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Biomedical subjects

B Mayer

Publications and source records attributed to B Mayer.

At least 55 records · Page 3Linked to original sources
PubMed2001

Effects of an onion-olive oil maceration product containing essential ingredients of the Mediterranean diet on blood pressure and blood fluidity.

Twenty-four patients with arterial hypertension (WHO class I) received either 4 capsules of an onion-olive oil maceration product, containing essential ingredients of the Mediterranean diet, or placebo daily over a period of one week. In order to investigate the acute effect on arterial blood pressure, measurements were performed before and 5 h after the administration of the first dose of 4 capsules verum or placebo, respectively. For the evaluation of the long term effect further blood pressure measurements were performed after one week's treatment with a daily dose of 4 capsules. After a wash-out phase of 2 weeks followed by a crossover, the second study phase, which was identical in design, was carried out. In addition patients were instructed to measure their blood pressure 4 times daily at home for the whole study period. Haemorheological parameters (platelet aggregation, erythrocyte aggregation, plasma viscosity and haematocrit) were also determined at the measuring points mentioned above. The onion-olive oil maceration product led to a significant decrease in systolic blood pressure. There was also a trend towards a decrease in diastolic blood pressure. The improved blood fluidity observed resulted from a decrease in haematocrit. All effects could be shown immediately and after one week's administration.

Adult↗
PubMed2001

Protein tyrosine nitration in mouse peritoneal macrophages activated in vitro and in vivo: evidence against an essential role of peroxynitrite.

Tyrosine nitration is considered a key reaction of peroxynitrite-triggered tissue injury in inflammatory diseases. We investigated the potential involvement of peroxynitrite in protein tyrosine nitration in isolated murine peritoneal macrophages activated either in vitro with interferon-gamma/lipopolysaccharide or in vivo by priming mice with Corynebacterium parvum (10 mgxkg-1). Both protocols led to release of NO and accumulation of nitrite accompanied by formation of protein-bound 3-nitrotyrosine. Oxidation of dihydrorhodamine 123, a measure of peroxynitrite release, remained close to basal levels upon in vitro activation of the macrophages but was increased approximately twofold in vivo. Tyrosine nitration in macrophages activated in vitro was inhibited by catalase and the time course of nitration correlated with nitrite accumulation, whereas superoxide (O2*-) and H2O2 release occurred at much earlier times. To address the contribution of O2*- and peroxynitrite to in vivo nitration, a O2*- scavenger (MnTBAP; 1 mgxkg-1) was given to C. parvum-primed mice. MnTBAP led to almost complete inhibition of C. parvum-triggered O2*- and peroxynitrite release, whereas nitrite accumulation and formation of 3-nitrotyrosine were less affected ( approximately 50% of controls). These results argue against an essential role of peroxynitrite in protein tyrosine nitration in vivo.

Animals↗
PubMed2001

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

Blotting, Southern↗
PubMed2001

Molecular mechanisms involved in the synergistic activation of soluble guanylyl cyclase by YC-1 and nitric oxide in endothelial cells.

YC-1 is a direct activator of soluble guanylyl cyclase (sGC) and sensitizes the enzyme for activation by nitric oxide (NO) and CO. Because the potentiating effect of YC-1 on NO-induced cGMP formation in platelets and smooth muscle cells has been shown to be substantially higher than observed with the purified enzyme, the synergism between heme ligands and YC-1 is apparently more pronounced in intact cells than in cell-free systems. Here, we investigated the mechanisms underlying the synergistic activation of sGC by YC-1 and NO in endothelial cells. Stimulation of the cells with YC-1 enhanced cGMP accumulation up to approximately 100-fold. The maximal effect of YC-1 was more pronounced than that of the NO donor DEA/NO (approximately 20-fold increase in cGMP accumulation) and markedly diminished in the presence of L-N(G)-nitroarginine, EGTA, or oxyhemoglobin. Because YC-1 did not activate endothelial NO synthase, the pronounced effect of YC-1 on cGMP accumulation was apparently caused by a synergistic activation of sGC by YC-1 and basal NO. The effect of YC-1 was further enhanced by addition of DEA/NO, resulting in a approximately 160-fold stimulation of cGMP accumulation. Thus, YC-1 increased the NO-induced accumulation of cGMP in intact cells by approximately 8-fold. Addition of endothelial cell homogenate increased the stimulatory effect of YC-1 on NO-activated purified sGC from 1.2- to 3.7-fold. This effect was not observed with heat-denatured homogenates, suggesting that a heat-labile factor present in endothelial cells potentiates the effect of YC-1 on NO-activated sGC.

Animals↗
PubMed2001

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells.

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.

Calcium Chloride↗
PubMed2001

Ansatz for dynamical hierarchies.

Complex, robust functionalities can be generated naturally in at least two ways: by the assembly of structures and by the evolution of structures. This work is concerned with spontaneous formation of structures. We define the notion of dynamical hierarchies in natural systems and show the importance of this particular kind of organization for living systems. We then define a framework that enables us to formulate, investigate, and manipulate such dynamical hierarchies. This framework allows us to simultaneously investigate different levels of description together with their interrelationship, which is necessary to understand the nature of dynamical hierarchies. Our framework is then applied to a concrete and very simple formal, physicochemical, dynamical hierarchy involving water and monomers at level one, polymers and water at level two, and micelles (polymer aggregates) and water at level three. Formulating this system as a simple two-dimensional molecular dynamics (MD) lattice gas allows us within one dynamical system to demonstrate the successive emergence of two higher levels (three levels all together) of robust structures with associated properties. Second, we demonstrate how the framework for dynamical hierarchies can be used for realistic (predictive) physicochemical simulation of molecular self-assembly and self-organization processes. We discuss the detailed process of micellation using the three-dimensional MD lattice gas. Finally, from these examples we can infer principles about formal dynamical hierarchies. We present an ansatz for how to generate robust, higher-order emergent properties in formal dynamical systems that is based on a conjecture of a necessary minimal complexity within the fundamental interacting structures once a particular simulation framework is chosen.

Chemical Phenomena↗
PubMed2001

Defense of the ansatz for dynamical hierarchies.

Gross and McMullin [Artificial Life, 7, 355-365] criticize the conclusions of our article on dynamical hierarchies [Artificial Life, 7, 329-353]. In this note we respond to their criticisms. After clarifying our ansatz, we argue that the simulations presented by Gross and McMullin present no evidence against the ansatz, in part because their simulations use a different simulation framework, and in part because their simulations are no less complex than ours. We also clarify why the micelles in our simulations are third-order emergent structures, and why we emphasize realism in our simulation.

Models, Theoretical↗
PubMed2001

The revised version of the Screen for Child Anxiety Related Emotional Disorders (SCARED-R): treatment sensitivity in an early intervention trial for childhood anxiety disorders.

OBJECTIVES: The revised version of the Screen for Childhood Anxiety Related Emotional Disorders (SCARED-R) is a recently developed self-report questionnaire for measuring DSM-IV defined anxiety disorder symptoms in children. The current study examined the treatment sensitivity of the SCARED-R. METHODS: Thirty-six children (aged 8-13 years) who fulfilled the criteria for generalized anxiety disorder, separation anxiety disorder, and/or social phobia received either group or individual cognitive-behavioural treatment. Children completed the SCARED-R and a traditional measure of childhood anxiety, the State-Trait Anxiety Inventory for Children (STAIC), at three points in time: (1) 6 months before treatment (i.e. baseline), (2) pre-treatment, and (3) post-treatment. RESULTS: Results showed that children's SCARED-R and STAIC scores did not change from baseline to pre-treatment, but significantly declined from pre-treatment to post-treatment. An additional finding of the present study was that group and individual CBT were equally effective in reducing children's anxiety symptoms. CONCLUSIONS: It can be concluded that the SCARED-R reliably taps treatment effects and thus should be regarded as a useful self-report index of childhood anxiety in clinical and research settings. Furthermore, the data support the notion that CBT should be generally regarded as an efficacious intervention for anxiety disordered children.

Adolescent↗
PubMed2000

Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase.

Nitric-oxide synthases (NOS) are homodimeric proteins and can form an intersubunit Zn(4S) cluster. We have measured zinc bound to NOS purified from pig brain (0.6 mol/mol of NOS) and baculovirus-expressed rat neuronal NOS (nNOS) (0.49 +/- 0.13 mol/mol of NOS), by on-line gel-filtration/inductively coupled plasma mass spectrometry. Cobalt, manganese, molybdenum, nickel, and vanadium were all undetectable. Baculovirus-expressed nNOS also bound up to 2. 00 +/- 0.58 mol of copper/mol of NOS. Diethylenetriaminepentaacetic acid (DTPA) reduced the bound zinc to 0.28 +/- 0.07 and the copper to 0.97 +/- 0.24 mol/mol of NOS. Desalting of samples into thiol-free buffer did not affect the zinc content but completely eliminated the bound copper (</=0.02 mol/mol of NOS). Most (> or =75%) of the bound zinc was released from baculovirus-expressed rat nNOS by p-chloromercuriphenylsulfonic acid (PMPS). PMPS-treated nNOS was strongly (90 +/- 5%) inactivated. To isolate functional effects of zinc release from other effects of PMPS, PMPS-substituted thiols were unblocked by excess reduced thiol in the presence of DTPA, which hindered reincorporation of zinc. The resulting enzyme contained 0.12 +/- 0.05 mol of zinc but had a specific activity of 426 +/- 46 nmol of citrulline.mg(-1).min(-1), corresponding to 93 +/- 10% of non-PMPS-treated controls. PMPS also caused dissociation of nNOS dimers under native conditions, an effect that was blocked by the pteridine cofactor tetrahydrobiopterin (H(4)biopterin). H(4)biopterin did not affect zinc release. Even in the presence of H(4)biopterin, PMPS prevented conversion of NOS dimers to an SDS-resistant form. We conclude that zinc binding is a prerequisite for formation of SDS-resistant NOS dimers but is not essential for catalysis.

4-Chloromercuribenzenesulfonate↗
PubMed2000

Low-temperature optical absorption spectra suggest a redox role for tetrahydrobiopterin in both steps of nitric oxide synthase catalysis.

To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.

Animals↗
PubMed2000

The role of tetrahydrobiopterin in the activation of oxygen by nitric-oxide synthase.

We have studied the reaction of reduced nitric-oxide synthase (NOS) with molecular oxygen at -30 degrees C. In the first reaction cycle (from L-Arg to hydroxy-L-Arg), an oxygen adduct complex formed rapidly. Experiments in the absence of the reductase domain demonstrated that this complex was then further reduced by one electron stemming from the cofactor tetrahydrobiopterin (BH4). Spectral evidence suggested an iron(IV) porphyrin pi-cation radical as an intermediate. The nature of the oxidized BH4 was identified by EPR as a BH3* radical. Within the second cycle (from hydroxy-L-Arg to citrulline and NO), an iron(III)-NO complex could be identified clearly by its spectral characteristics. The strict requirement of BH4 for its formation suggests that BH4 plays a redox role, although transient, also in the second reaction cycle.

Biopterins↗
PubMed2000

Inhibition of purified soluble guanylyl cyclase by L-ascorbic acid.

OBJECTIVE: L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide (NO)/cGMP signalling. The aim of the present study was to investigate the effect of vitamin C on the most prominent physiological target of endogenous and exogenous NO, i.e. soluble guanylyl cyclase (sGC). METHODS: To address this issue we used a highly purified enzyme preparation from bovine lung (from the slaughterhouse). Enzymic activity was measured by a standard assay based on the conversion of [alpha-32P]GTP to [32P]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified using a commercially available Clark-type electrode. RESULTS: Stimulation of sGC by the NO donor 2, 2-diethyl-1-nitroso-oxyhydrazine was inhibited by ascorbate with an IC(50) of approximately 2 microM. Maximal enzyme inhibition ( approximately 70%) was observed at 0.1-1 mM vitamin C. Stimulation of sGC by the NO-independent activator protoporphyrin-IX was also inhibited with similar potency. The effect of ascorbate on sGC was largely antagonised by reduced glutathione (1 mM) and the specific iron chelator diethylenetriaminepentaacetic acid (0.1 mM). Electrochemical experiments revealed that NO is potently scavenged by vitamin C. Consumption of NO by ascorbate was prevented by reduced glutathione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide dismutase (500 units/ml) whereas up to 5000 units/ml superoxide dismutase failed to restore sGC activity. CONCLUSIONS: Our results suggest that physiological concentrations of L-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC.

Animals↗
PubMed2000

Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.

Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.

Animals↗
PubMed2000

Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method for the determination of nitric oxide synthase activity is described. The method is based on the gas chromatographic-mass spectrometric measurement of L-[15N2]arginine-derived [15N]nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of [15N]nitrite formation by purified neuronal nitric oxide synthase revealed K(M) values of 3.1 microM by Hanes and 4.6 microM by Lineweaver-Burk for L-[15N2]arginine. The corresponding Vmax values were 0.204 and 0.228 micromol [15N]nitrite min(-1) mg(-1) NOS, respectively. N(G)-Nitro-L-arginine and N(G),N(G)-dimethylarginine (asymmetric dimethylarginine) were identified by this method as the most potent enzyme inhibitors. Nitric oxide synthase activity was also assessed in vivo by i.v. injection of L-[15N2]arginine in a rat and determination of plasma [15N]nitrite and [15N]nitrate. The assay described in this work allows for accurate, specific and highly sensitive determination of nitric oxide synthase activity in vitro and in vivo.

Animals↗
PubMed2000

Interaction of 4-hydroxybiphenyl with cyclodextrins: effect of complex structure on spectroscopic and photophysical properties.

The structures, spectroscopic and photophysical properties of the inclusion complexes between 4-OH-biphenyl and cyclodextrins (CD) in water were studied theoretically and experimentally. The complex structures were predicted using a dynamic Monte Carlo procedure including solvation effects and analyzed via computed and experimentally determined circular dichroism. The interpretation of the induced circular dichroism spectra indicated the formation of stable 2:2 complexes between the probe and alpha-CD, while 1:1 structures account for the circular dichroism induced by beta- and gamma-CD. Fluorescence emission quantum yields and lifetimes, measured as a function of the CD concentration, confirmed the formation of this higher order complex with alpha-CD. Prototropic equilibration in the singlet excited state was found to be depressed in 2:2 complexes due to the hydrophobic environment of the OH groups, while it remained unperturbed in 1:1 complexes, where the substituent is exposed to the aqueous environment. Triplet-triplet absorption and triplet quenching data supported this interpretation. The photophysical properties of both the 1:1 and the 2:2 complexes are characterized by a significant reduction of the nonradiative decay rates.

Biphenyl Compounds↗
PubMed2000

Nitric oxide-induced autoinhibition of neuronal nitric oxide synthase in the presence of the autoxidation-resistant pteridine 5-methyltetrahydrobiopterin.

Nitric oxide synthase (NOS) catalysis results in formation of NO or superoxide (O(2)(-.)) depending on the presence or absence of the cofactor tetrahydrobiopterin (BH4). In the absence of O(2)(-.) scavengers, net NO formation cannot be detected even at saturating BH4 concentrations, which is thought to be due to O(2)(-.) production by BH4 autoxidation. Because the N-5-methylated analogue of BH4 (5-Me-BH4) sustains NOS catalysis and is autoxidation-resistant, net NO formation by the neuronal isoform of NOS (nNOS) can be observed at saturating 5-Me-BH4 concentrations. Here we compare the effects of 5-Me-BH4 on L-citrulline formation, NADPH oxidation, H(2)O(2) production and soluble guanylate cyclase (sGC) stimulation. All activities were stimulated biphasically (EC(50) approx. 0.2 microM and more than 1 mM), with an intermediate inhibitory phase at the same pterin concentration as that required for net NO generation and sGC stimulation (4 microM). Concomitantly with inhibition, the NADP(+)/L-citrulline stoichiometry decreased from 2.0 to 1.6. Inhibition occurred only at high enzyme concentrations (IC(50) approx. 10 nM nNOS) and was antagonized by oxyhaemoglobin and by BH4. We ascribe the first stimulatory phase to high-affinity binding of 5-Me-BH4. The inhibitory phase is due to low-affinity binding, resulting in fully coupled catalysis, complete inhibition of O(2)(-.) production and net NO formation. At high enzyme concentrations and thus high NO levels, this causes autoinhibition. NO scavenging by 5-Me-BH4 at concentrations above 1 mM, resulting in the antagonization of inhibition of NOS, explains the second stimulatory phase. In agreement with these assignments 5-Me-BH4 was found to stimulate formation of a haem-NO complex during NOS catalysis. The observation of inhibition with 5-Me-BH4 but not with BH4 implies that, unless O(2)(-.) scavengers are present, a physiological role for NO-induced autoinhibition is unlikely.

Animals↗
PubMed2000

GABA(A) receptor assembly. Identification and structure of gamma(2) sequences forming the intersubunit contacts with alpha(1) and beta(3) subunits.

GABA(A) receptors are ligand-gated chloride channels composed of five homologous subunits that specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we constructed C-terminal truncated gamma(2) subunits, as well as mutated and chimeric fragments. From their ability to interfere with alpha(1)beta(3)gamma(2) receptor assembly and to associate with full-length subunits, we concluded that amino acid sequences gamma(2)-(91-104) and gamma(2)-(83-90) form the sites mediating assembly with alpha(1) and beta(3) subunits, respectively. Neural network-based secondary structure prediction, Monte Carlo optimization, and hydrophobicity analysis led to the conclusion that these sites also form the intersubunit contacts in the completely assembled receptor and provided important information on the benzodiazepine-binding site and structure of GABA(A) receptors.

Animals↗