No abstract

The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

HM-1:IMSS, a pathogenic strain of Entamoeba histolytica, and its mutant BG-3, identified by resistance to cytochalasin D, were tested for their capacity to: (i) secrete electron-dense granules; (ii) adhere and digest native type I collagen gels; and (iii) produce liver abscesses in new-born hamsters. The results demonstrate that the mutant has low adherence to collagen, low electron-dense granule secretion and collagenolytic activity, and low capacity to produce liver lesions in vivo, compared with the parental strain HM1:IMSS.

BackgroundDengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies.Results(1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut.ConclusionOur results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.

In this paper we describe the interaction of melatonin with calmodulin in MDCK cells. The double staining immunofluorescent method showed that calmodulin in control MDCK cells appeared as fluorescent spots at the cell periphery. In contrast, MDCK cells cultured with 10(-9) M melatonin for 6, 12, 24 hr or for 4 days showed that calmodulin fluorescent spots were distributed throughout the cytoplasm as well as in the nucleus. These calmodulin rearrangements were reversed 6 hr after melatonin withdrawal. Moreover, calmodulin radioimmunoassay showed that in 10(-9) M melatonin-treated cells, membrane-bound calmodulin content was increased by 78% whereas cytosolic calmodulin decreased by 60%. Simultaneous labeling of MDCK cells with specific antibodies against calmodulin and melatonin showed that not only does the indole enter the cell, but that it has the same subcellular distribution as calmodulin. Besides the early responses induced by the melatonin antagonism to calmodulin, the results suggest that the indole may also induce long-term cellular responses by changing calmodulin concentrations in specific cellular compartments.

To genetically characterize dengue 2 (DEN-2) viruses in Oaxaca, Mexico, the C protein, and a portion of the prM protein genes of 8 isolates from the 2001 DEN epidemic were sequenced. The sequences were compared to those of prototype DEN-2 viruses from various parts of the world. Phylogenetic analysis suggested that the 2001 isolates of DEN-2 were of the American/Asian genotype and were most similar to the Jamaica and Venezuelan isolates MARA3, LARD1996 and LARD1910. Molecular analyses confirmed the origin of the isolates. This study indicates that DEN-2 strains of American/Asian genotype probably from Southeast Asian are circulating in Oaxaca.

Dengue viruses are arthropod-borne, single-stranded RNA viruses. Aëdes aegypti and Aëdes albopictus are the principal vectors. In order to understand the molecular basis of dengue virus infections we explored the biochemical identity of dengue-2 (DEN-2) virus receptors in the Aëdes albopictus-derived cell line C6/36. We show here that DEN-2 interacts with two major polypeptides of 80 and 67 kDa. Polyclonal anti-C6/36 membrane antibodies block DEN-2 binding to intact C6/36 monolayers as well as to membrane extracts. Our results strongly suggest that the identified polypeptides are part of the DEN-2 receptors.

In vitro interaction of Entamoeba histolytica with collagen induces intracellular formation and release of electron-dense granules (EDG) and stimulation of collagenolytic activity. Purified EDG contain 1.66 U of collagenase per mg of protein. Thus, EDG may participate in tissue destruction during invasive amebiasis. Monoclonal antibodies (MAbs) L1.1 and L7.1 reacted specifically with EDG in enzyme-linked immunosorbent assay (ELISA) and immunofluorescence and immunoelectron microscopy. MAb L7.1 immunoprecipitated three polypeptides with molecular weights of 95,000, 68,000, and 28,000 from lysates of biosynthetically labeled E. histolytica. Both MAbs recognized the pathogenic E. histolytica axenic strains HM1:IMSS, HM38:IMSS, and HK-9 but failed to react in ELISA with Entamoeba moshkovskii, Entamoeba invadens, and E. histolytica-like Laredo. In addition, MAb L7.1 reacted with one E. histolytica isolate from a symptomatic patient but did not react with four of five isolates from asymptomatic patients. EDG antigens were detected by a MAb L7. 1-based ELISA in E. histolytica-containing fecal samples from symptomatic, but not asymptomatic, individuals. These results suggest that the EDG antigen detected with MAb L7. 1 may be differentially expressed in pathogenic and nonpathogenic E. histolytica.