equal number of neurons to that in adulthood. These data indicate that in the rat gut the onset of the mature neuronal phenotype is not yet achieved at birth and that the neurochemical differentiation is accomplished during the first month of postnatal life.

Animals↗

PubMed1997

Conditional reasoning and phobic fear: evidence for a fear-confirming reasoning pattern.

In two experiments we explored the role of subjects' reasoning performance in the persistence of phobic fear. More specifically, we investigated whether (phobic) subjects are prone to selectively search for danger-confirming information when asked to judge the validity of conditional rules in the context of general and phobic threats. In both experiments, participants were presented with Wason Selection Tasks (WST) pertaining to general and phobic threats. The WSTs contained safety rules (if P then no danger) and danger rules (if P then danger). In Experiment 1 participants were high (n = 20) and low (n = 20) spider fearful students, whereas in Experiment 2 participants were untreated (n = 38) and treated (n = 27) spider phobic women and a group of non-fearful controls (n = 27). Both experiments showed that in the context of general threat, subjects predominantly rely on confirming information regarding danger rules and on falsifying information regarding safety rules. This reasoning strategy was not particularly pronounced in high fear or phobic subjects. However, only clinically diagnosed spider phobics displayed a similar reasoning strategy in the context of phobia-relevant threats. Thus, the present data seem to suggest that the mere perception of threat is already sufficient to activate a danger-confirming reasoning strategy and in case of phobic threats such a reasoning pattern logically serves to maintain or even enhance phobic fears.

Adolescent↗

PubMed1997

Role of endothelin, nitric oxide and L-arginine release in ischaemia/reperfusion injury of rat heart.

OBJECTIVES: We tested the hypothesis that endothelin-1 (ET-1) aggravates ischaemia/reperfusion injury by stimulating cellular L-arginine depletion, which would result in reduced synthesis of nitric oxide (NO) and withdrawal of cardioprotection. METHODS: Five groups of rat hearts (n = 5 each) were perfused at 9 ml/min per g for 45 min, subjected to 15 min total global ischaemia and reperfused for 30 min; they received, from 5 min pre-ischaemia to end of reperfusion, either vehicle, L-arginine (1 mmol/l), the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 mumol/l), the inhibitor of NO formation NG-nitro-L-arginine (L-NNA; 200 mumol/l), or the ET receptor antagonist PD 142893 (200 nmol/l). Cardiac function and release of L-arginine, cyclic GMP and lactate dehydrogenase (LDH) into coronary effluent were measured. RESULTS: Systolic, diastolic, and coronary reperfusion function were consistently improved by L-arginine, SNAP, or PD 142893, but worsened by L-NNA (P < 0.05 in each case). L-arginine release was transiently increased up to 25-fold on reperfusion (vehicle); release was reduced by SNAP (mean: 68%) and entirely prevented by PD 142893. Despite the increased outflow of L-arginine, formation of cyclic GMP was not reduced, but enhanced in reperfusion (11-fold; vehicle), and SNAP further augmented this release, but L-NNA had no significant effect. Release of LDH was decreased by L-arginine, SNAP, and PD 142893 in reperfusion. Finally, release of ET-1 was inhibited by NO in normoxia as well as throughout reperfusion as evident from the stimulatory effect of L-NNA. CONCLUSION: In ischaemia, ET-1 cause cell necrosis and L-arginine outflow without compromising NO synthesis in this model.

Analysis of Variance↗

PubMed1997

Nitric oxide synthase-expressing neurons are area-specifically distributed within the cerebral cortex of the rat.

Neuronal nitric oxide synthase produces nitric oxide, a radical involved in neurotransmission as well as in cytotoxicity during stroke and neurodegenerative diseases. In the adult Wistar rat neuronal nitric oxide synthase-positive neurons are inhomogenously distributed along defined cortical areas, with highest densities (18 cells/mm2) in cingular area 1, piriform cortex, frontal motor area Fr 2 and in the medial visual association area Oc 2MM. A medium packing density of neuronal nitric oxide synthase neurons (10/mm2) characterizes primary sensory areas, whereas retrosplenial cortices contain lowest cell numbers (3-5/mm2). The data suggest that functions of certain cortical areas are more dependent on intracortically produced nitric oxide than others, and that cortical injury may cause more severe nitric oxide related cytotoxicity in areas with higher numbers of neuronal nitric oxide synthase-positive neurons.

Animals↗

PubMed1997

Interference of carboxy-PTIO with nitric oxide- and peroxynitrite-mediated reactions.

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Animals↗

PubMed1997

Biosynthesis and action of nitric oxide in mammalian cells.

Nitric oxide (NO) can act as a vasorelaxant, a modulator of neurotransmission and a defence against pathogens. However, under certain conditions, NO can also have damaging effects to cells. Whether NO is useful or harmful depends on its chemical fate, and on the rate and location of its production. Here, we discuss progress in NO chemistry and the enzymology of NO synthases, and we will also attempt to explain its actions in the cardiovascular, nervous and immune systems.

Animals↗

PubMed1997

The Rhesus D-negative phenotype is an independent predictor of poor prognosis in curatively (RO) resected gastric cancer patients.

Among gastric cancer patients, the Rhesus D-negative phenotype correlated with increased tumour recurrence [all patients, n = 83, P = 0.026; curatively (R0) resected patients, n = 51, P = 0.093] and reduced overall survival time (all patients, log-rank P = 0.0028; R0 patients, log-rank P = 0.0003) and was identified in multivariate analysis as the most important independent prognostic marker in the R0 patient group (relative risk 9.1, P = 0.0013).

Aged↗