The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides

Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm. A unique K+ ATPase in GCAM generates three gradients across this barrier. A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports. A greater than 1000-fold [H+] gradient (lumen alkaline) and a greater than 10-fold [K+] gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material. Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides. Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides. Following the demonstration by Sacchi et al. that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta. Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut.

From a mono to a digenetic life-cycle: how was the jump for flagellates of the family trypanosomatidae?

It has been found that species of the digenetic genus Trypanosoma as well as species of monogenetic trypanosomatids of insects can grow in the lumen of the scent glands of opossums reproducing the cycle they perform in the intestinal tract of their insect hosts. Based on these findings, speculations are made on the incompletely known cycles of many mammalian trypanosomes and on the evolution of the family Trypanosomatidae.

Trypanosomes of the subgenus Megatrypanum from armadillos (Xenarthra: Dasypodidae)

A new species of trypanosome, Trypanosoma (Megatrypanum) peba, is described from the peripheral blood of the armadillo Euphractus sexcinctus setosus from Bahia State, Brazil. Ten out of 29 specimens of the armadillo Dasypus novemcinctus from Pará State were found to have trypanosomes, including epimastigote forms, in impression smears of subcutaneous lymph nodes. The trypanosomes from D. novemcinctus are illustrated and were identified a idenrified as belonging to the subgenus Megatrypanum on the basis of their general appearance, although they failed to multiply is blood-agar culture medium and no bloodstream forms were seen. This is the first published record of trypanosomes of this subgenus from armdillos and the first demonstration of epimastigote trypanosomes in the mammalian host other than in the bloodstream, or in the anal glands of opssums.

Description of Leishmania equatorensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting arboreal mammals in Ecuador

Characterization is given of a new parasite, Leishmania equatoriensis sp.n. wich was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest onthe Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. brasiliensis complex. L. equatoriensis is cleary distinguishable form all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Deletion, insertion and translocation of DNA sequences contribute to chromosome size polimorphism in Plasmodium berghei

Extensive chromosome size polymorphism in Plasmodium berghei in vivo mitotic multiplication. Size differences between homologous chromosomes mainly involve rearrangements in the subtelomeric regions while internal chromosomal regions are more conserved. Size differences are almost exclusively due to differences in the copy number of a 2.3 kb subtelomeric repeat unit. Not only deletion of 2.3 kb repeats occurs, but addition of new copies of this repeat sometimes results in the formation of enlarged chromosomes. Even chromosomes which originally lack 2.3 kb repeats, can acquire these during mitotic multiplication. In one karyotype mutant, 2.3 kb repeats were inserted within one of the original telomeres of chromosome 4, creating an internal stretch oftelomeric repeats. Chromosome translocation can contribute to chromosome size polymorphism as well We found a karyotype mutant in which chromosome 7 with a size of about 1.4 Mb is translocated to chromosome 13/14 with a size of about 3 Mb, resulting in a rearranged chromosome, which was shown to contain a junction between internal DNA sequences of chromosome 13/14 and subtelomeric 2.3 kb repeats of chromosome 7. In this mutant a new chromosome of 1.4 Mb is present which consists of part of chromosome 13/14.

Raillietina (Raillietina) guaricana n. sp. (Cestoda-Davaineidae), parasite of wild rats from the environmental protection area of Guaricana, Paraná, Brazil

Raillietina (Raillietina) guaricanae n. sp. is described from the wild rats Oryzomys intermedius, O. nigripes and O. ratticeps, captured in the Environmental Protection Area of Guaricana, from November 1988 to December 1989. Raillietina (Railietina) guaricanae n. sp. is closely related to the Neotropical mammalian Raillietina, however it differs by the fewer number of rostellar hooks, and tests different number of eggs capsules and host species. The number of known species of Raillietina (Raillietina), parasites of mammals in the Neotropical Region, is increased to four.

Description of Leishmania (Leishmania) forattinii sp. n., a new parasite infecting opossums and rodents in Brazil

A new parasite species of Leishmania is described, L. (Leishmania) forattinii sp. n., which was isolated from a pooled triturate of liver and spleen of a opossum (Didelphis marsupialis aurita) and from skin samples from a rodent (Proechmys iheringi denigratus), captured in primary forest on the Atlantic Cost of Brazil. Our results on the basis of biological and molecular criteria indicate that this taxonomically distinct parasite ias a new species of the L. mexicana complex, but closely related to L. (L.) aristidesi Laison & shaw, 1979, as revelated by phenetic and phylogenetic numerical analyses of the enzyme data. L. forattinii was clearly distinguishable from other Leishmania species of the genus usisng enzyme electrophoresis, monoclonal antibodies, molecular karyotypes, analysis of restriction enzyme digestion patterns of kinetoplast DNA (kDNA), as well as the use of kDNA hybridization procedures.

Sialoglycoconjugates in Trypanosoma cruzi-host cell interaction: possible biological model - a review

A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialogycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system.

American cutaneous leishmaniasis in Pernambuco, Brazil: eco-epidemiological aspects in "Zona da Mata" region

American cutaneous leishmaniasis (ACL) has been increasing in Pernambuco, thus becoming an important problem for Public Health. The incindence is predominant in the region called "Zona da Mata", in the east of this state. This region corresponds geographically to the primitive area of the Atlantic forest. In order to characterize the eco-epidemiology expression of ACL in this region, two localities situated in the municipalities of Amaraji e Cortes have been selected by the criterion of higher incindence of human cases. Five stocks of patients were characterized and identified on the basis of enzyme profiles as a new variant of Leishmania (V.) braziliensis. A survey of wild and domestic animals was carried out by means of a parasitological and serological diagnosis. Through the analysis of the spleen and liver imprints, were detected amastigotes compatible with Leishmania in five Nectomys s. squamipes, five Bolomys l. pixuna, two Rattus r. alexandrinus and one Rattus r. frugivorus. For two years we carried out monthly sandflies captures using CDC light traps as well as manual captures. Lutzomyia whitmani was predominant, which accounted for 97.4 por cento of the total. These data indicate a strong evidence on the vector and the potential reservoirs of L. braziliensis in this region.

Characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza A viruses

Six clinical isolates of influenza A viruses were examined for hemagglutinin receptor specificity and neuraminidase substrate specificity. All of the viral isolates minimally passaged in mammalian cells demonstrated preferential agglutination of human erythrocytes enzymatically modified to contain NeuAc alpha 2,6Gal sequences, with no agglutination of cells bearing NeuAc alpha 2,3Gal sequences. This finding is consistent with the hemagglutination receptor specificity previously demonstrated for laboratory strains of influenza A viruses. The neuraminidase substrate specificities of the clinical isolates examined were also identical to that described for the N2 neuraminidase of recent laboratory strains of human influenza viruses. The H3N2 viruses all displayed the ability to release sialic acid from both alpha 2, 3 and alpha 2, 6 linkages. In addition, two clinical isolates of H1N1 viruses also demonstrated this dual neuraminidase substrate specificity, a characteristic which has not been previously described for the N1 neuraminidase. These results demonstrate that complementary hemagglutinin and neuraminidase specificities are found in recent isolates of both H1N1 and H3N2 influenza viruses.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Inter-relation of sylvatic and domestic transmission of Trypanosoma cruzi in areas with and without domestic vectorial transmission in Minas Gerais, Brazil

During the period 1980-1986, we captured triatomine bugs and mammalian reservoir hosts from sylvatic and domestic situations in different municipalities of the State of Minas Gerais. Trypanosoma cruzi was isolated from captured bugs, mammals and patients. After cultivation in LIT medium, the electrophoretic enzyme profiles were determined. We obtained atotal of 32 parasite isolates from regions with active domestic transmission, and 24 isolates form areas under control. For the first areas the results suggest introduction of T. cruzi from sylvatic habitats, through incursion of infected opossums and/or sylvatic T. sordida, which appears to have given rise to at least one acute human infection. Of particular interest is the finding of sylvatic opossums and a T. sordida nymph infected with ZB, that could indicate return of parasites from chronic human infections to sylvatic transmission cycles. For the areas under control we also interpret the results as interaction between sylvatic and domestic cycles of transmission, here through the invasion of houses by bugs carrying the Z1 zymodeme from the sylvatic environment. The Multivariate Correspondence Analysis gives a spatial description between the different parasite isolates and confirms the existence of a bridge in the opposite direction in the region with active vectorial transmission including the exporting of Z2 through the peridomestic environment into the sylvatic cycle. For the others areas this bridge corresponds especially to Panstrongylus megistus, importing Z1 into the domestic environment.

Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

A large number of Endotrypanum stocks (representing an heterogeneous population of strains) have been screened against a panel of monoclonal antibodies (MAbs) derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12) or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5) reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved) or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent), but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

On Leishmania enriettii and Other Enigmatic Leishmania Species of the Neotropics

There are 20 named species of the genus Leishmania at present recognized in the New World, of which 14 are known to infect man. The present paper discusses the biological, biochemical and ecological features, where known, of six species which have not till now been found to cause human leishmaniasis; namely, Leishmania (Leishmania) enriettii, L. (L.) hertigi, L. (L.) deanei, L. (L.) aristidesi, L. (L.) forattinii and L. (Viannia) equatorensis. A protocol is suggested for attempts to discover the natural mammalian host(s) and sandfly vector of L. (L.) enriettii. Doubt is cast on the validity of the species L. herreri, described in Costa Rican sloths. Following the concensus of opinion that modern trypanosomatids derive from monogenetic intestinal flagellates of arthropods, phlebotomine sandflies are best regarded as the primary hosts of Leishmania species, with mammals acting as secondary hosts providing a source of parasites for these insects. There are probably natural barriers limiting the life-cycle of most leishmanial parasites to specific sandfly vectors

Biological Parameters and Molecular Markers of Clone CL Brener - The Reference Organism of the Trypanosoma cruzi Genome Project

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28oC is 58±13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace´s medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37oC; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24Sa ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed