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Biomedical subjects

J A Hoffmann

Publications and source records attributed to J A Hoffmann.

At least 73 records · Page 4Linked to original sources
PubMed1995

Innate immunity of insects.

Insects are particularly resistant to microorganisms. Their host-defense system relies on several innate reactions: upon injury, the immediate onset of two proteolytic cascades leading to localized blood clotting and to melanization, the latter process involving production of cytotoxic molecules (namely reactive oxygen intermediates); the phagocytosis of bacteria and the encapsulation of larger parasites by blood cells; the induced synthesis by the fat body of a battery of potent antimicrobial peptides/polypeptides which are secreted into the hemolymph where they act synergistically to kill the invading microorganisms. The insect host defence system shares many of the basic characteristics of the mammalian acute phase response, especially at the level of the coordinate control of gene expression, where similar cis-regulatory and inducible transactivators appear to play key functions. The powerful techniques developed to study the genetics of Drosophila provide a unique opportunity to dissect the development and differentiation of this primordial immune system and may contribute to our understanding of the innate immune response in higher organisms.

Animals↗
PubMed1995

Insect immunity: isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti.

The injection of Escherichia coli and Micrococcus luteus into the hemocoel of Aedes aegypti induces a potent antibacterial activity in the hemolymph. We have purified and fully characterized three 40-residue antibacterial peptides from the hemolymph of bacteria-challenged mosquitoes that are absent in naive mosquitoes. The peptides are potently active against Gram-positive bacteria and against one of the Gram-negative bacteria that were tested. The amino acid sequences clearly show that the three peptides are novel isoforms of the insect defensin family of antibacterial peptides. They differ from each other by one or two amino acid residues. We present here the complete amino acid sequences of the three isoforms and the activity spectrum of the predominant Aedes defensin.

Aedes↗
PubMed1995

Structure of a protein in a kinetic trap.

We have determined the structure of a metastable disulphide isomer of human insulin. Although not observed for proinsulin folding or insulin-chain recombination, the isomer retains ordered secondary structure and a compact hydrophobic core. Comparison with native insulin reveals a global rearrangement in the orientation of A- and B-chains. One face of the protein's surface is nevertheless in common between native and non-native structures. This face contains receptor-binding determinants, rationalizing the partial biological activity of the isomer. Structures of native and non-native disulphide isomers also define alternative three-dimensional templates. Threading of insulin-like sequences provide an experimental realization of the inverse protein-folding problem.

Amino Acid Sequence↗
PubMed1994

Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides.

In response to a septic injury (pricking with a bacteria-soaked needle) larvae and adults of Drosophila produce considerable amounts of a 44-residue peptide containing 8 cysteines engaged in intramolecular disulfide bridges. The peptide is synthesized in the fat body, a functional homologue of the mammalian liver, and secreted into the blood of the insect. It exhibits potent antifungal activity but is inactive against bacteria. This novel inducible peptide, which we propose to name drosomycin, shows a significant homology with a family of 5-kDa cysteine-rich plant antifungal peptides recently isolated from seeds of Brassicaceae. This finding underlines that plants and insects can rely on similar molecules in their innate host defense.

Amino Acid Sequence↗
PubMed1994

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.

Animals↗
PubMed1994

Novel inducible antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus.

Insects belonging to the recent orders of the endopterygote clade (Lepidoptera, Diptera, Hymenoptera and Coleoptera) respond to bacterial challenge by the rapid and transient synthesis of a battery of potent antibacterial peptides which are secreted into their haemolymph. Here we present the first report on inducible antibacterial molecules in the sap-sucking bug Pyrrhocoris apterus, a representative species of the Hemiptera, which predated the Endoptergotes by at least 50 million years in evolution. We have isolated and characterized from immune blood of this species three novel peptides or polypeptides: (i) a 43-residue cysteine-rich anti-(Gram-positive bacteria) peptide which is a new member of the family of insect defensins; (ii) a 20-residue proline-rich peptide carrying an O-glycosylated substitution (N-acetylgalactosamine), active against Gram-negative bacteria; (iii) a 133-residue glycine-rich polypeptide also active against Gram-negative bacteria. The proline-rich peptide shows high sequence similarities with drosocin, an O-glycosylated antibacterial peptide from Drosophila, and also with the N-terminal domain of diptericin, an inducible 9 kDa antibacterial peptide from members of the order Diptera, whereas the glycine-rich peptide has similarities with the glycine-rich domain of diptericin. We discuss the evolutionary aspects of these findings.

Amino Acid Sequence↗
PubMed1994

Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin. A study in insect immunity.

Insect defensins are a family of 4-kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. We report the characterization of a novel defensin isoform from Drosophila and the cloning of the gene encoding a preprodefensin. The gene, which is intronless and present in a single copy/haploid genome, maps at position 46CD on the right arm of the second chromosome. The analysis of the upstream region of the gene reveals the presence of multiple putative cis-regulatory sequences similar to mammalian regulatory motifs of acute-phase-response genes. Transcriptional profiles indicate that the Drosophila defensin gene is induced by bacterial challenge with acute-phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. These and other data on the Drosophila defensin gene lead us to suggest that insect and mammalian defensins have evolved independently.

Amino Acid Sequence↗
PubMed1994

The inducible antibacterial peptides of insects.

Insects respond to bacterial challenge by the rapid and transient synthesis of a large number of potent antibacterial peptides that are active against many different bacteria. Two families of inducible antibacterial peptides are well characterized: the cecropins and the insect defensins. A rapidly increasing number of proline- and glycine-rich peptides are reported from various insect species together with cecropins and insect defensins. In this review, Stéphane Cociancich, Philippe Bulet, Charles Hetru and Jules A. Hoffmann give an update of our current information on the induced antibacterial peptides.

Journal Article↗
PubMed1993

Insect immunity: the diptericin promoter contains multiple functional regulatory sequences homologous to mammalian acute-phase response elements.

We are using the diptericin gene as a model system to study the control of expression of the genes encoding antibacterial peptides during the Drosophila immune reaction. In order to investigate the putative regulatory regions in the diptericin promoter, we performed DNaseI footprinting experiments combined with gel-shift assays in two inducible systems: the larval fat body and a tumorous Drosophila blood cell line. Our results confirm the importance of kappa B-like elements previously described in the immune response of insects and reveal for the first time the involvement of other regions containing sequences homologous to mammalian acute-phase response elements.

Acute-Phase Proteins↗
PubMed1993

Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus.

Insect defensins are cationic, cysteine-rich peptides (approximately 4 kDa) that appear after bacterial challenge or injury in the hemolymph of insects belonging to a large variety of orders. These peptides possess anti-Gram-positive activity and participate in the potent antibacterial defense reactions of insects. Using recombinant insect defensin and the strain Micrococcus luteus as a test organism, we have investigated the mode of action of this peptide. We show that defensin disrupts the permeability barrier of the cytoplasmic membrane of M. luteus, resulting in a loss of cytoplasmic potassium, a partial depolarization of the inner membrane, a decrease in cytoplasmic ATP, and an inhibition of respiration. Potassium loss is inhibited below the order-disorder transition of the lipid hydrocarbon chains. It is also inhibited by divalent cations and by a decrease in the membrane potential below a threshold of 110 mV. We propose that these permeability changes reflect the formation of channels in the cytoplasmic membrane by defensin oligomers. This proposal is supported by patch-clamp experiments that show that insect defensins form channels in giant liposomes.

Adenosine Triphosphate↗
PubMed1993

A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution.

One of the facets of the host defense of higher insects is the rapid and transient synthesis, following bacterial challenge or trauma, of a battery of potent antibacterial peptides (Steiner, H., Hultmark, D., Engström, A., Bennich, H., and Boman, H. G. (1981) Nature 292, 246-248). The best characterized of these peptides are the cecropins (ibid.), 4-kDa peptides devoid of cysteines, and the insect defensins (Hoffmann, J. A., and Hetru, C. (1992) Immunol. Today 13, 411-415), 4-kDa peptides with three intramolecular disulfide bridges. Several other inducible antibacterial peptides have been characterized only at the level of their amino acid sequences (Hoffmann, J. A., Dimarcq, J. L., and Bulet, P. (1992) Médecine & Sciences 8, 432-439). We report here the isolation of a novel 19-residue proline-rich inducible antibacterial peptide from Drosophila. In contrast to all previous reports on antibacterial peptides, this molecule carries a substitution as evidenced by molecular mass determinations; our data show that this reflects the O-glycosylation of a Thr residue by an N-acetylgalactosamine plus a galactose. A synthetic nonsubstituted peptide of identical amino acid sequence has an activity several times lower (5-10) than the native compound. Our data suggest that this substitution represents a post-translational modification essential for the full biological activity of this novel peptide.

Amino Acid Sequence↗
PubMed1993

The humoral antibacterial response of Drosophila.

Drosophila, like other insects, responds to the injection of bacteria by the rapid and transient synthesis of a battery of potent antibacterial peptides. Only a few of these peptides have been fully characterized to date. We review our recent data on the control of the expression of a gene encoding one of the induced peptides, i.e. diptericin. Our data highlight the role of proximal cis-regulatory motifs similar to regulatory elements binding NF-kappa B and NF-IL6 in promoters of some immune genes of mammals. We argue that the Drosophila host defense is homologous to the mammalian acute phase response.

Amino Acid Sequence↗
PubMed1993

Insect immunity. Two 17 bp repeats nesting a kappa B-related sequence confer inducibility to the diptericin gene and bind a polypeptide in bacteria-challenged Drosophila.

The Drosophila diptericin gene codes for a 9 kDa antibacterial peptide and is rapidly and transiently expressed in larvae and adults after bacterial challenge. It is also induced in a tumorous Drosophila blood cell line by the addition of lipopolysaccharide (LPS). The promoter of this gene contains two 17 bp repeats located closely upstream of the TATA-box and harbouring a decameric kappa B-related sequence. This study reports that the replacement of the two 17 bp repeats by random sequences abolishes bacteria inducibility in transgenic fly lines. In transfected tumorous blood cells, the replacement of both or either of the 17 bp motifs reduces dramatically LPS inducibility, whereas multiple copies significantly increase the level of transcriptional activation by LPS challenge. A specific DNA-protein binding activity is evidenced in cytoplasmic and nuclear extracts of induced blood cells and fat body. It is absent in controls. It is proposed that induction of the diptericin gene mediated by the two 17 bp repeats occurs via a mechanism similar to that of mammalian NF-kappa B.

Animals↗
PubMed1993

Expression and nuclear translocation of the rel/NF-kappa B-related morphogen dorsal during the immune response of Drosophila.

The rel/NF-kappa B-related morphogen dorsal is a maternally expressed gene which is involved in the control of the dorso-ventral axis during early embryogenesis of Drosophila. We show that this gene is also expressed in the fat body of larvae and adults of Drosophila as well as in a tumorous blood cell line: its expression is noticeably enhanced upon bacterial (or lipopolysaccharide) challenge. This challenge also induces within 15-30 min a nuclear translocation of the dorsal protein. The genes encoding inducible antibacterial peptides in Drosophila contain kappa B-related nucleotide sequences and we show that the dorsal protein can bind to such motifs and sequence-specifically transactivate a reporter gene in co-transfection experiments with a Drosophila cell line. However, in dl1 mutants, in the absence of dorsal protein, the genes encoding antibacterial peptides retain their inducibility, suggesting a multifactorial control. The results indicate that in addition to its role in embryogenesis, dorsal is involved in the immune response of Drosophila. They also strengthen the analogy between the mammalian acute phase response and the insect immune response.

Animals↗
PubMed1992

A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata).

The injection of low doses of bacteria into the aquatic larvae of dragonflies (Aeschna cyanea, Odonata, Paleoptera) induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated a 38-residue peptide from this hemolymph which is strongly active against Gram-positive bacteria and also shows activity against one of the Gram-negative bacteria which was tested. The peptide is a novel member of the insect defensin family of inducible antibacterial peptides, which had so far only been reported from the higher insect orders believed to have evolved 100 million years after the Paleoptera. Aeschna defensin is more potent than defensin from the dipteran Phormia, from which its structure differs in several interesting aspects, which are discussed in the paper.

Amino Acid Sequence↗
PubMed1992

(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.

Amino Acid Sequence↗
PubMed1992

Insect immunity: developmental and inducible activity of the Drosophila diptericin promoter.

Diptericins are 9 kDa inducible antibacterial peptides initially isolated from immune haemolymph of Phormia (Diptera). Following the isolation of a Drosophila cDNA encoding a diptericin homologue, we have now cloned a genomic fragment containing the Drosophila diptericin gene. To dissect the regulation of this gene, we have transformed flies with a fusion gene in which the reporter beta-galactosidase gene is under the control of 2.2 kb upstream sequences of the diptericin gene. We show that such a fusion gene is inducible by injection of live bacteria or complete Freund's adjuvant and respects the tissue specific expression pattern of the resident diptericin gene. Our analysis reveals at least four distinct phases in the regulation of this gene: young larvae, late third instar larvae, pupae and adults. This complexity may be related to the presence in the upstream sequences of multiple copies of response elements previously characterized in genes encoding acute phase response proteins in mammals (e.g. NK-kappa B, NF-kappa B related, NF-IL6 response elements).

Acute-Phase Proteins↗