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H Bennich

Publications and source records attributed to H Bennich.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1981

Sequence and specificity of two antibacterial proteins involved in insect immunity.

Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.

Amino Acid Sequence↗
PubMed1981

Anti-IgE antibodies in human serum: occurrence and specificity.

An IgG type of antibody directed against IgE has been studied in serum from healthy and allergic individuals. The technique used is based on adsorption of the IgG fraction in serum to protein A-Sepharose followed by reacting any specific antibody with 125I-IgE. The anti-IgE antibodies were partially blocked by endogenous IgE, indicating the presence of IgE-containing immune complexes in serum. Heating of serum to 56 degrees C disrupted the immune complexes, thereby facilitating the detection of anti-IgE antibodies. The antibodies were essentially directed against the heat-labile D epsilon 2 antigen. Significantly raised (p less than 0.001) levels of anti-IgE antibodies were found in patients suffering from allergic asthma, as compared with non-allergic blood donors.

Antibodies, Anti-Idiotypic↗
PubMed1980

IgE production in vitro by two rat myeloma tumor lines (IR2 and IR162).

Two IgE-synthesizing rat myeloma tumor lines (IR2 and IR162) were maintained in growing cultures that were viable for prolonged periods of up to three months in the presence of primary fibroblasts. The production of IgE by explanted tumor cells was studied in short-term and long-term cultures. The IR 162 tumor line retained expression of both IgE heavy (H) and light (L) chain synthesis for up to three months under in vitro conditions, while the IR2 tumor line initially lost H chain expression after approximately one month in culture, followed by subsequent loss of L chain about one month later. Total polysomal RNA extracted from IR2 cells, before and after loss of IgE-expression, was separated by sucrose gradient centrifugation, and gradient fractions were analysed for poly(A)-containing mRNA by a [3H]poly(U)binding assay. The results for the long-term IR2 cultures showed a disappearance of mRNA sedimenting at 18-20 S and 15 S, found to represent H and L chain mRNAs, respectively. Furthermore, no IgE L chain mRNA sequences could be detected in long-term IR2 cultures when analysed by hybridization assays, using as a probe a 3H-labelled DNA complementary to IgE L chain mRNA, L chain mRNA sequences were also absent from the nuclear RNA in long-term IR2 cultures. The loss of IgE synthesis in long-term cultures of IR2 cells therefore appears to be correlated with a concomitant loss of IgE mRNA sequences indicating a dedifferentiation in vitro modulated at the transcriptional level.

Animals↗
PubMed1980

Adjuvants in the induction and enhancement of rat IgE responses.

In Hooded Lister rats the primary IgE antibody response induced by immunization with antigen and adjuvant may be enhanced either specifically by a further dose of antigen (booster response) or non-specifically by infection with helminth parasites (potentiated response). The initial immunizing technique can influence the occurrence and level of these enhanced responses and here we describe the effect of using different adjuvants in the priming event. Although the level of the primary response was broadly similar following immunization with egg-albumin and the adjuvants Bordetella pertussis aluminium hydroxide or Freund's complete adjuvant, the booster response was inhibited and the potentiated response intensified in animals immunized with the latter two adjuvants. A significant IgE booster response could only be obtained if B. pertussis had been used in the initial immunization. When aluminium hydroxide was adsorbed to B. pertussis it was found to have the same inhibitory effect on subsequent booster responses as when it was adsorbed to antigen. These results are discussed in relation to the intricacies of IgE production in the present model and to more general mechanisms of adjuvant action.

Adjuvants, Immunologic↗
PubMed1979

IgA antibody response in acute rubella determined by solid-phase radioimmunoassay.

A solid-phase radioimmunoassay (RIA) for detecting rubella virus IgA serum antibodies was developed. Purified rubella virus grown in roller cultures of Vero cells was adsorbed onto polystyrene beads. The coated beads were then incubated with dilutions of serum, and rubella IgA antibodies which attached to the virus antigen on the solid-phase were subsequently detected with 125I-labelled anti-human-alpha antibodies. The specificity of the iodinated anti-human immunoglobulins was confirmed by RIA analysis of fractions obtained by chromatography of an early convalescent serum on an agarose column. A complete separation of IgM, IgA, and IgG was observed. A total of 144 serial serum specimens from 31 adult patients with an acute rubella infection were tested for rubella IgA antibodies, and the results were compared with the RIA IgG and IgM titres reported earlier from the same specimens. The RIA IgA response was detected in each of the 31 patients and the IgA antibodies appeared almost simultaneously with the IgG and IgM antibodies. The maximum titres, which were lower than the IgG and IgM titres, were reached in about 1 week after the onset of rash. In 6 patients out of 31 the IgA antibody response was transient and persisted approximately two months, while in the remaining 25 patients the IgA antibodies persisted throughout the study period of more than 5 months. The results obtained indicate that the presence of rubella IgA antibodies in serum is not an indication for a recent rubella infection.

Antibodies, Viral↗
PubMed1979

Analysis of the serum IgE levels in nonimmunized rats of various strains by a radioimmunoassay.

An assay, the paper radioimmunosorbent test (PRIST) for the measurement of total serum IgE in the rat, that allows the measurement of IgE down to a level of 0.25 ng/ml, is described. With this assay it has been demonstrated that significant differences exist in normal serum IgE levels in non-immunized rats. These differences are strain-dependent, and strains may be classified as to low (less than 50 ng/ml), medium (50--300 ng/ml) or high (greater than 300 ng/ml) serum levels. The serum IgE level is not sex-dependent. Environmental factors have a pronounced influence, optimal housing conditions resulting in low and stable levels while suboptimal conditions result in high levels with great fluctuations. Hybridization experiments suggest a multigenic control, with low IgE strains having a dominating suppressor effect.

Age Factors↗
PubMed1979

A radioimmunoassay for evaluation of the IgE and IgG antibody responses in the rat.

An assay, the paper radioallergosorbent test (PRAST), for the measurement of specific serum IgE antibody in the rat is described in detail. This assay has been used, in conjunction with a modified PRAST for the determination of relative specific serum IgG antibody and the PRIST assay for total serum IgE [13], to measure specific IgE and IgG and total IgE immune responses in normal parasite infected rats immunized using various protocols. The results indicate that there is a relationship between the basic IgE level and the immune response, i.e. a rat strain with a low constitutive IgE level demonstrates a weak response whereas a high level strain reacts strongly. When PRAST and passive cutaneous anaphylaxis (PCA) were compared, using standardized IgE antibody containing sera, the results were in good agreement. However, PRAST is the preferable assay as it shows less intrinsic variation, is more sensitive than PCA, and is not influenced by high serum IgE levels in the recipient animal.

Anaphylaxis↗
PubMed1979

Solid-phase radioimmunoassay of serum immunoglobulin A antibodies to respiratory syncytial virus and adenovirus.

A solid-phase radioimmunoassay for detecting respiratory syncytial virus and adenovirus serum immunoglobulin A (IgA) antibodies was developed. An antigen consisting of purified adenovirus type 2 hexons or a crude lysate of respiratory syncytial virus-infected cells was first adsorbed onto polystyrene beads. The coated beads were then incubated with dilutions of serum, and IgA antibodies which attached to the solid-phase virus antigen were subsequently detected with 125I-labeled anti-human alpha antibodies. The anti-human alpha antibodies used were isolated by immunosorbent chromatography from rabbit antiserum produced by immunization with IgA purified from serum of an IgA myeloma patient. A total of 46 serum specimens from 13 patients with respiratory syncytial virus infections and 10 patients with adenovirus infections were tested. Complement fixation, homologous IgG and IgM radioimmunoassay, and heterologous IgA radioimmunoassay testing were also done. Specific values higher than 10,000 cpm were often reached with convalescent serum specimens, and positive-to-negative serum binding ratios of 50 or more were frequently obtained with lower serum dilutions. IgA titers of convalescent sera were from 1,000 to 16,000, and with few exceptions a fourfold or greater rise in the IgA titer was detected in the homologous IgA radioimmunoassay.

Acute Disease↗
PubMed1979

The allergenic structure of allergen M from cod. III. Studies on the antigenicity of long-sequence peptides.

Fragments TM 1 (75 amino acid residues) and TM 2 (38 amino acid residues), and 3 other polypeptides (range 16-25 amino acid residues) of Allergen M from cod were shown to be active in rabbit IgG-mediated reactions. The same peptides were previously found to possess reactivity in IgE-mediated reactions, thus suggesting a structural relationship between their antigenic and allergenic determinants.

Absorption↗
PubMed1977

Failure of the putative IgE pentapeptide to compete with IgE for receptors on basophils and masts cells.

We have examined the claim that a pentapeptide resembling a portion of epsilon-chain of IgE can inhibit allergic reactions by competing with IgE for basophil-mast cell IgE receptors. Four laboratories with expertise in different areas collaborated. The following was found. (1) There was no evidence, by autoradiography, that the pentapeptide could block IgE fixation to basophils or displace IgE from these cells. (2) There was no evidence, by histamine release studies that the pentapeptide could block basophil sensitization or remove IgE from actively sensitized cells. The pentapeptide, also, did not impair antigen-induced histamine release. (3) There was no evidence that the pentapeptide could block the skin test response in Prausnitz-Küstner testing with the antigen injected intradermally or orally administered. Neither the correct pentapeptide sequence from the epsilon-chain nor the Fc" fragment of IgE impaired the P-K test. In each series of experiments, IgE myeloma protein, as a positive control, blocked the allergic response. The failure of the pentapeptide to block these reactions was observed at molar ratios of pentapeptide/IgE of 10(6) to 10(7). Our results, therefore, provide no evidence for competition between the pentapeptide and IgE. Even if there should be some weak affinity between the pentapeptide and IgE receptors on basophils or mast cells this would, by virtue of quantitative considerations, be of no clinical relevance.

Autoradiography↗
PubMed1977

Concanavalin A induced histamine release from human basophils in vitro.

The site of interaction for concanavalin A (Con-A)-induced histamine release from human basophils was studied in vitro. Blocking the epsilon one determinant (D leads to 1) of IgE with high concentrations of monomer (Fab) anti-Depsilon1 does not significantly inhibit the quantity of histamine released by suboptimum concentrations of Fc specific anti-IgE. This indicates that the monomer anti-Depsilon1 does not have the capacity to sterically hinder the bridging of all of the determinants in the Cepsilon3 and Cepsilon4 domains (Fc'-epsilon-region) of IgE. The monomer anti-Depsilon1 does effectively inhibit release induced by suboptimum concentrations of Con-A. The data indicate that for suboptimum concentrations, Con-A activation is IgE mediated and takes place in the proximity of Depsilon1 and not at the membrane receptor for IgE.

Basophils↗