RESUMEN
The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes. The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species. Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105). Expression of dolichol phosphate mannose synthase of S. mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms. Northern blot analysis revealed a single 1-kb band. Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.
Asunto(s)
Fosfatos de Dolicol/metabolismo , Manosiltransferasas/metabolismo , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Cromatografía en Capa Delgada , Cartilla de ADN/química , Femenino , Biblioteca de Genes , Masculino , Manosiltransferasas/genética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Mast cell hyperplasia can be causally related with chronic inflammation and liver fibrosis. Their survival and proliferation is dependent upon locally produced growth factors, the major one being the stem cell factor (SCF). Glucocorticoids can decrease mastocytosis, down-regulating the mast cell production of pro-inflammatory factors or inhibiting the expression of SCF in stroma. We compared dexamethasone effect on SCF expression in co-cultures of mast cells with NIH/3T3 fibroblasts or with primary cultures of activated hepatic stellate cells. Dexamethasone abrogated the NIH/3T3 stroma capacity to sustain mast cell proliferation, but not of hepatic stellate cells, at the post-transcriptional level. Mast cells reverted completely dexamethasone effect on hepatic stellate cells, increasing their SCF synthesis and transport. In both models, dexamethasone inhibited the mast cell spreading on the stroma, which was thus not required for mast cell survival and proliferation. Liver pathologies associated with mast cell hyperplasia are not expected to be sensitive to glucocorticoid treatments.
Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Regulación hacia Abajo/efectos de los fármacos , Mastocitos/efectos de los fármacos , Factor de Células Madre/efectos de los fármacos , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Animales , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/fisiología , Granuloma/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Factor de Células Madre/metabolismoAsunto(s)
Hemoproteínas/biosíntesis , Hemoproteínas/química , Schistosoma mansoni/metabolismo , Animales , Cristalización , Femenino , Hemo/análisis , Hemo/metabolismo , Hemoproteínas/análisis , Concentración de Iones de Hidrógeno , Masculino , Pigmentos Biológicos/análisis , Pigmentos Biológicos/biosíntesis , Pigmentos Biológicos/química , Schistosoma mansoni/química , Caracteres Sexuales , Solubilidad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The formation of mannolipid through catalysis by mannosyl transferase of adult females of Schistosoma mansoni was found to be 2-3-fold higher than male worms. In contrast, mannosyl transferase in immature females generated approximately the same amount of mannolipid as male worms, immature or not. Exogenous dolichol phosphate added to homogenates of male worms produced a stoichiometric increase in mannolipid formation. Saturating amounts of dolichol phosphate generated similar mannosyl transferase activities in male and female homogenates, showing that in S. mansoni, dolichol phosphate is the lipid intermediate in the glycosylation reaction and that this mannolipid is a rate-limiting substrate. Thin layer chromatography revealed that the mannolipid was identical in male and female worms. Adult males incubated with 14C-acetate synthesised several apolar compounds, one of which displayed a Rf identical to that of the mannolipid. When exposed to 14C-acetate treated males in vitro, untreated females were able to incorporate a compound, which partitioned in the same way as the mannolipid. The increased mannosyl transferase-dependent mannolipid formation in adult females may reflect a higher energy demand by these parasites, which is probably associated with oogenesis.
Asunto(s)
Fosfatos de Dolicol/metabolismo , Manosiltransferasas/metabolismo , Schistosoma mansoni/enzimología , Animales , Cricetinae , Femenino , Cinética , Masculino , MesocricetusRESUMEN
Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/genética , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Abejas/enzimología , Caenorhabditis elegans/enzimología , Bovinos , Clonación Molecular , Secuencia de Consenso , ADN Complementario , Complejo I de Transporte de Electrón , Biblioteca de Genes , Humanos , Mitocondrias Cardíacas/efectos de los fármacos , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Receptores Androgénicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma mansoni/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Partículas Submitocóndricas/efectos de los fármacos , Partículas Submitocóndricas/metabolismo , Testosterona/farmacologíaRESUMEN
The occurrence of a receptor for human LDL was investigated in the tegument of adult Schistosoma mansoni employing several approaches. Binding of LDL to SDS-PAGE fractionated tegument proteins was measured directly on nitro-cellulose membranes and visualised by an anti-human LDL antibody. Proteins with an Mr of 60, 35 and 14 kDa were evidenced. Affinity chromatography of 125I-labelled tegument proteins on a LDL-Sepharose column, revealed the same pattern of proteins observed in the immunoblot experiments. Finally, the binding of human LDL to the intact tegument was measured by microcalorimetry. Binding was shown to be an exothermic reaction, releasing approximately 2500 kcal/mol, it was saturable, and reproducibly displayed a biphasic curve suggesting that binding of LDL to S. mansoni might occur through a two step process, initiated by a nonspecific hydrophobic interaction followed by a specific high affinity ligand-receptor reaction. Pre-treatment of the tegument with trypsin reduced the binding of LDL to the tegument. Furthermore, albumin, which is an abundant lipid carrier protein in the serum and thus a potential ligand, failed to release any measurable heat when incubated with the tegument.