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Biomedical subjects

V Braun

Publications and source records attributed to V Braun.

At least 217 records · Page 12Linked to original sources
PubMed1985

Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids.

Iron acquisition via aerobactin enhances the virulence of Escherichia coli. Genes that specify functions for aerobactin synthesis and iron(III)-aerobactin transport have been identified on several ColV plasmids. Previously, we cloned the locus for aerobactin synthesis from pColV-K311 and assigned to three loci termed aerA, aerB, and aerC the functions for hydroxylation of lysine, acetylation of the 6-amino group of 6-hydroxy-lysine and coupling of N-acetyl-N-hydroxy-lysine with citrate, respectively (Gross et al. 1984). In this paper we show that aerA and aerB determine polypeptides with molecular weights of 50,000 and 35,000, respectively. We identified a fourth gene designated aerD that codes for a polypeptide with a molecular weight of 60,000, and which is required for the linkage of one residue of N-acetyl-N-hydroxy-lysine to citrate. The aerC gene product completes aerobactin synthesis by coupling the second N-acetyl-N-hydroxy-lysine to the monoacylated derivative citrate. The order of the genes in the operon was found to be aerD-aerB-aerC-aerA.

Base Sequence↗
PubMed1985

Hemolytic activity of Serratia marcescens.

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca2+ ions.

Electrophoresis, Agar Gel↗
PubMed1985

Functional domains of colicin M.

The structure of colicin M of Escherichia coli was studied with regard to its organization into functional domains. A proteolytic fragment with an Mr of 24,000 was isolated which comprised the carboxyterminal portion of the protein. It adsorbed to the outer membrane receptor protein and inhibited killing of cells by colicin M and by phage T5 that uses the same receptor. The fragment killed cells when the outer membrane was rendered permeable to macromolecules for a short time by the osmotic shock procedure. It is concluded that the fragment contains the receptor binding site and the active center but is lacking the sequence required for transport into cells. The carboxy-terminal amino acid sequence-Lys-Arg of the fragment was identical to that obtained from colicin M. Release of lysine and arginine led to inactivation of colicin M. The sequence of the first 39 amino acids of the amino terminal end of colicin M was determined.

Amino Acid Sequence↗
PubMed1985

Human B-cell differentiation induced by murein.

Like "true" polyclonal B-cell activators (PBA) for murine B cells, crude membrane preparations of Klebsiella pneumoniae (Klebs M) and some other enterobacteriaceae stimulate human B cells to mature into immunoglobulin (Ig) secreting cells without significant prior proliferation and in the absence of T cells. To investigate the biochemically defined membrane component with this unique PBA property, we studied lipoprotein and murein isolated from E. coli, since other components (e.g., a variety of lipopolysaccharide (LPS) fragments) failed to imitate Klebs M as a PBA. Mononuclear cells (MNC) and B cell-enriched cell populations from healthy blood donors were stimulated with various doses of lipoprotein and murein and, in comparison, Klebs M and pokeweed mitogen (PWM). Cell cultures exposed to either lipoprotein, murein, or Klebs M failed to incorporate [3H]thymidine significantly after 5 days in culture. In contrast, there was significant DNA synthesis (stimulation index greater than 3) when PWM was given to the same MNC population. All stimulants, with the exception of lipoprotein, induced B-cell differentiation in MNC cultures, as measured by an ELISA quantitating secreted Ig in the culture supernatants. In cultures with B cell-enriched cell populations, however, only Klebs M and murein were able to induce the production of significant amounts of IgM. Thus, the actual PBA moiety contained in the crude membrane fraction (Klebs M) appears to be associated with murein. It is important to note that murein induced considerably weaker Ig secretion than Klebs M did.

B-Lymphocytes↗
PubMed1985

Cell-bound and secreted proteases of Serratia marcescens.

Exoprotease of Serratia marcescens ATCC 25419 is exceptional among members of the family Enterobacteriaceae in that it is secreted in large amounts by viable cells into the culture medium. Labeling of cells with radioactive amino acids revealed no intracellular protein that could be precipitated with antibodies raised against purified exoproteases. With substances known to interfere with the excretion of some proteins--tosyl-L-lysine chloromethyl ketone, phenethyl alcohol, procaine, and sodium azide--and with rifampin, an intracellular form (apparent molecular weight, 52,000) larger than the major exoform (molecular weight, 51,000) was identified. Moreover, the 52,000-molecular-weight form was the main protein in immunoprecipitates of a cysteine-auxotrophic mutant starved for cysteine. Beside the major exoform, protease I, two additional exoproteases, termed II and III, appeared in the medium of stationary cultures. They were precipitated by antibodies against protease I, were identical in the Ouchterlony double-diffusion assay, and exhibited only a small difference, if any at all, in the peptide pattern after partial hydrolysis with protease V8 of Staphylococcus aureus. The amino- and carboxy-terminal amino acid sequences of protease I and II were determined and found to be identical, NH2-Ala-Ala-Thr-Gly-Gly-Tyr-Asp-Ala-Val-Asp and Phe-Ile-Val-COOH, respectively. The microheterogeneity of the isolated exoforms revealed by anion-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also observed in samples pulse-labeled with radioactive amino acids. It remains to be determined whether the different protease forms are the result of processing (modification) reactions or whether they constitute isoenzymes encoded by very similar genes.

Amino Acid Sequence↗
PubMed1985

[Are hydrophilic areas of polypeptide chains always identical with epitomes? Discussions on the problems related to immunoglobulin allotypes in humans].

Hydrophilic areas of human polypeptide chains are obvious antigenic determinants for the allotypes of the Km-and Gm-system if they are located within special exposed segments of the native molecule. Allotype associated exchanges of amino acids are observed likewise in fewer hydrophilic segments. Even in strongly hydrophilic areas of polypeptide chains, antigenic determinants, characteristic for known iso- and allotypes, have not so far been observed. The prediction of antigenic determinants, exclusively based on the hydrophily of segments of polypeptide chains seems to be doubtful.

Amino Acid Sequence↗
PubMed1984

Genetic and biochemical characterization of the aerobactin synthesis operon on pColV.

Certain ColV plasmids of Escherichia coli contain genes that specify functions for the acquisition of iron(III) via aerobactin. The locus for aerobactin synthesis of pColV-K311 was cloned into pBR322. Mutagenesis with the transposon Tn1000, and the generation of deletions with restriction enzymes resulted in multicopy plasmids which complemented pColV mutants impaired in various steps of aerobactin synthesis. The insertion and deletion mutants were mapped and assigned to three loci termed aerA, aerB, and aerC. It is proposed that these genes mediate the synthesis of aerobactin by specifying functions for hydroxylation (aerA), acetylation of the 6-amino group of 6-hydroxylysine (aerB), and the coupling of N-acetyl-N-hydroxy-lysine with citrate (aerC). The order and transcription polarity of the structural genes was found to be aerB, aerC, aerA.

Bacteriocin Plasmids↗
PubMed1984

Cloning and expression of the activity and immunity genes of colicins B and M on ColBM plasmids.

The activity and immunity genes for colicins B and M on two conjugative ColBM plasmids, pCl139 and pF166, were cloned into pBR322 and pACYC184, respectively. The colicin regions on both recombinant plasmids were identical with regard to restriction endonuclease sites and the arrangement of the genes. They map close to each other in the order cmi cma cbi cba, where cmi denotes the locus that determines immunity to colicin M, cma the structural gene for colicin M, cbi immunity to colicin B, and cba the structural gene for colicin B. With the use of mutants obtained by insertion of the transposon Tn5, and by translation in minicells, the transcriptional polarity of cma and cba was found to be from right to left. cma and cba code for polypeptides with molecular weights of 27,000 and 58,000, respectively. No evidence of biosynthetic precursors was obtained.

Bacteriocin Plasmids↗
PubMed1984

Murein hydrolase (N-acetyl-muramyl-L-alanine amidase) in human serum.

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and L-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetyl-muramyl-L-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed. The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.

Amidohydrolases↗
PubMed1984

Exogenous induction of the iron dicitrate transport system of Escherichia coli K-12.

Streptonigrin was used to select mutants impaired in the citrate-dependent iron transport system of Escherichia coli K-12. Mutants in fecA and fecB could not transport iron via citrate. fecA-lac and fecB-lac operon fusions were constructed with the aid of phage Mu dl(Ap lac). Strains deficient in ferric dicitrate transport which were mutated in fecB were as inducible as transport-active strains. They expressed the FecA outer membrane protein and beta-galactosidase of the fecB-lac operon fusions. In contrast, all fecA::lac mutants and fecA mutants induced with N-methyl-N'-nitro-N-nitrosoguanidine did not respond to ferric dicitrate supplied in the growth medium. tonB fecB mutants which were lacking all tonB-related functions were not inducible. We conclude that binding of iron in the presence of citrate to the outer membrane receptor protein is required for induction of the transport system. In addition, the tonB gene has to be active. However, iron and citrate must not be transported into the cytoplasm for the induction process. These data support our previous conclusion of an exogenous induction mechanism. Mutants in fur expressed the transport system nearly constitutively. In wild-type cells limiting the iron concentration in the medium enhanced the expression of the transport system. Thus, the citrate-dependent iron transport system shares regulatory devices with the other iron transport systems in E. coli and, in addition, requires ferric dicitrate for induction.

Carrier Proteins↗
PubMed1983

Plasmid and chromosomal mutants in the iron(III)-aerobactin transport system of Escherichia coli. Use of streptonigrin for selection.

Assimilation of iron via aerobactin by Escherichia coli requires functions encoded by certain ColV plasmids and by the chromosomal fhu region. Mutants obtained by insertion of the transposons Mu d1 (Ap lac) and Tn5 into ColV-K311 revealed that the aerobactin genes are transcribed from the locus that determines aerobactin synthesis to the gene encoding the outer membrane receptor protein for ferric aerobactin uptake. The antibiotic streptonigrin was used for selection of mutants impaired in iron assimilation. A simple assay was devised to identify mutants devoid of aerobactin synthesis. In addition, we performed complementation studies between fhu mutants and fhu genes cloned into pBR322. The genes fhuC,D,B were required for uptake of ferric aerobactin. Mutations in the fhuA gene usually decreased expression of the subsequent fhuC,D,B genes to such an extent that growth on ferric aerobactin as sole iron source was abolished. The results suggest that the genes on the chromosome and on the ColV plasmid are organized in two operons.

Biological Transport, Active↗
PubMed1983

Iron supply to Escherichia coli by synthetic analogs of enterochelin.

Synthetic analogs of enterochelin (enterobactin) were tested for their ability to support the growth of Escherichia coli K-12 under iron-limiting conditions. The cyclic compound MECAM [1,3,5-N.N'; N"-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene] and its N-methyl derivative Me3MECAM promoted growth, whereas the 2,3-dihydroxy-5-sulfonyl derivatives MECAMS and Me3MECAMS were inactive. The same results were obtained with TRIMCAM [1,3,5-tris(2,3-dihydroxybenzoylcarbamido)-benzene] and TRIMCAMS (the 2,3-dihydroxy-5-sulfonyl derivative of TRIMCAM). However, the sulfonic acid-containing linear compound LICAMS [1,5,10-N,N', N"-tris(5-sulfo-2,3-dihydroxybenzoyl)-triaza-decane] supported growth. In contrast, LIMCAMC, in which the sulfonyl groups at the five position of LICAMS are replaced by carboxyl groups at the four position, was inactive. The uptake of the active analogs required the functions specified by the fepB, fesB, and tonB genes. Surprisingly, growth promotion of mutants lacking the enterochelin receptor protein in the outer membrane was observed. Only MECAM protected cells against colicin B (which kills cells after entering at the enterochelin uptake sites) and transported Fe3+ at about half the enterochelin rate.

Biological Transport↗
PubMed1983

Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium.

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.

Aminopeptidases↗
PubMed1983

Cloning and expression of the fhu genes involved in iron(III)-hydroxamate uptake by Escherichia coli.

Each of the four hydroxamate compounds, ferrichrome, aerobactin, rhodotorulic acid, and coprogen, known to transport ferric iron into Escherichia coli requires a specific outer membrane receptor protein. In addition, common transport functions for all four ferric hydroxamate compounds have been identified in the 3.5-min region of the linkage map and designated fhu. The fhu region was cloned into pBR322. By restriction analysis, Tn5 insertion mutations, and complementation studies between plasmid fragments and chromosomal mutants at least four loci in the order fhuA fhuC fhuD fhuB were found. The genetic products were determined in maxicells and minicells. fhuA codes for the known 78,000-dalton receptor protein and the 81,000-dalton precursor in the outer membrane, fhuC codes for a 30,000-dalton protein, and fhuD encodes a 26,000-dalton protein in the cytoplasmic membrane. No protein(s) could be assigned to the fhuB region. Truncated proteins derived from partial fhuA genes (68,000, 42,000, and 39,000 daltons) and a partial fhuD gene (24,000 daltons) and the strong polar effect on the expression of the genes indicated the direction of transcription to be from fhuA to fhuD.

Bacterial Outer Membrane Proteins↗
PubMed1982

[Continuous registration of micropotentials of the human heart. Initial experiences with a new high resolution ECG amplifier system].

A new ECG-amplifier system for recording cardiac microvolt potentials from the body surface is described. The improvement in signal-to-noise ratio was achieved by using specially designed suction electrodes, which were isolated from each other; by applying parallel signal averaging from four electrode pairs via four low-noise amplifiers; and by conducting the registration in Faraday cage. in 14 normal subjects, 12 patients with coronary heart disease and one patient with surgically corrected ventricular septal defect and pulmonary stenosis, pre-P-potentials (possible sinus node activity), His bundle potentials and ventricular late potentials were recorded with differing degrees of success. Variations of the time intervals to the preceding QRS complex were observed within the S-T segment in six of nine patients with demonstrable ventricular late ventricular late potentials. The advantage of such continuously recording ECG system lies in the highly accurate registration of cardiac micropotentials, particularly with ventricular late potentials that are changing in time, whereas the signal-averaging technique does not provide such possibilities.

Adolescent↗
PubMed1982

[The significance of the spinal averaging technique in cardiology (author's transl)].

This paper gives a survey on the principles, methodology, and clinical applications of the signal-averaging technique. Bioelectric signals of the heart at the microvolt level are overwhelmed by basal noise in high-gain amplified EKG registrations. By summation and averaging of several hundreds of cardiac cycles, basal noise can be minimized and the signal of interest accumulates. This improvement of the signal-noise ratio primarily depends on the number of cycles averaged, e.g., by 10:1 when 100 cycles are averaged (approximately, the square root of the number of averaged cycles). At present, signal-averaging computers in use are rather variable with respect to data acquisition, filter settings, trigger mechanism/stability, and data processing. These properties have to be taken into account when the results of different groups using different types of averaging computers have to be compared. At present, several applications of the signal-averaging technique in clinical cardiology have been proposed: (1) Surface EKG--registrations of pre-P potentials, of His bundle potentials, and of ventricular late potentials, as well as analysis of the frequency spectrum of the QRS complex in patients with myocardial infarction; (2) intracardiac EKG--recordings of pre-P potentials and of A-V nodal potentials; (3) surface phonocardiogram--detection of low-amplitude, late diastolic signals; (4) Intracardiac pressure analysis--recording of average phasic pressure changes. The most promising and clinically proven application of the signal-averaging technique, at present, seems to be the registration of His bundle potentials as well as of ventricular late potentials from the body surface. Future efforts will need to be made to record cardiac microvolt potentials beat by beat from the body surface by means of specially designed EKG recorders.

Arrhythmias, Cardiac↗