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.

Analysis of fungal diversity of grapes by application of temporal temperature gradient gel electrophoresis - potentialities and limits of the method.

AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.

[Effect of the destruction of the pars intercerebralis on molting in Aeshne cyanes Müll. larva (Insecta, Odonata)]. / Effets de la destruction de la pars intercerebralis sur la mue chez la larve d'Aeshna cyanea Müll. (Insecte Odonate)

The cauterization of the pars intercerebralis blocks the moulting process in Aeshna. The critical period for this experiment has been determined in the 2 last instars. After this period brain cautery only delays moulting without preventing this process. Cauterization of the pars intercerebralis after the critical period of the prothoracic gland activity does not affect moulting in any way.

[Effects of O2 inhalation on the partial arterial-alveolar pressure of CO2 in healthy patients and in patients suffering from chronic bronchitis (author's transl)]. / Effets de l'inhalation d'O2 pur sur la différence artério-alvéolaire de pression partielle de CO2 chez le sujet normal et le bronchiteux chronique.

The increase in arterial-alveolar CO2 difference (D[a-A]CO2) on 100% O2 breathing was studied in healthy subjects and in chronic bronchitic patients with or without hypoxemia. This increase in D(a-A)CO2 showing the enhancement of the dead-space effect (i.e. of the ventilation/perfusion ratio; VA/Q,), resulted from increase in PaCO2 or/and from decrease in PACO2 and was only found in hypoxic bronchitic subjects. In such subjects D(a-A)CO2 increased by about 50%. This phenomenon seems to arise from the vasomotor effect of pure O2 on pulmonary circulation and the role played by the Haldane effect in increasing PaCO2 and thus D(a-A)CO2 in some subjects is very weak. In subjects where inhalation of pure O2 produced the greatest increase in D(a-A)CO2 and in VA/Q, the calculated value of the venous admixture (QSh/QT) after measurement of D(A-a)O2 at the 30th min of hyperoxia was overestimated. Indeed D(A-a) O2 was enlarged by increasing the dead-space effect under 100% O2 breathing.

The follicle cell epithelium of maturing ovaries of Locusta migratoria: a new biosynthetic tissue for ecdysone.

Follicle cells of maturing ovaries of Locusta migratoria are demonstrated to synthesize the moulting hormone ecdysone (2beta,3beta,14alpha,22R,25-pentahydroxy-5beta-cholest-7-en-6-one). Studies of secretory kinetics under in vitro conditions show that the intensity of hormone secretion is strictly dependent on the stage of maturation of the excised ovaries.

Surface IgG content of murine hybridomas: Direct evidence for variation of antibody secretion rates during the cell cycle.

Previous experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. (c) 1995 John Wiley & Sons Inc.

Immunocytochemical localization of Bombyx-PTTH-like molecules in neurosecretory cells of the brain of the migratory locust, Locusta migratoria. A comparison with neuroparsin and insulin-related peptide.

Using a monoclonal antibody directed against a synthetic pentadecapeptide corresponding to the N-terminus of the prothoracicotropic hormone (PTTH) of Bombyx mori, we report the presence of immunoreactive molecules in a large number of median neurosecretory cells of the pars intercerebralis of the migratory locust, Locusta migratoria. These cells correspond to the A1 cell type which we show to contain also neuroparsins, a family of predominant neurohormones of the migratory locust. In contrast, PTTH-like molecules are absent from A2 cells of the pars intercerebralis which contain Locusta insulin-related peptide (LIRP). Developmental studies show the presence of PTTH-related substances in neurosecretory cells of Locusta migratoria from late embryogenesis to adult development, including ageing vitellogenic female adults.

Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis.

We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.

Cloning of the gene encoding the antibacterial peptide drosocin involved in Drosophila immunity. Expression studies during the immune response.

A potent inducible antibacterial peptide carrying an O-glycosylated substitution has recently been isolated from Drosophila [Bulet, P., Dimarcq, J. L., Hetru, C., Lagueux, M., Charlet, M., Hegy, G., Van Dorsselaer, A. and Hoffmann, J. A. (1993) J. Biol. Chem. 268, 14893-14897]. Here we report cloning studies that show that Drosophila contains a single, intronless gene, located at position 51C1-6, which encodes the precursor protein from which drosocin is processed. The upstream and the downstream sequences of the drosocin gene contain putative cis-regulatory elements similar to mammalian regulatory motifs, namely three kappa B-related decameric sequences. The drosocin gene is silent in naive animals, and is strongly induced with acute phase kinetics after immune challenge in larvae and in adults. We have established several transgenic fly lines in which reporter genes were placed under the control of various drosocin promoter sequences. Our results indicate that 2.5 kb of upstream sequences confer inducibility and tissue specificity to the transgene, but that the level of its expression in the fat body after immune challenge is low. Addition of genomic regions downstream of the drosocin transcribed sequences results in increased transcription levels, which are similar for the fusion and the resident drosocin genes upon infection. Analysis of transgenic fly lines showed that the drosocin reporter gene is constitutively expressed in the oviducts of egg-laying females.

Antimicrobial activity spectrum, cDNA cloning, and mRNA expression of a newly isolated member of the cecropin family from the mosquito vector Aedes aegypti.

An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophila cecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naïve adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7-10 days.

Ecdysteroid biosynthesis and embryonic development are disturbed in insects (Locusta migratoria) reared on plant diet (Triticum sativum) with a selectively modified sterol profile.

Wheat seedlings germinating in the presence of the systemic fungicide fenpropimorph accumulate 9beta,19-cyclopropylsterols (95% of total sterols) in place of Delta(5)-sterols, which are normally produced in these plants. Adult females of the phytophagous insect Locusta migratoria show a dramatic decrease in their cholesterol content when reared on fenpropimorph-treated wheat. These females lay eggs with the ecdysteroid concentration reduced by up to 80% as compared to controls. Injection of fenpropimorph to the insects or feeding them on wheat coated with the fungicide (normal sterol composition) does not affect their sterol or ecdysteroid profiles; addition of cholesterol to fenpropimorph-treated wheat prior to feeding restores normal ecdysteroid titers in the insects. The severe reduction of the ecdysteroid content in eggs laid by females reared on fenpropimorph-treated wheat is associated with a series of developmental arrests and/or abnormalities. The results show that the dietary 9beta,19-cyclopropylsterols cannot be used by Locusta in place of Delta(5)-sterols for ecdysteroid biosynthesis. They suggest that the selective inhibition of specific enzymes in the sterol biosynthetic pathway of the plants can be used as a strategy to control insect development.

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.

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae.

Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.