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H Bennich

Publications and source records attributed to H Bennich.

At least 19 recordsLinked to original sources
PubMed1991

Influence of carrier molecules on the intensity of biomolecule ions in plasma desorption mass spectrometry.

The intensity of insulin and melittin singly charged molecule ions is suppressed when the sample is mixed with either a lysozyme carrier or bovine serum albumin (BSA) carrier. The suppression with a BSA carrier is shown to be dependent on the carrier concentration. In contrast, luteinizing hormone releasing hormone or glutathione when mixed with insulin or melittin does not result in suppression. These results suggest sample preparation procedures to increase the sensitivity with mass spectrometry.

Glutathione↗
PubMed1991

Plasma desorption mass spectrometry of phosphopeptides: an investigation to determine the feasibility of quantifying the degree of phosphorylation.

The intact ion yields of phosphotyrosine, phosphothreonine and mono- and diphosphoserine residue-containing peptides have been compared with the non-phosphorylated sequences using plasma desorption mass spectrometry. Equimolar mixtures of the phosphorylated (Mp) and non-phosphorylated peptides (M) were also analysed. The positive mass spectra of these mixtures show a higher intensity of the [M + H]+ compared with the [Mp + H]+. In the negative mass spectrum, the bias towards the [M - H]- compared with the [Mp - H]- was reduced, but the spectra generally did not accurately reflect the stoichiometry.

Amino Acid Sequence↗
PubMed1991

Purification of synthetic peptides. Immobilized metal ion affinity chromatography (IMAC).

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N-terminal metal-binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side-products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30-40 amino acid residues) were efficiently purified by this approach.

Amino Acid Sequence↗
PubMed1991

Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription.

Attacins are antibacterial proteins synthesized by pupae of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. In this report we show that the previously described, attacin-induced alteration in the structure and the permeability of the outer membrane of Escherichia coli is associated with a specific inhibition of the synthesis of several outer membrane proteins, including OmpC, OmpF, OmpA, and LamB. The inhibition is expressed as a reduction in the steady-state mRNA levels and is at least in part the results of a block in transcription of the corresponding genes. Transcription directed by the promoter of ompR, the positive regulator of ompC and ompF expression in response to environmental conditions, is also affected by attacin. The effects on mutant strains show that the primary activity of attacin is not mediated by the ompR-envZ regulatory system. Instead our data suggest the existence in E. coli of a previously unknown system for the transcriptional regulation of a large set of outer membrane proteins previously not known to be coordinately regulated. We propose that the activity of attacin is directed towards this system.

Animals↗
PubMed1989

Mass spectrometric identification of peptides present in immunized and parasitised hemolymph from honeybees without purification.

We propose that a substance, identified using mass spectrometry, present in the hemolymph of the honeybee (Apis mellifera) as a result of stimulating the insect immune system corresponds with Apidaecin I, an antibacterial peptide recently described. The plasma desorption mass spectra indicate that several other higher molecule mass substances are synthesised as a result of bacterial or parasite infection.

Animals↗
PubMed1989

Influence of sample concentration and adsorption time on the yield of biomolecule ions in plasma desorption mass spectrometry.

The yield of intact ions formed in 252Cf plasma desorption mass spectrometry has been investigated by analyzing melittin and bovine trypsin at different concentrations on a nitrocellulose surface. The yield of trypsin ions is shown to vary with the protein concentration and adsorption time. The singly charged ion of trypsin is observed when concentrated solution (10 microM to 1 mM) of bovine trypsin are applied for sufficient time.

Adsorption↗
PubMed1988

The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study.

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.

Anti-Bacterial Agents↗
PubMed1985

On the primary structures of lysozyme, cecropins and attacins from Hyalophora cecropia.

Diapausing pupae of Cecropia respond to a bacterial infection by the selective synthesis of RNA and 15-20 hemolymph proteins. Of these we have purified lysozyme and two classes of antibacterial proteins called cecropins and attacins. The primary structure has been determined for the lysozyme, one attacin and five cecropins. We have also prepared a cDNA bank, isolated and sequenced clones corresponding to the lysozyme, the two main attacins and one cecropin. The results of these structural studies are briefly summarized. Finally we review the solid phase synthesis of cecropin A and B and 9 analogs of cecropin A.

Amino Acid Sequence↗
PubMed1985

Structure and expression of kappa-chain genes in two IgE-producing rat immunocytomas.

The light chain expression in two IgE-producing rat immunocytomas, IR2 and IR162, was studied. Both immunocytomas produce light chains of the kappa type. The kappa chains were characterized at the protein level by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and amino acid (aa) sequencing. cDNA clones corresponding to the kappa-chain mRNA were also prepared and sequenced. The results showed that rat kappa chains have the same structure as their mouse counterparts with respect to signal sequence cleavage, somatic mutations in the V-J region and invariance of all the aa positions which are strongly conserved in the frame work regions of mouse V kappa chains (greater than 95% conservation). Results from studies on kappa-chain transcription lend support to the allelic exclusion model with only one functionally expressed light chain in each immunocytoma.

Amino Acid Sequence↗
PubMed1984

The antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli.

The attacins are antibacterial proteins which accumulate in the hemolymph of the giant silk moth, Hyalophora cecropia, in response to a bacterial infection. Here we show that the permeability barrier function of the outer membrane is affected shortly after addition of attacin to growing cultures of Escherichia coli. Specifically, the penetration through the outer membrane of beta-lactam antibiotics, chicken egg white lysozyme and the detergent Triton X-100 was found to be facilitated. The sensitivity of E. coli to cecropin B, another antibacterial protein present in the hemolymph of H. cecropia, was also found to be increased after treatment with attacin. The results suggest that the target of the attacins in E. coli is the outer membrane. Other effects of the attacins which have been observed are likely to be indirect consequences of the alteration in the properties of the outer membrane. These effects include changes in the cell shape, irregular patterns of cell division and lysis. The minimal concentration at which the attacins affected the growth of E. coli was 1 and 0.5 microM for the neutral (pI 7) and basic (pI 9) attacins, respectively, which corresponds to less than 2% of the concentration of the attacins in the hemolymph of infected pupae.

Animals↗
PubMed1984

Role of IgE receptors in effector function of human eosinophils.

After analysis of the technical parameters of the rosette assay with human IgE-coated erythrocytes, Fc epsilon receptors for IgE (Fc epsilon R) on human peripheral blood eosinophils were compared to Fc epsilon R on lymphocytes and monocytes. Antibodies directed against Fc epsilon R on lymphocytes and monocytes inhibited the IgE rosettes formed by eosinophils from hypereosinophilic patients, which suggests that Fc epsilon R on eosinophils were antigenically related to Fc epsilon R on lymphocytes and monocytes. Fc epsilon R on human eosinophils were shown to participate in the killing effect of Schistosoma mansoni schistosomula in vitro in the presence of purified eosinophils from highly hypereosinophilic patients (blood counts greater than 3000/mm3) and anti-schistosomula IgE antibodies present in S. mansoni-infected patient sera. Similar levels of inhibition of cytotoxicity were obtained after preincubation of eosinophils with aggregated human IgE or with anti-Fc epsilon R antibodies, whereas preincubation with aggregated IgG or with anti-C3b receptor antibodies did not decrease the killing effect for schistosomula targets. This IgE-dependent cytotoxic capacity seemed restricted to eosinophils with an abnormally low density ("hypodense" cells) present only in highly hypereosinophilic patients. These observations might be related to nonparasitic situations in which increased levels of IgE and tissue or blood eosinophils are observed.

Antilymphocyte Serum↗
PubMed1983

Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia.

Six closely related antibacterial proteins, attacins A-F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A-F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20-23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino-terminal sequences, attacins A-D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram-negative bacteria isolated from the gut of a silk worm but they did not act on other Gram-positive and Gram-negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.

Amino Acid Sequence↗
PubMed1982

Structure and evolution of the heavy chain from rat immunoglobulin E.

The nucleotide sequence of the rat epsilon-chain mRNA has been determined by sequencing cloned cDNA copies of the mRNA. The established sequence covers the coding region, the 3'-non coding region and most of the 5' non-coding region. A comparison with the nucleotide sequence of the human epsilon-chain constant region reveals that C3 and C4 are the most highly conserved domains. The rat epsilon-chain contains a C-terminal decapeptide which is not present in the human counterpart.

Amino Acid Sequence↗
PubMed1982

Characterization and molecular cloning of the mRNA for the heavy (epsilon) chain of rat immunoglobulin E.

We report a study of the mRNA for the heavy (epsilon) chain of rat IgE. Cytoplasmic RNA was prepared from the two rat immunocytomas IR2 and IR162 and fractionated by sucrose gradient centrifugation. An enriched fraction containing approximately 5% mRNA for the epsilon chain was obtained in this way. When translated in vitro, it produced a 59,000-dalton polypeptide, which in the presence of a membrane fraction yielded a 90,000-dalton polypeptide, presumably through posttranslational modification. Both polypeptides were precipitated by rabbit antisera that were monospecific for rat epsilon chains. The epsilon chain mRNA was estimated to be approximately 2200 nucleotides long and constitutes a minute fraction in the total mRNA both in the IR2 and the IR162 tumors, unlike the mRNA for light chains. Double-stranded cDNA copies prepared frm the RNA fraction, which was enriched for epsilon chain mRNA, were inserted into the Pst I cleavage site of the pBR322 vector. Twenty clones with inserts exceeding 1000 base pairs were used for selection of mRNA from the IR2 tumor. By in vitro translation of the selected mRNA, one clone was identified that yielded a polypeptide with the same size as the unprocessed epsilon chain. The nucleotide sequence was determined for part of the inserted cDNA in this candidate clone and was found to be homologous to a sequence in the constant region (C) of the human epsilon chain. In this communication we report a sequence from the C epsilon 3 domain of the rat IgE. When compared to the corresponding sequence of human IgE, 55% of the amino acids in the rat sequence were found to be conserved.

Amino Acid Sequence↗
PubMed1982

Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae.

We have investigated low molecular weight antibacterial proteins from the Cecropia moth. Hyalophora cecropia. In addition to the previously described cecropins A and B, five new antibacterial proteins were discovered, the cecropins C, D, E and F, and the factor G. A scheme for the purification of these factors is presented. Cecropin D is a major cecropin, its amino acid sequence, WNPFKELEKVGQRVRDAVISAGPAVATVAQATALAK, shows homology to cecropin A and B. Like these cecropins, cecropin D has a block C-terminal. The previously tentative C-terminal sequence of cecropin A is also confirmed. It is concluded that the three major cecropins, A, B and D, are products of three different genes that are derived from a common ancestor. The cecropins C, E and F were present in very low amounts, and thus their primary structures could not be fully elucidated. Cecropin C has an amino acid sequence that up to residue 37 is identical to the sequence of A, though it lacks the C-terminal blocking group. It may be a precursor or degradation product of cecropin A. The minor cecropin E shows a similar relation to cecropin D. Cecropin F has a single amino acid replacement (17 Asp leads to Asn) compared to cecropin D, and is probably a product of an allele that is present at a low frequency in the population. The primary structure of the factor G could not be determined, however its amino acid composition is different from that of the cecropins. All the major cecropins were found to be efficient against several gram-positive and gram-negative bacterial strains. No significant difference was found between them in their activity against Escherichia coli, though against some less susceptible bacteria the most basic cecropins were more effective, the activity falling in the series B greater than A much greater than D.

Amino Acids↗
PubMed1982

Insect immunity: isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth, Antheraea pernyi.

The immune system in the Chinese oak silk moth, Antheraea pernyi, has been compared with that of the Cecropia moth which has been characterized earlier. Antibacterial activity against Escherichia coli was induced in diapausing pupae by injection of viable E. coli or Enterobacter cloacae. The activity reached a maximum on day 7-8 after which the response gradually declined. The pupae produced a set of immune proteins with P4 and P5 as major labelled components similar to that earlier found in Cecropia. The major antibacterial factor in A. pernyi was cecropin D. A procedure is described for the isolation of cecropin B and D, which is in principle similar to the one used for the isolation of the corresponding cecropins from Cecropia pupae. Amino acid sequence analyses of the A. pernyi cecropins show the D form to contain 36 amino acid residues and that both cecropins have blocked C-termini. The general structure of cecropins having a charged N-terminal region (residues 1-21) followed by a long hydrophobic stretch (residues 22-32) is well conserved. Cecropin B and D from A. pernyi differ from the corresponding proteins in Cecropia by four and three conservative amino acid replacements, respectively. The homology between the cecropins from the two insects suggests that they orginate from a single ancestral gene. The antibacterial activity was tested against nine different bacterial species. Evolutionary aspects of the cecropins are discussed.

Amino Acids↗
PubMed1981

Purification and characterization of the messenger RNA for the heavy chain of rat immunoglobulin E.

The isolation and translational properties of rat immunoglobulin E (IgE) heavy chain mRNA are described. The mRNA has a sedimentation coefficient of approximately 18S, a chain length of about 2000 nucleotides and directs the synthesis in vitro of a polypeptide of 65000 molecular weight in an mRNA-dependent rabbit reticulocyte lysate. Inclusion of dog pancreatic microsomes in the cell-free translation system resulted in a heavy chain product of about 75000 molecular weight, presumably as a consequence of glycosylation in vitro. This species co-migrated in an SDS polyacrylamide gel with mature IgE heavy chain. Substantial purification of heavy chain mRNA was achieved by denaturing sucrose gradient centrifugation and agarose gel electrophoresis.

Animals↗