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Biomedical subjects

H Schütte

Publications and source records attributed to H Schütte.

At least 19 recordsLinked to original sources
PubMed1992

The Mycobacterium tuberculosis 38-kDa antigen: overproduction in Escherichia coli, purification and characterization.

The 38-kDa protein (Ag38) of the Gram+ bacterium, Mycobacterium tuberculosis H37Rv, is an immunodominant antigen of potential utility for diagnosis and vaccine development. Assessment of this potential requires large amounts of the purified protein that would be difficult, if not impossible, to obtain from M. tuberculosis itself. The gene coding for Ag38 had been previously cloned and in the present study was expressed as an unfused protein in Escherichia coli under the control of strong transcriptional (bacteriophage lambda pLpR) and translational (atpE) signals. Fermentation of the recombinant E. coli K-12 strain CAG629[pMS9-2], which is deficient in Lon protease and the heat-shock response, produced recombinant Ag38 (reAg38) at high levels (about 10% of total cellular protein). The reAg38, which accumulated as inclusion bodies, was completely solubilized in 6 M guanidine.HCl, refolded and purified to apparent homogeneity. The product showed the expected amino acid composition and M(r), and had similar reactivities as the native protein with three different mAb. Polyclonal antibodies raised against reAg38 reacted strongly with the native antigen in enzyme-linked immunosorbent assay. These results demonstrate that reAg38, which cannot be distinguished antigenically from the native protein of M. tuberculosis, can be prepared in quantity from E. coli.

Amino Acid Sequence↗
PubMed1992

Induction of severe vascular leakage by low doses of Escherichia coli hemolysin in perfused rabbit lungs.

S. aureus alpha-toxin and E. coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins. Both are established virulence factors and have been implicated in the development of septic lung failure. Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs. In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990). Here, we present results of a parallel study conducted with E. coli hemolysin. Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases. Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold. Perfusion was terminated when the total lung weight gain surpassed 20 gm. 0.12 HU/ml E. coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm). Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs. Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier. Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain). In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina. Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other.

Animals↗
PubMed1991

Incidence of abortion after genetic amniocentesis in twin pregnancies.

Fetal outcome after genetic amniocentesis (AC) in viable twin pregnancies was analysed in a retrospective study at three centres in order to estimate the rate of fetal loss after AC. The maternal age ranged from 33 to 45 years (mean 36.7 years). The gestational age varied between 15 and 20 weeks of gestation (mean 17.1). In 98 viable twin pregnancies with complete follow-up, spontaneous abortion of both fetuses occurred within 28 completed weeks of gestation in eight pregnancies and six women aborted within 20 completed weeks of gestation after AC, corresponding to a rate of fetal loss of 8.1 and 6.1 per cent, respectively (excluding the loss of five twins with viable outcome of the co-twin in five pregnancies).

Abortion, Spontaneous↗
PubMed1991

Neutrophil passage through isolated perfused rabbit lungs.

Kinetics of polymorphonuclear leukocyte (PMN) lung passage were investigated in ex vivo isolated and ventilated left rabbit lungs, perfused with buffer solution at physiological flow rate. 111In-labeled PMNs of rabbit or human origin were injected into the pulmonary artery, and the first fraction of PMNs that rapidly passed the lung together with coinjected erythrocytes, was collected separately for external radioactivity counting. Washout of initially retained PMNs from the lung was monitored by use of a sodium-iodide detector. Recirculation of cells was avoided by insertion of a filter in the perfusion circuit. A fraction of 16.6 +/- 1.3% of rabbit PMNs rapidly passed the lung vasculature, followed by an exponential washout of initially retained PMNs [half-time (t50) of lung transit 8.1 +/- 0.6 min]. Slightly higher t50 (12.2 +/- 1.0 min) was obtained upon use of human PMNs. Reduction in flow by 50% caused a marked prolongation of PMN transit (t50 = 27.8 +/- 5.1 min), whereas increase in flow to 150% only insignificantly decreased t50. Rise in pulmonary venous pressure to 5 and 8 mmHg caused retardation of PMN lung transit (t50 = 15.3 +/- 0.6 and 31.6 +/- 3.6 min). Preincubation of PMNs with 2 ng/ml endotoxin for 1 h induced marked delay in PMN washout (t50 = 26.1 +/- 2.8 min). In conclusion, single-pass PMN kinetics in isolated lungs correspond to in vivo studies previously reported, thus allowing elucidation of PMN-endothelial interactions in an intact lung vasculature under standardized conditions.

Animals↗
PubMed1990

Correlation of migration behavior in free-flow zone electrophoresis and electrophoretic titration curve.

The correlation of electrophoretic migration behavior in free-flow zone electrophoresis (FFZE) and electrophoretic titration curve (ETC) has been explored. It is shown that the ETC of a protein or a mixture of proteins can be used to predict the fraction numbers at which those proteins elute in a preparative scale FFZE experiment. The ETC is a quick and effective way to choose optimal buffer conditions in FFZE. FFZE is employed to determine the isoelectric points (pI) of proteins whose pIs lie beyond the range of IEF 3-9 gels. It is found that separations in FFZE are governed by the net surface charge of the proteins.

Buffers↗
PubMed1990

Separation of enzymes from Candida boidinii crude extract by continuous flow zone electrophoresis.

Separation of the enzymes formate dehydrogenase, formaldehyde dehydrogenase and methanol oxidase from Candida boidinii crude extract has been explored using continuous flow zone electrophoresis in the VaP-22 and the scaled-up VaP-220 electrophoresis apparatus. Yields up to 95% and purification factors between 3 and 7 were obtained, together with separation of cell debris from the enzymes. Multiple injections of sample were used to obtain a protein throughput of 46.2 mg/h in the VaP-22. A tenfold higher throughput was achieved using the VaP-220. Correlation of the electrophoretic mobility in continuous flow zone electrophoresis with the elution behavior in ion-exchange chromatography confirmed the primary role of net surface charge in the separation of biological molecules. Proteins and enzymes with differences greater than 0.05 M elution molarities in ion-exchange chromatography can be separated. This corresponds to a preparative scale (mg/h or g/h) separation of proteins and enzymes whose difference in apparent electrophoretic mobility is greater than 0.70 x 10(-5) cm2/(V.s).

Alcohol Oxidoreductases↗
PubMed1990

Pilot- and process-scale techniques for cell disruption.

Microorganisms are a source of protein with catalytic and/or biological activity, which are of increasing commercial interest for applications in industry or therapy. For the isolation of intracellular products cell disruption is necessary. In principle, chemical, biological, or physical means may be employed to release proteins from cells. These different approaches are reviewed with special emphasis on scale-up and possible industrial operation. Mechanical devices have been improved considerably in recent years and appear most universally suited to cell disintegration. Chemical extraction or enzymatic lysis offers improved selectivity but requires individual procedures for each product. For a final process design, product yield and cost of the unit operation must be considered as well as the possible implications for the subsequent steps in product recovery, especially on solid/liquid separation.

Cell Membrane Permeability↗
PubMed1989

[Early complications of caustic injuries of the digestive tract].

We retrospectively analyzed the gastrointestinal complications observed in 119 patients who had ingested corrosive agents. Hydrochloric acid and sodium hydroxide were the agents involved in 62% of patients. Women predominated over men (p less than 0.001); mean age was 36 for males and 29 for women (p less than 0.05). Endoscopy was performed in 55% of patients and revealed acute lesions in 69%. Complications were observed in 18% of patients requiring surgery in 12 (10%). Main complications included sepsis of abdominal or mediastinal origin and gastrointestinal bleeding. Mortality among these patients was 73%.

Adolescent↗
PubMed1988

Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase.

An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.

Amino Acid Oxidoreductases↗
PubMed1988

Common bile duct pressure in patients with common bile duct stones with or without acute suppurative cholangitis.

Resting common bile duct pressure was measured in three groups of patients: group 1, 53 patients with gallstones but without common duct stones; group 2, 35 patients with common bile duct stones unaccompanied by cholangitis; and group 3, 36 patients with common duct stones and acute suppurative cholangitis. A significantly higher pressure in the common bile duct was documented in patients with cholangitis when compared with the other two groups. Twenty-four patients with cholangitis had common duct pressure values above 20 cm H2O, the maximal values of normal. Additionally, patients with cholangitis with pressure values over 30 cm H2O (nine patients) showed absence of green bile in the extrahepatic biliary tract, suggesting cessation of bile excretion into biliary duct. In all these cases, an impacted stone at the distal end of the common bile duct was documented.

Acute Disease↗
PubMed1986

Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84.

The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis. Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity. The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C. The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline. The hydantoin-hydrolyzing activity could be reactivated by ferrous ions. Dihydrouracil was the most readily hydrolyzed substrate. The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30%. The only identified end product was N-carbamyl-D-valine.

Amidohydrolases↗
PubMed1985

Improved enzyme screening by automated fast protein liquid chromatography.

The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.

Alcohol Oxidoreductases↗
PubMed1984

Eight new restriction endonucleases fröm Herpetosiphon giganteus--divergent evolution in a family of enzymes.

Characterization of eight restriction endonucleases isolated from five strains of Herpetosiphon giganteus is described. HgiCI from strain Hpg9 recognizes and cleaves the degenerate sequence: GGPyPuCC, producing 5'-hexanucleotide protruding ends. Endonucleases HgiBI, HgiCII and HgiEI are isoschizomers of AvaII; HgiCIII and HgiDII are isoschizomers of SalI; and HgiDI and HgiGI are isoschizomers of AcyI. Based upon their closely related and in part overlapping recognition specificities a close evolutionary relationship is proposed for all known Hgi restriction endonucleases.

Base Sequence↗