Biomedical subjects

E Schulze

Publications and source records attributed to E Schulze.

At least 19 recordsLinked to original sources

PubMed1992

Insect proteins homologous to mammalian high mobility group protein 1. Characterization and DNA-binding properties.

Two chromosomal high mobility group (HMG) proteins from larvae of Chironomus thummi (Diptera) and from an epithelial cell line of Chironomus tentans were purified to homogeneity and chemically characterized. cDNA clones encoding these proteins were isolated from an expression library using an immunoscreening approach and were sequenced. The deduced amino acid sequences revealed their homology to HMG protein 1 of vertebrates. These insect proteins have therefore been designated cHMG1a and cHMG1b. They have a molecular mass of 12,915 and 12,019 kDa, respectively, and preferentially bind to AT-rich DNA. Indirect immunofluorescence microscopy with a polyclonal antibody showed the presence of cHMG1a and cHMG1b in condensed chromomeres but not in puffs, nucleoli, and cytoplasm. The cHMG1a and cHMG1b genes were both localized to a single band in region 14 of chromosome 1 of C. tentans and appear to be single copy genes. An immunologically related protein was purified from Drosophila melanogaster Kc cells. Its size and amino acid composition indicate that it is an HMG1 of D. melanogaster. On the other hand, our antibody did not recognize calf HMG1. The identification and characterization of HMG1 proteins in insects with polytene chromosomes opens new possibilities for studying function(s) of this group of chromosomal proteins.

Amino Acid Sequence↗

PubMed1992

Reconstitution of pyruvate dehydrogenase multienzyme complexes based on chimeric core structures from Azotobacter vinelandii and Escherichia coli.

Two unique restriction sites were introduced by site-directed mutagenesis at identical positions in the DNA encoding the dihydrolipoyltransacetylase (E2p) components of the pyruvate dehydrogenase complex from Azotobacter vinelandii and from Escherichia coli. In this manner each DNA chain could be cut into three parts, coding for the lipoyl domain, which consists of three lipoyl subdomains, the binding domain and the core-forming catalytic domain, respectively. Chimeric E2p components were constructed by exchanging the three domains between E2p from A. vinelandii and E. coli on gene level. The six chimeric E2p proteins were expressed and purified from E. coli TG2. All chimeras were catalytically active, 24-subunit E2p proteins. Interactions of the peripheral components E1p and E3 with the wild-type enzymes from A. vinelandii and E. coli and with the chimeric proteins were studied by gel-filtration experiments, analytical ultracentrifugation and reconstitution of the overall activity of the complex. A. vinelandii E3 interacts only with those chimeras that contain the A. vinelandii binding domain, whereas E. coli E3 interacts with all chimeras. Exchange of the lipoyl or catalytic domain did not influence the binding properties of E3. Recognition of E1p depends on the origin of both the binding domain and the catalytic domain. E. coli E1p interacts strongly with those chimeras in which both the binding domain and the catalytic domain were derived from E. coli E2p and weakly with chimeras that contained either the binding domain or the catalytic domain from E. coli E2p. No binding of E. coli E1p was observed when both domains were of A. vinelandii origin. A. vinelandii E1p recognizes E2p from A. vinelandii and E. coli, but strong interaction required that the binding and catalytic domain were of the same origin. Exchange of lipoyl domains had no effect on the binding properties of the E1p component. These observations confirm previous conclusions, based on site-directed mutagenesis of A. vinelandii E2p [Schulze, E., Westphal, A. H., Boumans, H., and de Kok, A. (1991) Eur. J. Biochem. 202, 841-848], that the binding site for E1p consists of amino acid residues derived from both the binding and the catalytic domain and extend these conclusions to E. coli E2p. Dissociation of the 24 subunit E2p core was only detected when the chimeric E2p proteins contained the catalytic domain from A. vinelandii E2p. Dissociation depends on the binding of peripheral components to the E1p-binding sites, pointing to differences in the inter-trimer contacts between the E2p proteins from both species.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter vinelandii↗

PubMed1992

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.

Acetyltransferases↗

PubMed1992

Atomic structure of the cubic core of the pyruvate dehydrogenase multienzyme complex.

The highly symmetric pyruvate dehydrogenase multienzyme complexes have molecular masses ranging from 5 to 10 million daltons. They consist of numerous copies of three different enzymes: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and lipoamide dehydrogenase. The three-dimensional crystal structure of the catalytic domain of Azotobacter vinelandii dihydrolipoyl transacetylase has been determined at 2.6 angstrom (A) resolution. Eight trimers assemble as a hollow truncated cube with an edge of 125 A, forming the core of the multienzyme complex. Coenzyme A must enter the 29 A long active site channel from the inside of the cube, and lipoamide must enter from the outside. The trimer of the catalytic domain of dihydrolipoyl transacetylase has a topology identical to chloramphenicol acetyl transferase. The atomic structure of the 24-subunit cube core provides a framework for understanding all pyruvate dehydrogenase and related multienzyme complexes.

Amino Acid Sequence↗

PubMed1992

Effect of energy metabolism on 13C/12C-ratios in milk fat and lactose of cows.

1. The ratios of stable carbon isotopes 13C/12C in milk constituents of Holstein dairy cattle were investigated by mass spectrometry. 2. Under physiological feeding conditions the natural abundance of 13C in lactose was greater than in milk fat. 3. Reduction of energy intake diminished the abundance of 13C in lactose resulting in values similar to those of fat. 4. It is suggested that by comparing the 13C/12C ratios in milk fat and lactose the metabolic energy state of cows may be rated.

Animals↗

PubMed1992

[The occurrence of aeromonads in a drinking water supply system].

This study concerns with the occurrence of aeromonads, coliforms and colony counts in a drinking water supply. Aeromonas contents were detected in the range of 15.0 to greater than 2,400/100 ml in the raw water samples of the man made lake. After the drinking water treatment process including fast sand filtration and chlorination aeromonads indicated in comparison to total coliforms and colony counts early and significant an after-growth of maximal 240 aeromonads/100 ml in the peripheric drinking water supply. Drinking water samples characterized by a higher water temperature resulted in the highest contents of aeromonads. The Aeromonas-Species Aeromonas sobria and Aeromonas hydrophila were isolated most frequently with 56.9 and 37.4 percent, respectively. The role of aeromonads as an indicator of after-growth in drinking water supplies is discussed.

Aeromonas↗

PubMed1991

Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Binding of the peripheral components E1p and E3.

Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.

Acetyltransferases↗

PubMed1991

The catalytic domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli. Expression, purification, properties and preliminary X-ray analysis.

Partial sequences of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine-proline-rich sequence between a putative binding region for the peripheral components and the catalytic domain (apa-4). Most of the catalytic domain from A. vinelandii without the apa-4 sequence was degraded intracellularly, probably due to incorrect folding. Fusion proteins of six amino acids from beta-galactosidase, the apa-4 region and the catalytic domains of A. vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24-subunit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild-type E2p. This can be explained by intracellular degradation of over-expressed wild-type E2p, whereas the catalytic domains are stable against proteolysis in vivo and in vitro. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose-6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable. Crystals of the catalytic domain of A. vinelandii E2p could be grown to a maximum size of 0.6 x 0.6 x 0.4 mm. They diffract up to a resolution of 0.28 nm.

Acetyltransferases↗

PubMed1991

Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0 degree C, do not bind to E2p. Lipoamide dehydrogenase from A. vinelandii contains a C-terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C-terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site-directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C-terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 microM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed. It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete.

Acetyltransferases↗

PubMed1991

Hepatorenal reflex regulating kidney function.

In anesthetized male rats, infusion of glutamine (2 mumol/min) into the superior mesenteric vein at a rate known to induce liver cell swelling leads to marked decreases in renal glomerular filtration rate, renal para-aminohippurate clearance and urinary flow rate. Glutamine infused at identical rates into the jugular vein does not elicit any of these effects. The effect of glutamine is mimicked by serine but not by glutamate. Spinal transection, renal denervation or section of the vagal hepatic nerves abolishes the effect of mesenteric venous glutamine infusion. Mesenteric application of glucagon (1 ng/min) or of both glutamine and glucagon enhances glomerular filtration rate and urinary flow rate. Infusion of 1 ng/min glucagon through the jugular vein does not significantly alter glomerular filtration rate or urinary flow rate. The data disclose a powerful liver-borne mechanism regulating kidney function that is mediated by the hepatorenal innervation.

Animals↗

PubMed1991

Glucose entry rate in dairy cattle as determined by stable isotope 13C-labelled glucose at different stages of reproduction.

1. Uniformly labelled stable 13C-glucose was used to study glucose entry in high yielding Holstein cows (n = 8) under normal production conditions. 2. The single injection technique was repeated at three different reproductive phases. A two compartment model was applied to calculate mean entry rates of glucose resulting in: (1) Terminal phase of pregnancy (2 weeks a.p.): 0.41 g/hr/kg0.75; (2) Peak lactation (6 weeks p.p.): 0.97 g/hr/kg0.75; (3) End of lactation (37 weeks p.p.): 0.61 g/hr/kg0.75. 3. Data from studies using radioactively labelled tracers are in good agreement with our results obtained without any restrictions implied by the handling with radioactive substances.

Animals↗

PubMed1991

[The hygienic situation of the central drinking water supply in the former East Germany--an evaluation of the annual reports on water hygiene from 1984 to 1989].

In the former GDR an annual report on the situation in the field of communal hygiene had to be elaborated and submitted to the minister of public health. One part of this was the report on the hygienic situation in water supply, worked out by the Reference Laboratory for Water Hygiene in Bad Elster. After the political changes in autumn 1989 it became possible to analyse these reports as a whole. In this paper the reports from 1984 to 1989 are interpreted. The results of the laboratory measurements and field controls by the State Sanitary Inspectorates are shown in 17 graphics and compared to the bacteriological and chemical limits in drinking water standards. Special issue is drawn on the estimation of the number of inhabitants concerned by reduced drinking-water quality or any hazardous situations. The special problems of the different districts are compared in some graphics. The hygienic safety of the central drinking-water supply units is assessed basing on the results of the field controls by the State Sanitary Inspectorate. The Sanitation of the central drinking-water supply facilities in the new federal countries of the FRG is connected with the solution of a variety of technological problems, particularly the improvement of the water treatment techniques and the restoration of the pipe systems. The use of surface waters from extremely polluted rivers generates a high hygienic risk and requires the sanitation of the rivers. The high number of existing protection zones in the catchment areas for drinking-water is a valuable precondition to ensure the hygienic safety of the drinking-water supply in the new federal countries.

Fresh Water↗

PubMed1991

[The occurrence and survival of Campylobacter in sewage sludge].

Campylobacter was found only in raw sewage sludge samples of the high-rate municipal sewage treatment plant studied. Campylobacters were isolated from 28.6% of the sewage sludge samples of grit tank and primary settling tank. 40% of the settled activated sludge samples contained Campylobacter. Campylobacter was never found in digested sludge of a mean sludge age of more than 90 days. The survival times of Campylobacter in sewage sludge samples ranged between some hours and 10 days. Therefore a low risk of the spread of Campylobacter in the environment due to agricultural application of digested sewage sludge is assumed.

Campylobacter↗

PubMed1991

Environmental aspects of Campylobacter infections.

Epidemiological data indicate high incidence of campylobacteriosis. Improperly prepared poultry-products, unpasteurized milk as well as non-chlorinated drinking water were shown to be the main vehicles of Campylobacter transmission to man. There is a lack of knowledge concerning the role of various environments in transmission of Campylobacter. The review summarizes the present knowledge about occurrence and survival of Campylobacters in various environments (sewage, sludge, surface water, drinking water). In conclusion risk assessment for public health is discussed.

Animals↗

PubMed1991

[Fetal triploidy--diagnosis and sequelae].

A pregnancy in the 22nd week of gestation with severe asymmetric retardation of growth without malformations by ultrasonography is described. A triploidy was found by prenatal cytogenetic investigation. Possible signs for triploidy in pregnancy and the necessary conclusions are discussed.

Abnormalities, Multiple↗

PubMed1990

Time-resolved fluorescence studies on mutants of the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

Fluorescence anisotropy decays were measured for the wild-type dihydrolipoyl transacetylase (E2) component of pyruvate dehydrogenase complex from Azotobacter vinelandii and E. coli and for E2-mutants from A. vinelandii in which the alanine-proline-rich sequence between the binding domain and the catalytic domain is partially or completely deleted. In both E2-mutants the rotational mobility of the lipoyl domain and the overall activity after reconstitution of the complex are significantly decreased indicating the important role of the deleted sequence for the movement of the lipoyl domain and the transfer of substrates between the different active sites within the complex.

Acyltransferases↗

PubMed1990

The 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. 1. Molecular cloning and sequence analysis of the gene encoding the 2-oxoglutarate dehydrogenase component.

The nucleotide sequence of the gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. The protein-coding sequence consists of 2832 bp (944 codons, including the AUG start codon and the UAA stop codon). The predicted molecular mass (105,687 Da) is in good agreement with that published for the isolated enzyme. The E1o gene is separated from the gene encoding the E2o component by a 42-bp intergenic region. No Escherichia-coli-like promoter sequences are found in the sequenced 97 bp upstream from the E1o gene. A putative ribosome-binding site is located 10-16 bp upstream from the start codon of the E1o gene. No terminator sequences could be detected downstream from the stop codon. Together with the identical situation for the E2o gene and the presence of terminating sequences downstream of the E3 gene, it can be assumed that all three genes of the 2-oxoglutarate dehydrogenase multienzyme complex are transcribed as a single mRNA transcript under the control of a promoter, located more than 100 bp upstream of the E1o gene, analogous to the pyruvate dehydrogenase complex in E. coli. The similarity with the sucA gene of E. coli is high with 59% identity.

Amino Acid Sequence↗

PubMed1990

Type III collagen aminopropeptide and laminin P1 levels in serum of patients with silicosis-associated and idiopathic systemic scleroderma.

A group of 191 patients with systemic scleroderma and 12 patients with silicosis-associated scleroderma were investigated for connective tissue turnover. The serum levels of type III collagen aminopropeptide (P-III-P), the laminin PI (Lam PI) fragment and the acid lysosomal beta-galactosidase (beta-Gal) were determined by specific radioimmunoassays and spectrofluorometry, respectively. Increased levels of type III collagen aminopropeptide strongly correlated with enhanced activity of beta-galactosidase. Both parameters correlated with the clinical course in idiopathic systemic scleroderma and in silicosis-associated scleroderma. Serum levels of Lam PI were also found to be elevated in both groups, although there was no correlation with the severity of the disease. Autoantibodies directed against the DNA topoisomerase Scl-70 and against centromeric proteins were found in a similar range in patients with idiopathic systemic and silicosis-associated scleroderma. These results suggest that P-III-P, Lam PI and beta-Gal are useful serological markers of fibrotic activity and demonstrate similarities between idiopathic systemic scleroderma and scleroderma associated with silica-dust exposure.

Aged↗