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Biomedical subjects

B Schmidt

Publications and source records attributed to B Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed2001

Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase.

The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs. A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface. A non-redox-active Mg(2+) site is located in the interior of this region, and our previous studies [Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G. T., and Ferguson-Miller, S. (1998) in Phototropic Prokaryotes (Peschek, G. A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York] have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with (2)H(2)O. Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze-quench and electron spin echo envelope modulation (ESEEM) analysis of Mn(2+)-substituted cytochrome c oxidase, which retains full activity. In the oxidized enzyme at room temperature, deuterium exchange at the Mn(2+) site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s(-1). The extent of deuterium substitution is dependent on the concentration of (2)H(2)O in the sample, indicative of rapid equilibrium, with three inner sphere (2)H(2)O exchanged per Mn(2+). This indicates that the water ligands of the Mn(2+)/Mg(2+) site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover.

Animals↗
PubMed2001

A naturally occurring nonpolymerogenic mutant of alpha 1-antitrypsin characterized by prolonged retention in the endoplasmic reticulum.

The classical form of alpha 1-antitrypsin (alpha 1-AT) deficiency is associated with a mutant alpha 1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) Z allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of alpha 1-AT in a family in which three affected members had severe liver disease associated with alpha 1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI Z allele and a second allele, the PI Z + saar allele, bearing the mutation that characterizes alpha 1-ATZ as well as the mutation that characterizes alpha 1-AT Saarbrucken (alpha 1-AT saar). The mutation in PI saar introduces a premature termination codon resulting in an alpha 1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the alpha 1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of alpha 1-AT saar and alpha 1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant alpha 1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in alpha 1-AT deficiency.

Alleles↗
PubMed2001

Frequency of genotypic and phenotypic drug-resistant HIV-1 among therapy-naive patients of the German Seroconverter Study.

Genotypic and phenotypic resistance of viral reverse transcriptase (RT) and protease (PR) was determined for 64 therapy-naive, HIV-1-infected seroconverters of the German Seroconverter Study coordinated by the Robert Koch-Institut, Berlin. The date of seroconversion of patients and the laboratory, clinical, and therapeutic follow-up data were documented. Samples were collected between 1996 and 1999. Phenotypic resistant HIV-1 were found in 8 (13%) seroconverters; in most cases resistance was weak and mainly directed against RT inhibitors (4 nucleoside reverse transcriptase inhibitors [NRTIs], 2 nonnucleoside reverse transcriptase inhibitors [NNRTIs], 1 combination NRTI/NNRTI). Only one infection with a weak PR inhibitor resistance was identified. Transmission of multidrug-resistant HIV-1 has not yet been observed. Frequently at least one or more amino acid mutations associated with antiretroviral drug resistance were detected by genotypic analysis. The mean number of resistance-associated mutations in the RT of the transmitted virus has increased significantly since 1996. Studies have shown the improved benefit of initial antiretroviral therapy if based on genotypic resistance data. In view of the considerably high level of transmission of resistant HIV-1 in Germany, which is also seen in other studies in Europe and the United States, we suggest determining the genotypic resistance pattern before starting therapy of newly HIV-1-infected patients.

Anti-HIV Agents↗
PubMed2001

Prion protein binds copper within the physiological concentration range.

The prion protein is known to be a copper-binding protein, but affinity and stoichiometry data for the full-length protein at a physiological pH of 7 were lacking. Furthermore, it was unknown whether only the highly flexible N-terminal segment with its octarepeat region is involved in copper binding or whether the structured C-terminal domain is also involved. Therefore we systematically investigated the stoichiometry and affinity of copper binding to full-length prion protein PrP(23-231) and to different N- and C-terminal fragments using electrospray ionization mass spectrometry and fluorescence spectroscopy. Our data indicate that the unstructured N-terminal segment is the cooperative copper-binding domain of the prion protein. The prion protein binds up to five copper(II) ions with half-maximal binding at approximately 2 microm. This argues strongly for a direct role of the prion protein in copper metabolism, since it is almost saturated at about 5 microm, and the exchangeable copper pool concentration in blood is about 8 microm.

Animals↗
PubMed2001

Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis.

Arylsulfatase A (ASA) belongs to the sulfatase family whose members carry a C(alpha)-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The crystal structures of two arylsulfatases, ASA and ASB, and kinetic studies on ASA mutants led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. The structures of two ASA mutants that lack the functional C(alpha)-formylglycine residue 69, in complex with a synthetic substrate, have been determined in order to unravel the reaction mechanism. The crystal structure of the inactive mutant C69A-ASA in complex with p-nitrocatechol sulfate (pNCS) mimics a reaction intermediate during sulfate ester hydrolysis by the active enzyme, without the covalent bond to the key side-chain FGly69. The structure shows that the side-chains of lysine 123, lysine 302, serine 150, histidine 229, the main-chain of the key residue 69 and the divalent cation in the active center are involved in sulfate binding. It is proposed that histidine 229 protonates the leaving alcoholate after hydrolysis.C69S-ASA is able to bind covalently to the substrate and hydrolyze it, but is unable to release the resulting sulfate. Nevertheless, the resulting sulfation is low. The structure of C69S-ASA shows the serine side-chain in a single conformation, turned away from the position a substrate occupies in the complex. This suggests that the double conformation observed in the structure of wild-type ASA is more likely to correspond to a formylglycine hydrate than to a twofold disordered aldehyde oxo group, and accounts for the relative inertness of the C69S-ASA mutant. In the C69S-ASA-pNCS complex, the substrate occupies the same position as in the C69A-ASA-pNCS complex, which corresponds to the non-covalently bonded substrate. Based on the structural data, a detailed mechanism for sulfate ester cleavage is proposed, involving an aldehyde hydrate as the functional group.

Alanine↗
PubMed2001

Metabolism of the herbicide glufosinate-ammonium in plant cell cultures of transgenic (rhizomania-resistant) and non-transgenic sugarbeet (Beta vulgaris), carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium).

The metabolism of the herbicide glufosinate-ammonium was investigated in heterotrophic cell suspension and callus cultures of transgenic (bar-gene) and non-transgenic sugarbeet (Beta vulgaris). Similar studies were performed with suspensions of carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium). 14C-labelled chemicals were the (racemic) glufosinate, L-glufosinate, and D-glufosinate, as well as the metabolites N-acetyl L-glufosinate and 3-(hydroxymethylphosphinyl)propionic acid (MPP). Cellular absorption was generally low, but depended noticeably on plant species, substance and enantiomer. Portions of non-extractable residues ranged from 0.1% to 1.2% of applied 14C. Amounts of soluble metabolites resulting from glufosinate or L-glufosinate were between 0.0% and 26.7% of absorbed 14C in non-transgenic cultures and 28.2% and 59.9% in transgenic sugarbeet. D-Glufosinate, MPP and N-acetyl L-glufosinate proved to be stable. The main metabolite in transgenic sugarbeet was N-acetyl L-glufosinate, besides traces of MPP and 4-(hydroxymethylphosphinyl)butanoic acid (MPB). In non-transgenic sugarbeet, glufosinate was transformed to a limited extent to MPP and trace amounts of MPB. In carrot, D stramonium and D purpurea, MPP was also the main product; MPB was identified as a further trace metabolite in D stramonium and D purpurea.

Aminobutyrates↗
PubMed2001

[Virtual bronchoscopy].

Flexible bronchoscopy represents a clinically well-established invasive diagnostic tool. Virtual bronchoscopies, calculated from thin-slice CT sections, allow astonishing immitations of reality although principal differences exist between both technologies: the fact that colour representation is artificial and concommitant interventions are impossible limits the clinical use of virtual bronchoscopy. However, its value increases when calculations can be attained within minutes due to technological advancements, and when virtually any chest CT is suitable for further postprocessing. Indications, findings and the clinical role of virtual bronchoscopy are discussed.

Bronchoscopy↗
PubMed2001

Endotracheal balloon dilatation and stent implantation in benign stenoses.

BACKGROUND: Tracheal reconstruction is the treatment of choice in nontumorous tracheal stenoses, but recurrences and concomitant medical conditions limit this approach. We investigated the outcome after balloon dilatation and silicone stent implantation. METHODS: Forty-two patients with inoperable tracheal stenoses underwent balloon dilatation and afterward silicone stent implantation. Patients were divided into two groups, in group A 24 patients received tracheal stents as a temporary treatment. In group B, definitive stenting was done in 18 patients with severe concomitant medical conditions that did not allow for stent removal. RESULTS: Immediate results were satisfactory in all patients. In group A, stents could be removed in 12 patients after a mean interval of 20 months. Restenting was not required during the following 18.9 months. Twelve patients are still waiting for stent removal after a mean follow-up of 20 months. In group B, mean follow-up is now 48.4 months. Complications included retained secretions, dislocation, and granuloma formation. CONCLUSIONS: Stenting after balloon dilatation is safe and effective in benign tracheal stenoses. After temporary use, stents can be removed when local and general conditions permit. In all other patients, stenting proved beneficial for 5 years as more definitive treatment.

Adult↗
PubMed2001

Gene transfer into solid tumours--is a special application device beneficial?

The replacement of inactivated tumour suppressor genes is a promising approach in cancer therapy. The aim of this study was to evaluate the influence of technical determinants on the efficiency of adenoviral-mediated gene transfer into solid tumours. Therefore, we compared the efficacy of two different injection needle types, a conventional needle and a modified needle characterised by perforations at the side of the shaft in vivo. The total amount of adenoviral vector DNA and the activity of the transferred reporter gene were quantitatively analysed by polymerase chain reaction (PCR) specific for the E4 region of the Ad vector genome and the beta-galactosidase assay, respectively. The levels of adenoviral DNA, as well as the total beta-galactosidase activity, varied widely, but were not significantly different for the two groups. These results suggest, that the efficiency of intratumoral gene transfer cannot be improved by the design of the application device.

Adenoviridae↗
PubMed2001

1.3 A structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family.

BACKGROUND: Sulfatases constitute a family of enzymes with a highly conserved active site region including a Calpha-formylglycine that is posttranslationally generated by the oxidation of a conserved cysteine or serine residue. The crystal structures of two human arylsulfatases, ASA and ASB, along with ASA mutants and their complexes led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. RESULTS: The crystal structure of a bacterial sulfatase from Pseudomonas aeruginosa (PAS) has been determined at 1.3 A. Fold and active site region are strikingly similar to those of the known human sulfatases. The structure allows a precise determination of the active site region, unequivocally showing the presence of a Calpha-formylglycine hydrate as the key catalytic residue. Furthermore, the cation located in the active site is unambiguously characterized as calcium by both its B value and the geometry of its coordination sphere. The active site contains a noncovalently bonded sulfate that occupies the same position as the one in para-nitrocatecholsulfate in previously studied ASA complexes. CONCLUSIONS: The structure of PAS shows that the resting state of the key catalytic residue in sulfatases is a formylglycine hydrate. These structural data establish a mechanism for sulfate ester cleavage involving an aldehyde hydrate as the functional group that initiates the reaction through a nucleophilic attack on the sulfur atom in the substrate. The alcohol is eliminated from a reaction intermediate containing pentacoordinated sulfur. Subsequent elimination of the sulfate regenerates the aldehyde, which is again hydrated. The metal cation involved in stabilizing the charge and anchoring the substrate during catalysis is established as calcium.

Arylsulfatases↗
PubMed2001

Complete compression ultrasound for the diagnosis of proximal and distal deep venous thrombosis--a retrospective outcome study.

BACKGROUND: Compression ultrasound is considered the preferred test for the diagnosis of deep vein thrombosis of the leg (DVT). Since sensitivity for distal thrombosis is low-additional tests are required. We developed a protocol of complete compression ultrasound of all venous segments of the leg (CCUS). A retrospective outcome study was performed to get an estimate of the rate of indeterminate results necessitating repeated testing as well as for the clinical safety of CCUS in a cohort of consecutive, unselected patients. PATIENTS AND METHODS: Case records of all patients referred for clinical suspicion of deep vein thrombosis within a three months period were reviewed. Patients with negative CCUS were followed directly or via the general practitioner in order to know whether an episode of venous thromboembolism had been documented since the initial CCUS. RESULTS: 132 inpatients and 154 outpatients were identified. Clinical probability was high in 50 patients, medium in 142, and low in 94. The first CCUS was negative in 209 cases. Five patients (1.8%) had repeated CCUS within the next 7 days because of incomplete visualisation of the distal veins and turned out to be negative as well. Of all 214 patients with negative CCUS a clinical follow-up information was obtained after 168 +/- 25 days. Five patients had died, none due to pulmonary embolism. In two patients deep vein thrombosis had been documented (0.9% [95% CI: 0.1-3.3%]) 148 and 172 days after CCUS, respectively. CONCLUSION: CCUS for diagnosis of DVT needs to be repeated in very few cases only. Clinical safety seems to fall into the same range as with combined algorithms and should be tested in a prospective design. Patients with medium and high probability showed a very low incidence of DVT within three months following CCUS; therefore, they may be included in a prospective outcome study.

Adolescent↗
PubMed2001

Lipid mediator class switching during acute inflammation: signals in resolution.

Leukotrienes (LTs) and prostaglandins (PGs) amplify acute inflammation, whereas lipoxins (LXs) have unique anti-inflammatory actions. Temporal analyses of these eicosanoids in clinical and experimental exudates showed early coordinate appearance of LT and PG with polymorphonuclear neutrophil (PMN) recruitment. This was followed by LX biosynthesis, which was concurrent with spontaneous resolution. Human peripheral blood PMNs exposed to PGE2 (as in exudates) switched eicosanoid biosynthesis from predominantly LTB4 and 5-lipoxygenase (5-LO)-initiated pathways to LXA4, a 15-LO product that "stopped" PMN infiltration. These results indicate that first-phase eicosanoids promote a shift to anti-inflammatory lipids: functionally distinct lipid-mediator profiles switch during acute exudate formation to "reprogram" the exudate PMNs to promote resolution.

Animals↗
PubMed2001

Isolation and polymerase chain reaction typing of Borrelia afzelii from a skin lesion in a seronegative patient with generalized ulcerating bullous lichen sclerosus et atrophicus.

A 64-year-old woman presented with bullous and ulcerating lichen sclerosus et atrophicus (LSA) on the neck, trunk, genital and perigenital area and the extremities. Histology of lesional skin showed the typical manifestations of LSA; in one of the biopsies spirochaetes were detected by silver staining. Despite treatment with four courses of ceftriaxone with or without methylprednisone for up to 20 days, progression of LSA was only stopped for a maximum of 1 year. Spirochaetes were isolated from skin cultures obtained from enlarging LSA lesions. These spirochaetes were identified as Borrelia afzelii by sodium dodecyl sulphate--polyacrylamide gel electrophoresis and polymerase chain reaction (PCR) analyses. However, serology for B. burgdorferi sensu lato was repeatedly negative. After one further 28-day course of ceftriaxone the lesions stopped expanding and sclerosis of the skin was diminished. At this time cultures for spirochaetes and PCR of lesional skin for B. afzelii DNA remained negative. These findings suggest a pathogenetic role for B. afzelii in the development of LSA and a beneficial effect of appropriate antibiotic treatment.

Borrelia↗
PubMed2001

[Dose reduction in spiral-CT: detection of pulmonary coin lesions with and without anatomically adjusted modulation of tube current].

PURPOSE: To investigate the potential of projection angle-dependent anatomical adaptation of tube current to reduce dose in spiral CT examinations of the thorax without loss in detectability of pulmonary nodules and image quality. PATIENTS AND METHODS: Dose can be reduced for non-circular patient cross-sections without an increase in noise if the tube current is reduced at those angular positions where the patient diameter and, consequently, attenuation are small. The examinations were dose with SOMATOM Plus 4 (Siemens AG). CT projection data were analysed to determine the optimum tube current for each projection angle in real time. We compared image quality, done, and detection rates of pulmonary nodules for thoracic spiral CT examinations with and without online anatomically adapted tube current control in a group of 38 patients. Three radiologists counted all intrapulmonary nodules in consensus separated in three different groups (< 5 mm, 5-10 mm, > 10 mm). Image quality was evaluated in a scale from 1-3 (1 = very good, 2 = good, 3 = worse). RESULTS: On average, the dose was reduced by 21% (15-34%). With a constant tube current, 704 pulmonary nodules were detected, with tube current modulation 707 pulmonary nodules. For three patients we saw more pulmonary nodules (five) with tube current modulation, for two patients we saw less pulmonary nodules (two). In a direct comparison, the missed intrapulmonary nodules were also detected in the respective method. Thus, the detection rate of intrapulmonary nodules was uninfluenced. In general, no deterioration of image quality was observed. CONCLUSION: On average, 21% dose reduction was achieved by an anatomically adapted tube current modulation in spiral CT examinations of the thorax without a loss in detectability of pulmonary nodules and image quality. This method is an important contribution to dose reduction in spiral CT.

Diagnosis, Differential↗