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V Braun

Publications and source records attributed to V Braun.

At least 199 records · Page 11Linked to original sources
PubMed1988

Comparison of four hemolysin-producing organisms (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) for release of inflammatory mediators from various cells.

We investigated the role of various hemolysin-producing strains (Escherichia coli, Serratia marcescens, Aeromonas hydrophila, and Listeria monocytogenes) in induction of inflammatory mediators, e.g., histamine release from rat mast cells as well as the chemiluminescence response and the release of lipoxygenase transformation products from human polymorphonuclear neutrophils. Our data show that the hemolysin-positive bacteria as well as the hemolysin-positive culture supernatants were active in inducing the chemiluminescence response, leukotriene (LTB4 and LTC4) release from human granulocytes, and histamine release from rat mast cells. The degree of leukotriene release was dependent on the hemolysin type and on the expression of hemolysin activity. The E. coli alpha-hemolysin and the aerolysin-producing A. hydrophila were the most potent stimuli whether washed bacteria or bacterial supernatant was used. Bacteria expressing the S. marcescens hemolysin and the listeriolysin were only poor inducers of leukotriene generation. In contrast to leukotriene generation, all hemolysin-positive strains induced nearly the same histamine release in a dose-dependent manner. Our data suggest a potent role for various hemolysins as virulence factors in inducing the release of inflammatory mediators.

Aeromonas↗
PubMed1987

Nucleotide sequence of the fhuC and fhuD genes involved in iron (III) hydroxamate transport: domains in FhuC homologous to ATP-binding proteins.

The transport of Fe3+ into cells of Escherichia coli occurs via siderophores and the uptake through the outer membrane of three Fe3+-siderophore compounds containing hydroxamate residues requires three specific receptor proteins. In contrast, transport through the cytoplasmic membrane is catalysed by three common proteins encoded by the fhuB, fhuC and fhuD genes. The nucleotide sequence of a DNA fragment containing the fhuC and fhuD genes has been determined: the open reading frame of fhuC contains 795 nucleotides which encode a polypeptide with a molecular weight of 29,255 and the largest open reading frame of the fhuD region comprises 888 nucleotides. However, we propose that translation of fhuD initiates at the fourth potential start codon resulting in a polypeptide with a molecular weight of 28,282. Both proteins are moderately nonpolar and membrane-bound. They lack obvious signal sequences. Segments of the FhuC protein display strong homology to ATP-binding proteins, suggesting a function in Fe3+ uptake similar to the ATP-binding proteins of transport systems that depend on periplasmic proteins. This study completes the nucleotide sequence of the fhu operon which consists of the four genes fhuA fhuC fhuD fhuB arranged in this order on the E. coli chromosome and transcribed from fhuA to fhuB.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of the exbB gene of Escherichia coli K-12.

The exbB locus of Escherichia coli is involved in the uptake of certain iron(III) siderophore compounds, of vitamin B12 and of certain colicins. Outer membrane receptor proteins are essential constituents of the corresponding uptake systems. The DNA carrying the exbB locus was cloned into pACYC184 and subcloned into pUC18. With the use of insertion mutagenesis employing transposon Tn1000 and by deletion analysis, the exbB locus was confined to a 1.9 kb DNA fragment. An in vitro transcription/translation system and minicells programmed by exbB+ plasmids expressed a protein with an apparent molecular weight of 26,000. One plasmid, designated pKE7, expressed this protein to an extent that it became a prominent band in the membrane fraction of transformants. In contrast, chromosomally encoded ExbB protein could not be detected. The plasmid-encoded ExbB protein was mainly localized in the cytoplasmic membrane. Ferrichrome transport in exbB mutants was restored by exbB+ plasmids. Moderate overexpression of ExbB resulted in an enhanced ferrichrome transport, strong overexpression reduced the transport rate compared to a wild-type strain. The ExbB function shares some properties with the TonB function.

Bacterial Proteins↗
PubMed1987

Role of cell-bound hemolysin as a pathogenicity factor for Serratia infections.

The hemolytic activities of clinical isolates of Serratia marcescens, of Serratia liquefaciens, and of Escherichia coli strains containing a cloned hemolysin gene of S. marcescens were determined. Hemolysis was induced only by cells and not by spent media. The hemolytically active bacteria induced the release of the leukotriene C4 and of much less leukotriene B4 from polymorphonuclear leukocytes, the release of histamine from rat mast cells, and chemoluminescence of neutrophils. The hemolytic activity was correlated with the response of the leukocytes, but quantitative differences were recorded with regard to the release of the inflammatory mediators. Therefore, other factors in addition to the hemolysin contribute to the stimulation of leukotriene generation and histamine release. It is concluded that the hemolysin via these inflammatory mediators can increase vascular permeability, edema formation, and granulocyte accumulation and thus contributes to the pathogenicity of Serratia species.

Animals↗
PubMed1987

Sequence, expression, and localization of the immunity protein for colicin M.

Escherichia coli strains carrying the cmi locus on plasmids are immune against colicin M, which primarily inhibits murein biosynthesis, followed by lysis of cells. The nucleotide sequence of the cmi region was determined. It contains an open reading frame for a polypeptide with a molecular weight of 19,227. However, the major protein band observed on polyacrylamide gels after transcription and translation in an in vitro system or in minicells had an apparent molecular weight between 15,000 and 16,000. The nucleotide sequence contained internal ATG codons, two of which could serve for the synthesis of polypeptides with molecular weights of 15,349 and 15,996, respectively. A subclone with a DNA fragment that encoded these two shorter polypeptides exhibited full immunity. The colicin M immunity protein was found in the cytoplasmic membrane. The colicin M activity and immunity genes were transcribed in opposite directions. Both properties are typical of the channel-forming colicins and are in contrast to the colicins with endonuclease activities. However, colicin M does not form channels and exhibits no structural similarity to channel-forming colicins.

Amino Acid Sequence↗
PubMed1987

Ferric-coprogen receptor FhuE of Escherichia coli: processing and sequence common to all TonB-dependent outer membrane receptor proteins.

Iron transport via siderophores requires outer membrane receptor proteins and the TonB protein. The FhuE protein of Escherichia coli functions as the receptor for ferric coprogen and ferric-rhodotorulic acid. A chromosomal DNA fragment bearing the fhuE gene was cloned into pACYC184. The gene was localized by insertion mutagenesis by using the transposon Tn1000. Expression in minicells revealed a FhuE precursor with an apparent molecular weight of 82,000 and a FhuE protein with a molecular weight of 76,000. The transcription polarity of the fhuE gene was deduced from the size of truncated polypeptides derived from Tn1000 insertions, which were mapped by restriction analysis. The processing of truncated precursors that were synthesized by insertion mutants was strongly reduced even when the insertion site was close to the carboxy terminus of the FhuE protein. It is concluded that either the efficient insertion of proFhuE into the cytoplasmic membrane or the rate of cleavage of the signal peptide requires a particular conformation of the proFhuE protein, which is only formed by the complete primary structure. The amino-terminal amino acid sequence deduced from the nucleotide sequence was confirmed by gas-phase sequencing of the precursor and the mature form, which were separated by electrophoresis on polyacrylamide gels. The precursor contained an unusually long signal peptide of 36 amino acids. The amino-terminal end of the mature form contained the sequence Glu-Thr-Val Ile-Val. A pentapeptide starting with either Glu or Asp, followed by Thr, and two uncharged residues ending with Val were found in all outer membrane receptor proteins that were constituents of TonB-dependent transport systems.

Amino Acid Sequence↗
PubMed1987

Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli.

A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50L::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hly, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxy-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (Mr 504) to maltoheptaose (Mr 1,152) and was totally abolished by dextran 4 (Mr 4,000). This result and the observed influx of [14C]sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.

Bacterial Proteins↗
PubMed1987

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.

Amino Acid Sequence↗
PubMed1987

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.

Amino Acid Sequence↗
PubMed1986

Structural and functional properties of colicin B.

Colicin B was isolated in pure form from cells of Escherichia coli that contained the colicin activity and immunity genes cloned on a multi-copy plasmid. Active colicin B consisted of a single polypeptide with Mr of about 60,000. The sequence of 44 amino acids from the amino-terminal portion is presented. The isoelectric point of the protein was at 4.5. Colicin B inhibited the membrane potential-dependent transport of proline and enhanced the uptake of alpha-methylglucoside via the phosphoenolpyruvate-dependent phosphotransferase system. Colicin B formed small, ion permeable channels with an average single-channel conductance of 13.7 pS (1 pS = 10(-12) siemens) in 1 M KCl. Channel formation was voltage-dependent in the pH range between 4.5 and 6. At pH 7 the channels were voltage independent. Voltage-dependent channels were only formed when the trans compartment (the protein was added to the cis compartment) was negative by at least 70 mV. Evidence for an asymmetric single channel conductance was obtained. With KCl a hyperbolic conductance-concentration relationship was observed. The conductance for monovalent cations was minimal for Li+ and was maximal for NH+4. The single channel conductance of colicin B was larger than that of colicin A as judged from lipid bilayer experiments under otherwise identical conditions.

Amino Acid Sequence↗
PubMed1986

Iron hydroxamate transport of Escherichia coli: nucleotide sequence of the fhuB gene and identification of the protein.

The genes fhuA, fhuC and fhuD encode products involved in the transport of ferrichrome into cells of Escherichia coli. The nucleotide sequence of an additional locus contiguous to fhuD and formerly designated fhuB was determined. It contains an open reading frame for a polypeptide that consists of 659 amino acids. Expression of plasmids containing the fhuB region in an in vitro transcription/translation system and in E. coli minicells revealed a polypeptide with an electrophoretic mobility on SDS-polyacrylamide gels extremely dependent on the experimental conditions employed. This property could be explained by the very hydrophobic nature of the protein and it was concluded that the fhuB locus consists of a structural gene that encodes a membrane protein with a molecular weight of 82,181. The fhuB gene is required for all iron transport systems which operate via hydroxamate compounds. The order of the genes in the fhu operon is fhuA fhuC fhuD fhuB, and transcription proceeds from fhuA to fhuB.

Amino Acid Sequence↗
PubMed1986

Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation, properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane.

The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37 degrees C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued. The precipitate formed was sedimented by centrifugation at 8,000 x g. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of the phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.

Bacterial Outer Membrane Proteins↗
PubMed1986

Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12.

A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1.

Bacterial Outer Membrane Proteins↗
PubMed1986

[Heparin dosage in long-term intravenous administration based on the example of vascular surgery patients].

For prophylaxis of deep-vein thrombosis, 23 male and 7 female patients received intravenous 15 000 IE/24 h sodium-heparin continuously after different operations for blood-vessel surgery. The plasma heparin concentration was measured with chromogenic substrate at 0, 1, 2, 4, 8 and 12-18 hours after beginning of heparin application. Each patient's hematocrit, sex, height, weight and parameters of liver and renal function were measured at the same time. The aim of this investigation was to find a general model for intravenous heparin application which would allow the best individual therapeutic plasma heparin concentration to be obtained. At the latest eight hours after the beginning of heparin-infusion the steady state of plasma heparin concentration was reached in 100%, but only in 56.67% it equalled the therapeutical factor Xa inhibition-level of 0.10 IE/ml to 0.20 IE/ml (= 0.077 USP-E/ml to 0.154 USP-E/ml). It was found that the plasma heparin concentration correlates negatively with the plasma volume. The heparin halftime depends on the dosage. Plasma heparin concentrations from 0.04 IE/ml to 0.20 IE/ml (= 0.031 USP-E/ml to 0.154 USP-E/ml) correlate to heparin halftimes of 9 min to 38 min. With this results formulae for calculating the quantity of the bolus and the daily heparin application were obtained. As opposed to the liver function the kidney function does not have any significant effect on the plasma concentration of biological active heparin. Furthermore it was found that the actual weight is unreliable for determination of the dosage. Instead calculated weight (height minus 100) is to be used.

Adult↗
PubMed1985

Nucleotide sequence of the iron regulatory gene fur.

The fur gene of Escherichia coli is involved in all iron-regulated transcriptions hitherto studied. The nucleotide sequence of an 868 basepair fragment containing the fur gene was determined. There was only a single longer reading frame. The amino acid sequence derived from the nucleotide sequence comprised 148 amino acids that together made a polypeptide of 16,795 daltons. The amino acid sequence was confirmed by determination of the amino acid composition, the carboxy-terminal lysine residue and the internal Lys-Lys and Lys-Arg sequences of the isolated Fur protein. The nucleotide sequence contains typical initiation and termination sites for transcription and translation.

Amino Acid Sequence↗