Effects of destruxins, cyclic depsipeptide mycotoxins, on calcium balance and phosphorylation of intracellular proteins in lepidopteran cell lines.

The influence of the destruxin E, cyclodepsipeptidic mycotoxin produced by the filamentous entomopathogenic fungus Metarhizium anisopliae, on calcium fluxes and protein phosphorylation of in vitro cultivated lepidopteran cell lines have been studied. The use of the fluorescent calcium indicator Fura 2 AM did not show a fast increase in the level of cytosolic calcium in lepidopteran and human cell lines treated with dtx E. In contrast, 45Ca+2 assays detected a late but significant calcium influx in insect cells exposed to dtx E and A for at least 30 min. This effect was not inhibited by calcium channel blockers with the exception of nifedipine 25 microM. A viral treatment potentiated the effect of dtx E on calcium balance. Dtx E also induced a strong, fast (10 min), transient phosphorylation of high molecular weight (250 kDa) cellular proteins. The activation of phosphorylation was only partially inhibited by EDTA. This study provides the first direct evidence of dtx E influence on calcium fluxes and protein phosphorylation.

A titration procedure of the Junonia coenia densovirus and quantitation of transfection by its cloned genomic DNA in four lepidopteran cell lines.

A sensitive and reproducible tissue culture biossay method was developed based on indirect immunofluorescence to titrate virus suspensions of the Junonia coenia densovirus (JcDNV) and to quantify transfections by its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodoptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells derived from Spodoptera litura ovaries and hemocytes, respectively, and the SC-LD 135 from Lymantria dispar were compared for their efficiency to support viral replication. The viral titres expressed as TCID50/ml averaged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were above 10(7) for SPC-PL 65 cells. Even with this most sensitive cell line, the rate of infected cells did not exceed 75% and decreased progressively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant plasmid encompassing an infectious sequence of JcDNV genome. SPC-SL 52 cells were found to be the most sensitive, and the lipofection method resulted in about a 5-fold increase compared to the calcium phosphate precipitation protocol. The rescued virions proved to be infectious and the restriction profiles of their DNA were identical to that of wild type virions.

Establishment and characterization of three cell lines from Aedes triseriatus (Diptera: Culicidae).

Three cell lines (A.t. GRIP-1, 2, and 3) were established from Aedes triseriatus (Say) embryonated eggs or neonate larvae and their morphology, growth, karyotype, and isozyme pattern were studied. The isozyme alleles observed in the 3 cell lines also were found in adults of the original mosquito colony. Each cell line differed in enzymatic, morphological, and karyotypical patterns. La Crosse encephalitis (LAC) and snowshoe hare (SSH) viruses, members of the California encephalitis virus group, were able to replicate in these 3 cell lines. Furthermore, these cell lines, especially A.t. GRIP-1, were more sensitive than the Aedes aegypti (L.) (ATC 10) cell line for detection of small amounts of delta-endotoxin of Bacillus thuringiensis serovar. israelensis (de Barjac).

Insecticidal and cytotoxic effects of natural and hemisynthetic destruxins.

The insecticidal and cytotoxic effects of 13 natural and hemisynthetic destruxins have been studied. DE shows insecticidal effects similar to those of DA, while DE and DA are more active than all the other natural compounds and analogues tested. Brominated destruxin is a relatively active analogue displaying particular modalities of cytotoxic effects which reflect a certain originality of its mode of action. The linear molecule resulting from the opening of the DA cycle is not toxic. The most hydrophilic destruxins showing e.g. charged radicals (COO-) appear the least toxic probably because they do not penetrate easily the cellular membranes.

Synthesis and biological activity of a destruxin analogue: D-Lac-6 destruxin E.

A general strategy for the synthesis of destruxin analogues is described and applied to a particular example, D-Lac-6 destruxin E. The tetrapeptide Boc-Ile-N-MeVal-N-MeAla-beta-Ala-OMe (2) was chosen as the basic starting compound, and its preparation was optimized. This fragment was then coupled with the compound (D)Lac-Pro, and the resulting peptide was cyclized by the DEPC or DPPA/HOBt/DMAP methods at 21 and 30% yield, respectively. The biological activity of the analogue obtained was established by injection to an insect host.

Toxicity and pathology of crude broth filtrate produced by Hirsutella thompsonii var. thompsonii in shake culture.

Metabolic products secreted by the fungal mycelia of Hirsutella thompsonii var. thompsonii (CBS 556.77D) in a defined culture broth in shake culture were tested for toxicity to Galleria mellonella larvae and Drosophila melanogaster adults via injection and per os application, respectively. In addition, the toxic effect of broth filtrate was observed in vitro in a cell line of Bombyx mori. Czapek-Dox broth fortified with 1% yeast extract stimulated more rapid mycelial growth and correspondingly more toxin production in time. At 25-30 degrees C, metabolic toxin(s) was detected in broth via bioassay at about 4-5 days postinoculation when mycelial biomass reached 5 mg/ml (dry wt). At these temperatures, biological activity of the filtrate peaked at about 8-10 days when mycelial growth reached a maximum (10 mg/ml, dry wt). This suggests a positive relationship between toxic metabolite and mycelial production. After 10 days, the toxicity of the filtrate appeared to decline gradually. Pathogenicity symptoms of the metabolites developed slowly in both G. mellonella and D. melanogaster. Early signs of lethargy appeared at 4 days postinjection and cumulative mortality of G. mellonella larvae was low after 1 week; however, the percentage of mortality reached 98-100% after 14 days. At death, G. mellonella larvae displayed small dark spots on a brownish cuticle. Histopathological effects were observed in the larval midgut, malpighian tubules, hypodermis, fat body, hemocytes, muscle, and silk glands. Cellular change consisted of pycnosis of the nucleus and a reduction in cytoplasm density. Highest mortality (78.8%) to adult D. melanogaster occurred after 10 days post-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytometric study of the effects of destruxin E on leukemic cells in mice]. / Etude cytométrique des effets de la destruxine E sur des cellules leucémiques de souris.

The activity of destruxin E, a cryptogamic toxin isolated from the hyphomycete Metarhizium anisopliae, was studied on mouse leukemia cells (L 1210 and P 388) in culture. Besides a cytotoxic effect, a cytostatic effect (increase of diploïd cells proportion) was observed with all doses for P 388 (from 10 micrograms/ml to 0.001 microgram/ml) and to a concentration of 0.1 microgram/ml for L 1210. At the lower doses, the effect on cellular DNA content appears distinctly.

[Experimental infection of an invertebrate cell line with a mollicute-like procaryote inducing the "lethargy of coleoptera" (author's transl)]. / Infection expérimentale d'une lignée cellulaire d'invertébré par le procaryote intravacuolaire de type mollicute, agent de la "léthargie des coléoptères"

In vitro multiplication of a pathogenic intravacuolar mollicute-like procaryote from Melolontha melolontha L. was experimentally obtained in an insect cell line. The elongated and pleomorphic forms observed in the insect-host are reproduced in cell cultures. A third peculiar giant form is missing, showing that it does not play any role in the multiplication of the germ. The intrinsic potentialities of the germ are maintained during the successive passages, as proved by reinfection of the insect and by immunology. The original syndrome including the giant form is reproduced in the insect. The immunserum prepared from the wild germ isolated by density gradient is positive with the in vitro mollicute. The germs are intravacuolar, both in the cultured cells and in the insect host. Clearly the microorganism multiplies within the vacuoles. A cytopathogenic effect is noticed in the cultured cells overcrowed with germs. The germs become extracellular when they are released in the culture medium by disaggregation of the cell membranes. It seems that this work shows the first model of an intravacuolar mollicute-like procaryote experimentally multiplied in cultivated cells.

[In vivo and in vitro studies of a cytoplasmic polyhedrosis virus isolated from Euxoa scandens, Riley (Noctuidae, Agrotinae)(author's transl)]. / Caractérisation d'une polyédrose cytoplasmique chez le lépidoptère Euxoa scandens, Riley (Noctuidae, Agrotinae). Etudes in vivo et in vitro.

A virus isolated from the larvaes of Euxoa scandens (Noctuidae, Agrotinae) was studied by histological and histochemical techniques, and by electron microscopic observation of virus suspensions and ultrathin sections of infected tissues. Our studies demonstrated that the virus in icosahedral in shape, measures 70 nm and belongs to the cytoplasmic polyhedrosis virus group. Only the midgut cells of the larvae were found to be infected and the virus particles were localized in inclusion bodies most of which had a paraspherical, polyhedral shape. It was possible to infect Lymantria dispar L. cells in vitro with free virus or with extracts obtained from the diseased larvae. The virogenic stroma and the first small inclusion bodies in this stroma were detected in the cytoplasm at 18 and 36 hours post infection, respectively. These polyhedra had a cubical shape, reached their maximum size after 48 hours and were located in the cytoplasm of over 90 percent of the cells.

[Electron microscopic study of the reticulated cellular structures induced in Lepidoptera by baculovirus]. / Etude en microscopie électronique des structures cellulaires réticulées induites chez les Lépidoptères par des Baculovirus

Five of six Baculovirus species studied induce cytoplasmic and nuclear fibrous sheets, observed by electron microscopy, within infected cells of lepidopterous. The reticulated structures, revealed by fluorescence microscopy, assimilate the fibrillar substance induced by these viruses thanks to the affinity they show for globulins of healthy rabbits.

[Development and pathogenesis of an entomopoxvirus in lepidoptera cell culture]. / Développement et pathogenèse d'un entomopoxvirus en culture cellulaire de Lépidoptère.

Cell cultures of Lymantria dispar (Lepidoptera) have been successfully infected with the Entomopoxvirus of Amsacta Moorei (Lepidoptera). The different steps of viral morphogenesis and pathogenesis have been precisely detailed by light and electron microscopy of infected cells.

[The effect in vitro of the toxins of the mushroom Metarrhizium anisopliae (Metsch.) Sorok. on the hemocytic reaction of Oryctes rhinoceros L. (Coleoptera)]. / Effet in vitro de substances toxiques produites par le champignon Metarrhizium anisopliae (Metsch.) Sorok. sur la réaction némocytaire du Coléoptére oryctes rhinoceros L.

The filtrate of Metarhizium cultures contains toxic-substances inhibiting the process of formation of granuloma by Oryctes in vitro. This inhibition results from marked alterations of both nucleus and cytoplasm of hemocytes under the action of the fungal toxins.