http://www.dspace.espol.edu.ec/handle/123456789/5862 2026-05-20T09:57:31Z http://www.dspace.espol.edu.ec/handle/123456789/5879 Fotos laboratorío biomedicina Cardenas Medranda, Washington Bolívar FOTOS LABORATORIO BIOMEDICINA FOTOS LABORATORIO BIOMEDICINA 2009-07-08T00:00:00Z http://www.dspace.espol.edu.ec/handle/123456789/5877 Curriculo vitae Cardenas Medranda, Washington Bolívar 2009-01-01T00:00:00Z http://www.dspace.espol.edu.ec/handle/123456789/5875 Cresolase, catecholase and laccase activities in haemocytes of the red swamp crayfish Cardenas Medranda, Washington Bolívar; Dankert, John R. Phenoloxidase activity in crayfish haemocyte lysates and extracts of haemocyte membranes were studied using native PAGE and SDS-PAGE gels and staining for cresolase, catecholase and laccase activities. The activation of the proenzyme, prophenoloxidase to phenoloxidase, in native PAGE was demonstrated following exposure to SDS. By staining samples separated in SDSPAGE followed by renaturation, a high molecular mass phenoloxidase activity was identified in both the soluble and membrane fractions of haemocyte preparations. The membrane-associated activity appeared at only relatively high molecular mass (>300 kDa), and could easily be eluted from membranes using detergents or NaCl. Further, this membrane-associated activity has a catecholase activity but not the cresolase activity seen in the soluble preparations. In addition, several other phenoloxidase enzymes were identified with di#erent relative mobilities (250, 80, 72 and 10 kDa). Crayfish haemocytes also contained laccase activity, thought to be restricted to cuticle sclerotisation in the integument. Laccase activity in haemocytes might aid in the formation of capsule used to contain pathogens. 1999-05-31T00:00:00Z http://www.dspace.espol.edu.ec/handle/123456789/5876 Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides Cardenas Medranda, Washington Bolívar; Dankert, John R.; Jenkins, Jill A. Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P%0:05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicating common innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertebrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds. 2004-01-01T00:00:00Z