Biomedical subjects

B Mayer

Publications and source records attributed to B Mayer.

At least 109 records · Page 6Linked to original sources

PubMed1998

Sensitivity of flavin fluorescence dynamics in neuronal nitric oxide synthase to cofactor-induced conformational changes and dimerization.

The fluorescence intensity of the two flavin prosthetic groups, FMN and FAD, in neuronal nitric oxide synthase (nNOS) was found to decay highly nonexponentially, being best described by four fluorescence lifetimes. This excited state heterogeneity is the result of multiple flavin quenching sites which are due to several flavin microenvironments created mainly by stacking with aromatic amino acids. Investigating nNOS in the absence of one or more of Ca2+/calmodulin, tetrahydrobiopterin, and heme revealed an influence of these cofactors on the microenvironments of the flavin prosthetic groups. Similar effects on the flavin rotational dynamics were found by analyzing the fluorescence anisotropy decay of the holo and of the different apo forms of nNOS. Since the tetrahydrobiopterin and the heme are located in the N-terminal oxygenase domain of nNOS, their effect on the flavins in the C-terminal reductase domain is explained by a folding back of the reductase domain onto the oxygenase domain. Thereby a domain-domain interface is created containing the FAD, FMN, heme, and tetrahydrobiopterin prosthetic groups which allows for efficient electron transfer during catalysis. The heme group, which is known to be essential for homodimerization of nNOS, was also found to be essential for the formation of the domain-domain interface.

Animals↗

PubMed1998

Lack of tyrosine nitration by peroxynitrite generated at physiological pH.

Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of .NO and O-2 at physiological pH. In the present study, we measured the nitration of free tyrosine by .NO and O-2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]- dia zen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mM) to tyrosine (1 mM) at pH 7.4 yielded 36.77 +/- 1.67 microM 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from .NO and O-2, which were generated at equal rates ( approximately 5 microM x min-1) from 1 mM spermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mM hypoxanthine was much less efficient (0.67 +/- 0.01 microM; approximately 0.07% of total product flow). At O-2 fluxes exceeding the .NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 microM). Nitration was most efficient (approximately 0.3%) with the .NO donor alone, i.e. without concomitant generation of O-2. Nitration by .NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1-2 mM), and was not enhanced by NaHCO3 (up to 20 mM), indicating that it was mediated by .NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from .NO and O-2 as a mediator of tyrosine nitration in vivo.

Carbon Dioxide↗

PubMed1998

Isoform-specific effects of salts on nitric oxide synthase activity.

We investigated the effects of salts on the properties of the neuronal, endothelial, and inducible isoforms of nitric oxide synthase (nNOS, eNOS, and iNOS), and found pronounced isoform-specific effects on NOS-catalyzed L-citrulline formation. Salts inhibited iNOS monotonously, whereas nNOS and eNOS were stimulated up to 3-fold at low, and inhibited at high (>/=0.1-0.2 M) salt concentrations. The effectivities of different ions mostly followed the Hofmeister series, indicating that the effects can for a large part be ascribed to changes in protein solvation. Km(Arg) increased in the presence of NaCl, demonstrating the importance of charge interactions for substrate binding. The coupling of NADPH oxidation to NO production was not affected by KCl. Salts (</=1 M) had no major impact on the tertiary and quaternary structure, or on the state of the heme. Extrapolation of these results to commonly applied experimental conditions for in vitro activity assays suggests that true specific activities of nNOS and eNOS may, in some cases, be underestimated as much as 3-fold.

Animals↗

PubMed1998

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases.

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774-777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC50 for nNOS was 18 +/- 6 microM GST-PIN with 63 nM nNOS after 30 min at 37 degrees C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K(M) for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.

Animals↗

PubMed1998

Haem insertion, dimerization and reactivation of haem-free rat neuronal nitric oxide synthase.

The nitric oxide synthases are dimeric enzymes in which the intersubunit contacts are formed by the P-450-haem-containing, tetrahydrobiopterin-dependent oxygenase domain. The dimerization of the neuronal isoenzyme was shown previously to be triggered by Fe-protoporphyrin IX (haemin). We report for the first time the reactivation of the haem-deficient neuronal isoenzyme (from rat, expressed in a baculovirus/insect cell system) after haem reconstitution. We further examined the reconstitution of the enzyme with protoporphyrin IX (PPIX) and its Mn and Co complexes. All of these porphyrins inserted into the haem pocket, as assessed by quenching of intrinsic protein fluorescence. In addition to haemin, MnPPIX stimulated dimerization, as measured by gel filtration and by cross-linking with glutaraldehyde. In contrast, neither CoPPIX nor PPIX stimulated dimerization. The absorbance spectra of the reconstituted enzymes were measured and compared with published results on P-450 enzymes reconstituted with the same metals. The results suggest that those metalloporphyrins which caused dimerization were able to acquire a thiolate ligand from the protein, and we propose that this ligation is the trigger for dimerization. Substrate and tetrahydrobiopterin binding sites only emerged with the metalloporphyrins that caused dimerization.

Animals↗

PubMed1998

Reaction of neuronal nitric-oxide synthase with oxygen at low temperature. Evidence for reductive activation of the oxy-ferrous complex by tetrahydrobiopterin.

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at -30 degreesC. In the absence of substrate, the complex formed between ferrous NOS and O2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 (lambda max = 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than -20 degreesC. In the presence of substrate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production of Nomega-hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blue-shifted intermediate and of Nomega-hydroxy-L-arginine required the presence of tetrahydrobiopterin (BH4). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH4. These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH4 in NOS catalysis.

Animals↗

PubMed1998

Effects of pH on the structure and function of neuronal nitric oxide synthase.

We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomerleft and right arrow dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (lambdamax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (lambdamax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.

Animals↗

PubMed1998

Electrochemical determination of S-nitrosothiols with a Clark-type nitric oxide electrode.

Low-molecular-mass thiols and nitric oxide (NO) form S-nitrosothiols (thionitrites) in the presence of oxygen. Thionitrites play an integral role in a variety of NO-dependent physiological processes. This study describes a sensitive analytical method for the quantitative determination of thionitrites. The method is based on the Cu+-catalyzed homolytic cleavage of thionitrites and electrochemical detection of the released NO with a Clark-type electrode. Cu+ was generated by addition of Cu(NO3)2 to samples containing 1 mM GSH or 4 mM L-cysteine as reducing agents. The effect of Cu(NO3)2 on the release of NO from GSNO was concentration-dependent. In the presence of 1 mM GSH, the EC50 for Cu(NO3)2 was 1.34 +/- 0.08 mM. Using cysteine instead of GSH, NO release was quantitative at much lower concentrations of Cu(NO3)2 (EC50 = 8.5 +/- 2.8 microM. NO release was not significantly affected by pH (7.0-9.0) and was inhibited by the Cu+-selective chelator neocuproine, whereas the Cu2+ chelator cuprizone was approximately 16-fold less potent. Calibration of the method with GSNO, S-nitroso-N-acetyl-penicillamine, or S-nitrosated bovine serum albumin yielded linear plots of initial rates of NO release versus thionitrite concentration from 50 nM to 5 microM. This method may be useful for the quantitative determination of thionitrites in biological samples.

Animals↗

PubMed1998

Reaction of peroxynitrite with HEPES or MOPS results in the formation of nitric oxide donors.

We investigated the effects of organic buffers on the NO-like biological activities of ONOO-. In HEPES buffer (50 mM), ONOO- (1 mM) induced a 20-fold increase in endothelial cGMP accumulation and the effect was comparable to that elicited by a maximally active concentration of the NO donor DEA/NO. ONOO- produced a 12-fold increase of cGMP in MOPS buffer (50 mM), but was virtually inactive in phosphate buffer (50 mM). Electrochemical detection of NO showed that the biological effects of ONOO- in HEPES or MOPS were due to accumulation of compounds that released NO in the presence of copper ions. CuCl2-induced formation of NO was completely blocked by the Cu(I) chelator neocuproine but unaffected by the Cu(II) chelator cuprizone, pointing to a Cu(I)-catalyzed decomposition pathway. Formation of NO from ONOO- was not detectable in phosphate buffer, in agreement with the lack of effect of ONOO- on cGMP accumulation in this buffer. These data demonstrate that certain buffer components present in cell culture media may yield artificial results in experiments with authentic ONOO-.

Animals↗

PubMed1998

Neuronal nitric-oxide synthase interaction with calmodulin-troponin C chimeras.

Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS. The magnitude by which each chimera activated NO synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to approximately 80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron reduction. We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.

Amino Acid Sequence↗

PubMed1998

A new pathway of nitric oxide/cyclic GMP signaling involving S-nitrosoglutathione.

Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from L-arginine and O2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O-2). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3',5'-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 +/- 0.01 and 0.19 +/- 0.06 micromol of cGMP x mg-1 x min-1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 +/- 0.60 micromol of cGMP x mg-1 x min-1). The NO/O-2 donor SIN-1 caused only a slight accumulation of cGMP in the absence of GSH but was almost as effective as DEA/NO in the presence of the thiol. The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a maximally active concentration of 200 ng/0.1 ml, NOS increased sGC activity to 1.22 +/- 0.12 and 8.51 +/- 0.88 micromol of cGMP x mg-1 x min-1 in the absence and presence of GSH, respectively. The product of NOS and GSH was identified as the thionitrite GSNO, which activated sGC through Cu+-catalyzed release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O-2-triggered pathway was very efficient (25-45% GSNO) and insensitive to CO2. Cu+-specific chelators inhibited bradykinin-induced cGMP release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as intermediates of NO/cGMP signaling in mammalian tissues.

Animals↗

PubMed1998

Nitric oxide synthases: catalytic function and progress towards selective inhibition.

Biosynthesis of nitric oxide (NO) is performed by the dimeric, heme-containing enzyme nitric oxide synthase, which requires the flavins FAD and FMN, as well as the pteridine cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin) in order to catalyze the NADPH-dependent oxidation of L-arginine. The three major isoforms of nitric oxide synthase (NOS), although identical in that they contain a carboxy-terminal reductase and an amino-terminal oxygenase domain, fulfill diverse physiological functions, according to their differing expression patterns and mechanisms of activation. The pteridine H4biopterin, which affects both the conformational stability and activity of NOS, demonstrates anticooperative binding which results in the stoichiometric production of NO and O2-. Physiological mechanisms involving superoxide dismutase and reduced glutathione exist to avoid the subsequent formation of the potent oxidant peroxynitrite. With regard to inhibition of NO production, novel isoform-selective inhibitors are proving useful not only for dissecting the physiological functions of NOS, but also in the development of novel therapeutic agents.

Animals↗

PubMed1998

De novo expression of the cell adhesion molecule E-selectin on gastric cancer endothelium.

BACKGROUND AND AIMS: Angiogenesis and the molecular phenotype of the tumor vasculature determine tumor growth and metastasis. PATIENTS/METHODS: In a series of 58 gastric cancer patients, vascular density and the antigenic profile of endothelial cells in normal, inflamed and malignant gastric tissues were compared using immunohistochemistry. RESULTS: In both benign gastric mucosa and primary gastric cancer vascular density was inflammation-independent. However, increased vascularity in primary tumors was positively associated with a high tumor cell density suggesting tumor-induced angiogenesis (P=0.00001 ). P-selectin was expressed in most of the gastric mucosa samples on a small fraction of vessels and increased in the presence of moderate to strong leukocyte infiltrate. VCAM-1 positive mucosal vessels were rare and showed no association with inflammation. E-selectin and the EN 7/44 antigen defining budding vessels were absent on normal and inflamed endothelium. In contrast, in primary gastric cancer de novo expression of both E-selectin and the EN 7/44 antigen was observed. E-selectin positive vessels were preferentially found in vascular-rich tumor areas (P=0.0043) independently of leukocyte infiltration. Upregulation of VCAM-1 on tumor-associated endothelium was closely related to inflammation (P=0.019), while P-selectin expression resembled that in benign mucosa. CONCLUSIONS: Differentially expressed vascular molecules may influence the functional characteristics of extravasating leukocytes and represent new targets in anti-gastric cancer therapy.

Capillaries↗

PubMed1998

The Screen for Child Anxiety Related Emotional Disorders (SCARED) and traditional childhood anxiety measures.

The Screen for Child Anxiety Related Emotional Disorders (SCARED) is a self-report questionnaire that measures symptoms of DSM-IV linked anxiety disorders in children. This article presents two studies that investigated the relationship between the SCARED, on the one hand, and two other widely used anxiety measures for children, namely the Revised Children's Manifest Anxiety Scale (RCMAS) and the Fear Survey Schedule for Children-Revised (FSSC-R), on the other hand. Results indicate that SCARED scores are positively and in a theoretically meaningful way related to RCMAS and FSSC-R scores, and thus provide evidence for the concurrent validity of the SCARED.

Age Factors↗

PubMed1998

Hypochondriasis: the role of fear-confirming reasoning.

We investigated whether hypochondriacal patients are prone to selectively search for danger-confirming information when asked to judge the validity of conditional rules in the context of general and health threats. Therefore, hypochodtriacal patients (n = 27) and a healthy control group (n = 27) were presented with modified Wason Selection Tasks (WSTs) pertaining to general and health threats. The WSTs contained safety rules (If P then safe) and danger rules (If P then danger). Subjects adopted a verificationistic strategy in case of danger rules and tended to look for falsifications in case of safety rules. This danger-confirming reasoning pattern was similar for both types of contexts and not particularly pronounced in hypochondriacal persons. These findings argue against the idea that such a reasoning pattern directly causes hypochondriasis. Yet, in the presence of anxiogenic (hypochondriasis related) convictions such danger-confirming reasoning pattern logically serves to maintain or even enhance hypochondriacal complaints.

Adult↗

PubMed1998

Transient changes in the presence of nitric oxide synthases and nitrotyrosine immunoreactivity after focal cortical lesions.

Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus. These findings indicate that no perilesional changes in the number of neurons expressing nitric oxide synthase I occur, but that a transient increase in nitric oxide synthase II does take place in the aftermath of small cortical lesions. The results suggest that increased nitric oxide production is limited to certain post-lesional intervals in this experimental model. It is also obvious that the vast majority of nitric oxide synthase-positive cells are nitric oxide synthase II-containing astrocytes three days after lesioning, suggesting that astrocyte-derived nitric oxide plays a significant role in delayed neuronal death. Such a condition points to an important aspect of post-lesional astrocytosis.

Animals↗

PubMed1998

Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76.

Tyrosine phosphorylation of linker proteins enables the T cell antigen receptor (TCR)-associated protein tyrosine kinases to phosphorylate and regulate effector molecules that generate second messengers. We demonstrate here that the SLP-76 linker protein interacts with both nck, an adaptor protein, and Vav, a guanine nucleotide exchange factor for Rho-family GTPases. The assembly of this tri-molecular complex permits the activated Rho-family GTPases to regulate target effectors that interact through nck. In turn, assembly of this complex mediates the enzymatic activation of the p21-activated protein kinase 1 and facilitates actin polymerization. Hence, phosphorylation of linker proteins not only bridges the TCR-associated PTK, ZAP-70, with downstream effector proteins, but also provides a scaffold to integrate distinct signaling complexes to regulate T cell function.

Actins↗

PubMed1998

Differential effects of antihypertensive drugs on neurohormonal activation: insights from a population-based sample.

OBJECTIVES: The clinical course of hypertension or heart failure may be modified by the extent of concurrent neurohormonal activation. Factors that regulate neurohormones in patients with these conditions are complex. In the present study, we examined the relative contribution of antihypertensive therapy to the variability of neurohormonal levels in a well defined population based sample. DESIGN AND SETTING: Cross-sectional study of a mixed urban and rural population. SUBJECTS: Middle-aged individuals (n = 646) were analysed in order to elucidate determinants of neurohormone levels by uni- and multivariate comparisons. The assessment included anthropometric, echocardiographic and, if appropriate, genotype information. RESULTS: The intake of antihypertensive drugs was related to significant alterations of neurohormone levels that, in part, exceeded the contribution of all other variables studied. Multivariate analyses revealed that renin levels were independently related to the intake of beta blockers (n = 80; -8.4 mU L-1; P = 0.001), angiotensin-converting enzyme (ACE)-inhibitors (n = 39; +15.9 mU L-1; P = 0.0001), diuretics (n = 62; +14.3 mU L-1; P = 0.0001), and calcium channel blockers (n = 45; +5.9 mU L-1; P = 0.05). Aldosterone levels were related to ACE-inhibition (-156.5 pmol L-1; P = 0.04) and diuretic treatment (+422.4 pmol L-1; P = 0.0001) in an opposite fashion whereas beta blockers and calcium channel blockers had no significant independent effects. The levels of the atrial natriuretic peptide were significantly related to the use of beta blockers (+3.9 pmol L-1; P = 0.002) and calcium channel blockers (+3.1 pmol L-1; P = 0.05). Finally, serum angiotensinogen levels and ACE activity were not found to be significantly affected by antihypertensive medication but were rather related to gender or genotype. CONCLUSIONS: The data emphasize that antihypertensive treatment with different classes of drugs may modulate serum levels of neurohormones substantially resulting in distinct patterns of activation. These drug-related effects may require consideration when neurohormonal activation is of functional relevance or when neurohormones serve as prognostic predictors in patients with cardiovascular disorders.

Adrenergic beta-Antagonists↗