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Biomedical subjects

W Bautsch

Publications and source records attributed to W Bautsch.

At least 55 records · Page 3Linked to original sources
PubMed1994

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE).

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.

Base Sequence↗
PubMed1993

Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures.

Monoclonal antibodies were isolated which reacted specifically with the complement cleavage products C3a, C3adR, C5a, and C5adR but not with the parent molecules C3 or C5. In both cases the mAbs showed a higher affinity towards the desArg forms. These mAbs were used as capture antibodies in immunoassays for C3a/C3adR and C5a/C5adR. The immunoassays are based on the ABICAP technology which ensures for a rapid measurement. Due to the large binding capacity and the very short diffusion pathways in the gel-matrix the binding equilibrium between capture antibodies and the antigen is reached whilst the sample is flowing through the column. Therefore this test represents an endpoint assay offering the possibility of using a single calibration curve for a large number of measurements. With the C3adR assay concentrations down to 16 ng/ml C3adR can be detected. The lower detection limit of the C5adR assay is 1 ng/ml C5adR. The tests for C3a/C3adR, and C5a/C5adR can be performed in 20 to 25 min and this rapid processing of plasma samples should permit the application of these parameters for diagnostic purposes and patient management.

Amino Acid Sequence↗
PubMed1993

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome.

A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NheI revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the AluI-methylase six NheI recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior AluI methylation, respectively. All nine AluI-methylase/NheI and 14 NheI restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.

Base Sequence↗
PubMed1993

Chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a.

The receptor for the inflammatory peptide C3a has scarcely been examined on human cells. This work demonstrates that human tumor-derived basophilic granulocytes express C3a receptors, and presents parts of the hitherto unknown C3a-signal transduction. When incubated with IL-3, these cells specifically liberated histamine on C3a stimulation. Independent from IL-3, 240,000 +/- 100,000 receptors per cell with a Kd of 5.6 +/- 0.9 nM were determined. [Ca2+]i increased from 120 +/- 35 nM to 300 +/- 80 nM after a C3a challenge, as measured by digital imaging fluorescence microscopy, and rested at its basal level in the presence of C3a-desArg, the immediate catabolic product of C3a in vivo. This [Ca2+]i increase could be completely desensitized homologously by C3a as well as inhibited by up to 75% by pertussis toxin. Thus, tumor-derived basophils are suitable for cloning of the human C3a receptor.

Anaphylatoxins↗
PubMed1993

Evaluation of the C-terminal C5a effector site with short synthetic C5a analog peptides.

Biological activities have been determined for a series of 18 peptides based on the C-terminal sequence of human or rat C5a. Lysosomal enzyme release was tested in two cell types, the promyelotic leukemia cell line U937 and polymorphonuclear leukocytes. In addition, an ATP-release assay with guinea pig platelets was performed. It was demonstrated that the C-terminal octapeptide 67-74 of human C5a represents the minimal sequence required to induce a measurable biological signal in all assays. Extending this peptide to a length of 21 amino acids produced at best only a slight enhancement of potency. Amino acid replacements with either tryptophanyl or phenylalanyl residues in positions between 65-69 either increased potency (at position 67), or abrogated potency (at position 66) in the two lysosomal enzyme assays. N-terminal acylation with the fluorenylmethoxy-carbonyl-aminohexanoyl group slightly enhanced C5a potency. In desensitization experiments with guinea pig platelets all peptides with a C5a activity were able to desensitize not only the C5a but also the C3a responses.

Acetylglucosaminidase↗
PubMed1993

Tryptophan mutants of human C5a anaphylatoxin: a fluorescence anisotropy decay and energy transfer study.

Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 +/- 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

Amino Acid Sequence↗
PubMed1992

C3a receptor on dibutyryl-cAMP-differentiated U937 cells and human neutrophils: the human C3a receptor characterized by functional responses and 125I-C3a binding.

The anaphylatoxic peptide C3a is part of a basic immunological defense mechanism, the complement system. Research on the human C3a receptor and signal transduction is hampered by the lack of a suitable human cell or cell line. We screened tumor cell lines and human blood cells for a C3a-dependent increase in cytosolic Ca2+ ([Ca2+]i) and analyzed this reaction in a fura-2/AM fluorescence assay for cells in suspension. U937 cells, when differentiated with dibutyryl-cAMP (Bt2cAMP), and purified human neutrophils reacted in a dose-dependent fashion to C3a and a C3a analogue synthetic peptide. We found complete homologous desensitization of this response and no heterologous desensitization to human C5a. Pertussis toxin totally blocked the increase in [Ca2+]i, indicating the possible involvement of a G-protein. Single-cell analysis by digital imaging fluorescence microscopy indicated that neutrophilic granulocytes responded to C3a. In binding studies with Bt2cAMP-differentiated U937 cells and human granulocytes, the 125I-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (Kd) in the low nanomolar range. We identified myo-inositol 1,4,5-trisphosphate (IP3) as the second messenger possibly causing the [Ca2+]i increase and the release of N-acetyl-beta-D-glucosaminidase as one secretory cell response. By functional and binding studies we demonstrated the expression of the C3a receptor on Bt2-cAMP-differentiated U937 cells and human neutrophils and characterized parts of the C3a signal pathway. Our data support a physiological concept in which C3a might be more important than presently thought.

Acetylglucosaminidase↗
PubMed1992

A recombinant hybrid anaphylatoxin with dual C3a/C5a activity.

By site-directed mutagenesis of a human complement factor C5a cDNA clone, we have designed a hybrid anaphylatoxin in which three amino acid residues in the C-terminal sequence of human C5a were exchanged to create the native C-terminal human C3a (hC3a) sequence Leu-Gly-Leu-Ala-Arg. This hybrid anaphylatoxin rC5a-(1-69)-LGLAR exhibited true C3a and C5a activity when tested in the guinea pig ileum contraction assay. Quantitative measurements of ATP release from guinea pig platelets revealed about 1% intrinsic C3a activity for this hybrid, while the C5a activity was essentially unchanged. Competitive binding assays confirmed that the rC5a-(1-69)-LGLAR mutant was able to displace radioiodinated rhC5a with a KI of approx. 40 nM and hC3a with a KI of approx. 3.7 microM from guinea pig platelets. Since the C-termini of both human C3a and C5a anaphylatoxins are known to interact with their respective receptors, we conclude that the same peptidic sequence, LGLAR, is able to bind to and activate two different receptors, the C3a receptor as well as the C5a receptor. This clone provides a novel tool for the identification of further receptor-binding residues in both anaphylatoxins, since any mutants may be tested for altered C3a and C5a activity simultaneously.

Adenosine Triphosphate↗
PubMed1992

Synthetic peptides as antagonists of the anaphylatoxin C3a.

Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.

Amino Acid Sequence↗
PubMed1992

Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

Amino Acid Sequence↗
PubMed1992

Functional activities of synthetic anaphylatoxic peptides in widely used biological assays.

A comparison study was carried out between the modern ATP release assay (ARA) with guinea-pig platelets and the traditional guinea-pig ileum contraction assay (ICA). The biological activities of the anaphylatoxin C3a and synthetic C3a analogue peptides were determined in both assays. In dose-response curves with C3a, a human C3a peptide with the last 21 amino acids of the C terminus (C3a 56-77) and a peptide with 13 amino acids which was acylated N-terminal with the aromatic fluorenylmethoxycarbonyl group and an aminohexanoyl group (Fmoc-Ahx YRRGRAAALGLAR) were tested. The ARA turned out to be 100 times more sensitive than the ICA. In contrast to previous reports the 21 amino acid long C3a analogue peptide did not exhibit full C3a activity but only 7% (ARA) or 12% (ICA). The potentiation of biological activity in the ARA by coupling non-peptide acyl-residues N terminal to peptide C3a analogues could be confirmed with Fmoc-Ahx-YRRGRAAALGLAR in the ICA. In addition, the tri-peptide Fmoc-Ahx-LAR displayed C3a specific activity in the ICA demonstrated by desensitization experiments.

Adenosine Triphosphate↗
PubMed1991

Functional expression of a human C5a receptor clone in Xenopus oocytes requires additional RNA.

cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes.

Animals↗
PubMed1991

Characterization of the C5a receptor on guinea pig platelets.

Guinea pig (gp) platelets react to nanomolar doses of the complement-derived anaphylatoxin C5a with a shape change, aggregation and release of biogenic amines and nucleotides from their granules. We have investigated the specific receptor for C5a on gp platelets which mediates these biological effects. Competitive binding studies with 125I-labeled guinea pig C5a (125I-gpC5a) revealed approx. 4000 binding sites/cell with Kd = 6 x 10(-9) M. The more than 60-fold higher biological activity (ATP-release from gp platelets) of gpC5a versus recombinant human C5a (rhuC5a) and the different binding behavior of gpC5a and rhuC5a point to a species restriction in the gp platelet system. Cross-linking of 125I-gpC5a to gp platelets (250 microM DSS) and analysis by SDS-PAGE under reducing conditions resulted in labeling of a single band with a molecular mass of 32 kDa (ligand-receptor complex). Because of these characteristics, the C5a receptor on gp platelets clearly differs from all previously described C5a receptors.

Adenosine Triphosphate↗
PubMed1990

Reevaluation of the C3a active site using short synthetic C3a analogues.

In 1980 the C-terminal pentapeptide LGLAR (C3a 73-77) was described (Caporale, L. H. et al. J. Biol. Chem. 1980, 255: 10758) as the minimal sequence inducing a C3a-specific activity. We have synthesized C3a-analogue peptides connected to non-peptidic acyl residues known to potentiate biological activity. Starting from the acylated hexapeptide fluorenylmethoxycarbonyl(Fmoc)-aminohexanoyl(Ahx)-ALGLAR+ ++, a related series of shorter peptides was synthesized. C3a-specific activity was measured as ATP release from guinea pig platelets. Even the tripeptide LAR, acylated with Fmoc-Ahx, exhibited C3a-specific activity. With 0.34% C3a activity, it was even more potent than the native LGLAR sequence which has 0.01% activity. N-terminal extension of the acylated tripeptide LAR by adding one to three alanines increased activity tenfold up to 3.26% (Fmoc-Ahx-AAALAR), while N-terminal addition of three glycine residues (Fmoc-Ahx-GGGLAR) only increased activity to 0.83% of native C3a. Furthermore, a stimulus-specific desensitization could be observed. Fmoc-Ahx-R and Fmoc-Ahx-AR exhibited neither activity nor desensitizing capacity, but the addition of four alanines to the dipeptide AR led to a sequence (Fmoc-Ahx-AAAAAR) with a C3a-specific activity of 0.14%. Even arginine prolonged N-terminally with five glycines (Fmoc-Ahx-GGGGGR) exhibited some C3a-specific activity so that for biological activity only the appropriate presentation of arginine seems to be essential.

Adenosine Triphosphate↗
PubMed1989

A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.

Base Sequence↗
PubMed1989

Selective expression of an amiloride-inhibitable Na+ conductance from mRNA of respiratory epithelium in Xenopus laevis oocytes.

Poly(A)+RNA was prepared from primary cultures of human nasal polyp epithelia and from native bovine tracheal epithelia. Six to fifty nanograms mRNA were injected into prophase-arrested immature Xenopus laevis oocytes. One to four days later the oocytes were probed with electrophysiological techniques for induction of novel ion conductances. Oocytes injected with mRNA had lower membrane potentials (Vm) and resistances (Rm) than controls. By use of step changes in extracellular Na+ concentration and applying amiloride, a Na+ conductance could be identified in mRNA-injected oocytes, which was inhibited by submicromolar concentrations of amiloride with the same kinetics (Ki = 1.3 X 10(-7) mol/l) as in the original tissue. After complete inhibition of this conductance by 10(-5) mol/l amiloride, Vm and Rm approached the respective values of controls. The data indicate that oocytes express functional epithelial Na+ channels but apparently no other epithelial ion conductances from injected mRNA of respiratory epithelium.

Amiloride↗