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S Pfeiffer

Publications and source records attributed to S Pfeiffer.

At least 73 records · Page 4Linked to original sources
PubMed1997

Allosteric modulation of rat brain nitric oxide synthase by the pterin-site enzyme inhibitor 4-aminotetrahydrobiopterin.

We investigated the functional and allosteric effects of the 4-amino analogue of tetrahydrobiopterin, (6R)-2,4-diamino- 5,6,7,8-tetrahydro-6-(L-erythro-1,2-dihydroxypropyl) pteridine (4-amino-H4biopterin) on pteridine-free rat neuronal nitric oxide synthase. In the presence of added (6R)-5,6,7,8-tetrahydro-L-erythrobiopterin (H4biopterin; 10 microM), 4-amino-H4biopterin completely inhibited the conversion of both L-arginine and NG-hydroxy-L-arginine with half-maximally effective concentrations of 1.1+/-0.09 and 1.3+/-0.09 microM, respectively. Inhibition was reversible, as shown by a time-dependent restoration of citrulline formation upon dilution of the inhibitor-treated enzyme (t1/2=3.0 min). Binding of 4-amino-H4biopterin led to a complete conversion of the haem from low-spin to high-spin state, and to the formation of stable homodimers which partially survived electrophoresis under denaturating conditions. These results show that oxidation of both L-arginine and NG-hydroxy-L-arginine is pteridine-dependent, and that the allosteric effects of H4biopterin do not fully explain the essential role of the pteridine cofactor in nitric oxide biosynthesis.

Allosteric Regulation↗
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

Limits of NMR structure determination using variable target function calculations: ribonuclease T1, a case study.

Limits of NMR structure determination using multidimensional NMR spectroscopy, variable target function calculations and relaxation matrix analysis were explored using the model protein ribonuclease T1 (RNase T1). The enzyme consists of 104 amino acid residues and has a molecular mass of approximately 11 kDa. Primary experimental data comprise 1856 assigned NOE intensities, 493 3J coupling constants and 62 values of amid proton exchange rates. From these data, 2580 distance bounds, 168 allowed ranges for torsional angles and stereospecific assignments for 75% of beta-methylene protons as well as for 80% of diastereotopic methyl groups were derived. Whenever possible, the distance restraints were refined in a relaxation matrix analysis including amid proton exchange data for improvement of lower distance limits. Description of side-chain conformations were based on various models of motional averaging of 3J coupling constants. The final structure ensemble was selected from the starting ensemble comparing the global precision of structures with order parameters derived from 15N relaxation time measurements. Significant differences between the structure of RNase T1 in solution and in the crystal became apparent from a comparison of the two highly resolved structures.

Binding Sites↗
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

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

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

Dendritic cells generated from the blood of patients with multiple myeloma are phenotypically and functionally identical to those similarly produced from healthy donors.

Using a combination of GM-CSF, SCF, flk-2/flt-3 ligand, and IL-4, dendritic cells (DC) have been generated in vitro from the adherent fraction of mononuclear cells isolated from the blood of patients with MM. Analysis of cell yield showed no significant difference in DC yield (numbers or percentage of leucocytes) or total number of leucocytes generated in myeloma cultures compared to similar cultures prepared using mononuclear cells from the blood of healthy donors. The mean number of DC produced after 10d of culture were 8.19 x 10(5) and 9.87 x 10(5) cells (41% and 51% of all leucocytes) for the myeloma and normal cultures respectively. Flow cytometry investigation of phenotypic markers including CD1a, HLA-DR, CD80 (BB1/B7.1) and CD86 (B70/B7.2), and functional status (stimulatory potential in allogeneic mixed leucocyte reactions (MLR)) confirmed the generation of cells phenotypically identified as cultured DC. In addition, these cells were more effective than PBMC at stimulating allogeneic PBMC proliferation. These data demonstrate no difference between DC generated from patients with MM and healthy donors. This study was considered a prerequisite for future investigations directed towards developing effective immunotherapies for myeloma.

Adult↗
PubMed1997

AAMC STAT: a brief description.

AAMC STAT is a new, weekly electronic newsletter produced by the American Association of Medical Colleges. It provides coverage of national news related to academic medicine. This article briefly reviews the electronic publication and gives directions for subscribing to AAMC STAT.

Computer Communication Networks↗
PubMed1996

Structural and functional analogs of CuZn superoxide dismutase inhibit rat brain nitric oxide synthase by interference with the reductase (diaphorase) domain.

Copper complexes with superoxide dismutase (SOD) activity show a wide range of pharmacological activities. We have investigated the effect of ([N,N'-bis(2-pyridylmethylene)-1,4-butanediamine]-(N,N',N", N"')]-Cu(II)-chloride (Cu-PuPy) and ([N,N'-bis(2-pyridyl-phenyl)methylene-1,4-butanediamine]-(N,N',N", N"'))-Cu(II)-chloride (Cu-PuPhePy) on the multiple catalytic functions of rat brain NO synthase (NOS). Both drugs inhibited the formation of L-citrulline as well as the enzymatic reduction of cytochrome c. The uncoupled oxidation of NADPH, catalyzed by neuronal NOS in the absence of L-arginine, was inhibited by Cu-PuPy but stimulated by Cu-PuPhePy, suggesting that the phenyl-substituted compound acts as a parasitic electron acceptor. Our data identify copper complexes with SOD mimicking activity as a novel class of neuronal NOS inhibitors blocking the reductase (diaphorase) activity of the enzyme.

Animals↗
PubMed1996

Characterization of heme-deficient neuronal nitric-oxide synthase reveals a role for heme in subunit dimerization and binding of the amino acid substrate and tetrahydrobiopterin.

Neuronal nitric-oxide (NO) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multifunctional oxidoreductase catalyzing oxidation of L-arginine to L-citrulline and NO, reduction of molecular oxygen to superoxide, and electron transfer to cytochromes. To investigate how binding of the prosthetic heme moiety is related to enzyme activities, cofactor, and L-arginine binding, as well as to secondary and quaternary protein structure, we have purified and characterized heme-deficient neuronal NO synthase. The heme-deficient enzyme, which had preserved its cytochrome c reductase activity, contained FAD and FMN, but virtually no tetrahydrobiopterin, and exhibited only marginal NO synthase activity. By means of gel filtration and static light scattering, we demonstrate that the heme-deficient enzyme is a monomer and provide evidence that heme is the sole prosthetic group controlling the quaternary structure of neuronal NO synthase. CD spectroscopy showed that most of the structural elements found in the dimeric holoenzyme were conserved in heme-deficient monomeric NO synthase. However, in spite of being properly folded, the heme-deficient enzyme did bind neither tetrahydrobiopterin nor the substrate analog N(G)-nitro-L-arginine. Our results demonstrate that the prosthetic heme group of neuronal NO synthase is requisite for dimerization of enzyme subunits and for the binding of amino acid substrate and tetrahydrobiopterin.

Animals↗
PubMed1996

Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine.

1. The L-arginine derivatives NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2. A bolus of L-NAME and L-NOARG (0.25 micromol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 +/- 0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs 4.2 min). The time-dependent onset of the inhibitory effect of L-NAME was paralleled by the appearance of L-NOARG in the coronary effluent. 3. Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 microM) than L-NOARG (IC50 = 1.4 microM), but the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L-NAME closely correlated with hydrolysis of the drug to L-NOARG. 4. Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365 +/- 11.2 min in buffer (pH 7.4), 207 +/- 1.7 min in human plasma, and 29 +/- 2.2 min in whole blood (n = 3 in each case). When L-NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG in blood cells. 5. These results suggest that L-NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.

Animals↗
PubMed1996

[Radical lung surgery interventions with special reference to lymphadenectomy].

Intraoperative lymph node staging plays an essential part in the surgical treatment of lung cancer. The mean number of resected lymph nodes in 237 patients was 17, with a minimum of 1 and a maximum of 60. The largest number was found in N1 regions as expected. The number of resected nodes in the paratracheal, tracheobronchial and subcarinal region was 4, in the paraoesophageal and ligament region 1.2. Twenty-two percent of the whole group showed an N2 situation. In 45% of the patients with N2 invasion, we found a lymph node skipping and in 25% only one positive N2 node. Thus, it is obvious that without a systematic lymph node dissection an exact staging is not possible.

Carcinoma, Bronchogenic↗
PubMed1996

[Thoracic surgery in advanced age].

From 1990 through 1992, 70 patients 70 years of age or older (mean 72.8 years) underwent surgery for lung cancer at our hospital (47 males/23 females). 68 patients took part in a follow-up. Operative treatment consisted of standard lung resections in 42 cases. 17 patients underwent enlarged resections. In the postoperative period, complications occurred in 32 cases (47%). The overall operative mortality rate of 13% was mainly due to concomitant cardiovascular diseases and enlarged lung resections. Critical preoperative evaluation of the patients' functional status and selection of the proper operative procedure promise a successful treatment of lung cancer in the elderly.

Aged↗
PubMed1995

Brief communication: cortical remodeling data are affected by sampling location.

It has been argued that techniques for estimating adult age-at-death from cortical histology are deleteriously affected by sampling location. This study uses nine complete femoral midshaft cross-sections to test the effect of sampling site on measurement of a standard histological variable, percent remodeled bone. Circumferential periosteal fields from four anatomically defined locations (anterior, posterior, medial, lateral) and four mechanically defined locations (maximum and minimum moments of area) were evaluated. Locations deviating from the periosteal surface toward the endosteal surface were also compared. Significant differences were found for both location and field placement. The anatomical axes exhibited greater variability than the mechanical axes, in particular the anterior location, a standard sampling site for age-at-death estimation techniques. More endosteal fields tended to show elevated levels of percent remodeled bone. This study demonstrates that circumferential and radial sampling locations are important considerations in deriving and applying predictive equations based on cortical remodeling.

Adult↗
PubMed1993

Family involvement in residential treatment of children with psychiatric disorder and mental retardation.

OBJECTIVE: The goals of the study were to determine the extent of family involvement with children in residential treatment settings, to examine whether involvement diminishes over time, and to examine the relationship between involvement and the child's diagnosis as well as the demographic characteristics of the child and the family. METHODS: Staff rated the level and frequency of family involvement with 234 children, aged five to 19 years, living in three residential treatment centers. The children were grouped according to three diagnostic categories: psychiatric disorder, mental retardation, and dual diagnosis of psychiatric disorder and mental retardation. RESULTS: Almost one-third of the children had no family contact, and about 50 percent had three or fewer contacts per year. The level of family involvement was lower with dually diagnosed children than with children who had only psychiatric disorder or mental retardation. Level of involvement was also related to the driving time between the family home and the residential facility and to socioeconomic status, variables that seemed to account for the lower level of involvement of families with dually diagnosed children. CONCLUSIONS: Greater distance between the family home and the residential treatment center and low socioeconomic status greatly limit a family's accessibility for involvement with children in out-of-home placements. These obstacles are more likely to occur in families of dually diagnosed children.

Adolescent↗
PubMed1993

Evaluation of morphological and histological adult skeletal age-at-death estimation techniques using ribs.

Adult age-at-death estimation standards were applied to an independent sample (N = 50, 55) of documented ages 17.5 to 95 years. Estimates derived from the sternal rib end morphological and from the cortical rib histological techniques were compared with each other and to the documented ages. Comparisons to the documented ages reveal no statistically significant differences between the techniques. However, the comparison of each individual's estimates show a poor correlation (r = 0.54) despite the equal performance of the age estimations on the entire sample. Averaging of the two rib age estimates results in an estimate with a stronger Pearson's r (0.86) and a lower standard error of the estimate (7.5 years).

Adolescent↗