Browsing by Author "Rosemblatt, M"

Now showing 1 - 7 of 7
  • No Thumbnail Available
    Item
    Complete sequence of the genome of the human isolate of Andes virus CHI-7913
    (2003) Tischler, ND; Fernández, J; Müller, I; Martínez, R; Galeno, H; Villagra, E; Mora, J; Ramírez, E; Rosemblatt, M; Valenzuela, PDT
    We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M. and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7%. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98% overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92%. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed.
  • No Thumbnail Available
    Item
    Hantavirus Gc glycoprotein: evidence for a class II fusion protein
    (2005) Tischler, ND; Gonzalez, A; Perez-Acle, T; Rosemblatt, M; Valenzuela, PDT
    Hantavirus cell entry is promoted by its envelope glycoproteins, Gin and Gc, through cell attachment and by fusion between viral and endosomal membranes at low pH. However, the role of Gn and Gc in receptor binding and cell fusion has not yet been defined. In this work, a sequence presenting characteristics similar to those of class II fusion peptides (FPs) of alphavirus Ell and flavivirus E proteins is identified within the hantavirus Gc glycoprotein. A three-dimensional comparative molecular model based on crystallographic data of tick-borne encephalitis virus E protein is proposed for the Andes virus (ANDV) Gc ectodomain, which supports a feasible class II fusion-protein fold. In vitro experimental evidence is provided for the binding activity of the ANDV FP candidate to artificial membranes, as demonstrated by fluorescence anisotropy assays. Taken together, these results support the hypothesis that the Gc glycoprotein of hantaviruses and of other members of the family Bunyaviridae directs the viral fusion activity and that it may be classified as a class II viral fusion protein.
  • No Thumbnail Available
    Item
    Histamine reduces gap junctional communication of human tonsil high endothelial cells in culture
    (2004) Figueroa, XF; Alviña, K; Martínez, AD; Garcés, G; Rosemblatt, M; Boric, MP; Sáez, JC
    The regulation of gap junctional communication by histamine was studied in primary cultures of human tonsil high endothelial cells (HUTECs). We evaluated intercellular communication, levels, state of phosphorylation, and cellular distribution of gap junction protein subunits, mainly connexin (Cx)43. Histamine induced a time-dependent reduction in dye coupling (Lucifer yellow) associated with reduction in connexin43 localized at cell-cell appositions (immunofluorescence), without changes in levels and phosphorylation state of connexin43 (immunoblots). These effects were prevented with chlorpheniramine, an H-1 receptor blocker; indomethacin, a cyclooxygenase blocker; or GF109203X, a protein kinase C inhibitor. Treatment with phorbol myristate acetate, a protein kinase C activator, and 4bromo (4Br)-A23187, a calcium ionophore, mimicked the histamine-induced effects on dye coupling. 8Bromo-cAMP doubled the dye coupling extent and prevented the histamine-induced reduction in incidence of dye coupling. After 24-h histamine treatment, known to desensitize H, receptors, reapplication of histamine increased cell coupling in a way prevented by ranitidine, an H-2 receptor blocker. Thus, activation of H-1 and H-2 receptors, which increase intracellular levels of free Ca2+ and cAMP, respectively, may affect gap junctional communication in opposite ways. Stabilization of actin filaments with phalloidine diminished but did not totally prevent histamine-induced cell shape changes and reduction in dye coupling. Hence, the histamine-induced reduction in gap junctional communication between HUTEC is mediated by cytoskeleton-dependent and -independent mechanisms and might contribute to modulate endothelial function in lymphoid tissue. (C) 2004 Elsevier Inc. All fights reserved.
  • No Thumbnail Available
    Item
    Human and rodent humoral immune responses to Andes virus structural proteins
    (2005) Tischler, ND; Galeno, H; Rosemblatt, M; Valenzuela, PDT
    In the present work we identified B-cell epitopes recognized by sera of humans and rodents naturally infected with Andes virus, a hantavirus present in Chile and Argentina. Analysis of patient and rodent sera with overlapping peptides revealed 21 human and rodent epitopes on the three structural proteins. Whereas in the nucleoprotein the region comprising aa 248-260 was shown to be the key determinant of human sera, the major antigenic site of rodent antibody reactivity is located at aa 326-338. In G1, the main epitope recognized by human sera was mapped to aa 14-26, while rodent antibodies bound predominantly to aa 599-611. In contrast, humans and mice had strong responses to three regions in G2 (aa 691-703, aa 918-930, aa 955-967), of which the last two are associated with neutralization of Hantaan virus. This insight affords important information for the development of immunotherapies for the acute phase of hantavirus cardiopulmonary syndrome. (c) 2005 Elsevier Inc. All rights reserved.
  • No Thumbnail Available
    Item
    Isolation and expression of the genes coding for the membrane bound transglycosylase B (MltB) and the transferrin binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis
    (2004) Wilhelm, V; Morales, C; Martínez, R; Rosemblatt, M; Burzio, LO; Valenzuela, PDT
    We have isolated and sequenced the genes encoding the membrane bound transglycosylase B (MltB) and the transferring binding protein B (TbpB) of the salmon pathogen Piscirickettsia salmonis. The results of the sequence revealed two open reading frames that encode proteins with calculated molecular weights of 38,830 and 85,140. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from phylogenetically related microorganisms. Partial sequences coding the amino and carboxyl regions of MltB and a sequence of 761 base pairs encoding the amino region of TbpB have been expressed in E. coli. The strong Immoral response elicited by these proteins in mouse confirmed the immunogenic properties of the recombinant proteins. A similar response was elicited by both proteins when injected intraperitoneally in Atlantic salmon. The present data indicates that these proteins are good candidates to be used in formulations to study the protective immunity of salmon to infection by P. salmonis.
  • Thumbnail Image
    Item
    Lymphoid B cells induce NF-kappa B activation in high endothelial cells from human tonsils
    (OXFORD UNIV PRESS, 2006) Naves, R; Reyes, LI; Rosemblatt, M; Jacobelli, S; Gonzalez, A; Bono, MR
    Immune surveillance depends on still poorly understood lymphocyte-endothelium interactions required for lymphocyte transendothelial migration into secondary lymphoid organs. The nuclear factor kappa B (NF-kappa B) regulatory system and its inhibitory I kappa B proteins control the inducible expression of adhesion molecules, cytokines and chemokines involved in endothelial activation and lymphocyte transmigration. Here we present results showing the activation of this system in response to the interaction of high endothelial cells from human tonsils (HUTEC) with human B and T lymphoid cell lines and primary tonsillar lymphocytes. Western blot and electrophoretic mobility shift assays show that adhesion of different lymphoid cells induce varying levels of NF-kappa B activation in HUTEC, with Daudi cells, tonsil-derived B cell line 10 (TBCL-10) and primary tonsillar B lymphocytes causing the strongest activation. The main NF-kappa B protein complexes translocated to the nucleus were p65/p50 and p50/p50. Results from reverse transcription-PCR and flow cytometry analysis of HUTEC indicate that the interaction with Daudi cells induce an increased expression of IL-6 and IL-8 mRNA and cell-surface expression of intercellular adhesion molecule-1, all of which were prevented by sodium salicylate, an inhibitor of NF-kappa B activation. Transwell experiments show that NF-kappa B activation and the response of HUTEC to the interaction of Daudi cells does not depend on direct cell-cell contact but rather on the production of soluble factors that require the presence of both cell types. These results suggest that lymphocytes and high endothelium establish a cross talk leading to NF-kappa B-mediated expression of cytokines and adhesion molecules, inducing endothelial cell activation.
  • No Thumbnail Available
    Item
    Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis
    (2005) Wilhelm, V; Soza, C; Martínez, R; Rosemblatt, M; Burzio, LO; Valenzuela, PDT
    We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.