RESUMEN
The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.
Asunto(s)
Bacteriocinas/genética , ADN Bacteriano/genética , Lactococcus lactis/genética , Plásmidos/genética , Análisis de Secuencia de ADN , Secuencia de Aminoácidos , Bacteriocinas/biosíntesis , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/análisis , Lactococcus lactis/metabolismo , Datos de Secuencia Molecular , FilogeniaRESUMEN
The induction of cell death in leukemic HL-60 cells by the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH(3) induced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH(3) also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). ET-18-OCH(3) analogues unable to induce apoptosis failed to disrupt DeltaPsi(m) and to activate caspase-3. ET-18-OCH(3)-resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo DeltaPsi(m) disruption upon ET-18-OCH(3) incubation. Cyclosporin A partially inhibited DeltaPsi(m) dissipation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented DeltaPsi(m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2, 6-dichlorobenzoyloxymethylketone prevented ET-18-OCH(3)-induced PARP proteolysis and DNA fragmentation, but not DeltaPsi(m) dissipation. ET-18-OCH(3) did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH(3)-induced apoptosis did not require protein synthesis. Our data indicate that DeltaPsi(m) dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET-18-OCH(3), and that the sequence of events in the apoptotic action of ET-18-OCH(3) on human leukemic cells is: DeltaPsi(m) disruption, caspase-3 activation and internucleosomal DNA degradation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Leucemia/patología , Mitocondrias/fisiología , Éteres Fosfolípidos/farmacología , Caspasa 3 , Inhibidores de Caspasas , Caspasas/genética , Fragmentación del ADN , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Leucemia/enzimología , Leucemia/fisiopatología , Leucemia Mieloide Aguda/patología , Leucemia-Linfoma de Células T del Adulto/patología , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
In this study, superoxide dismutase (SOD) and catalase activities were determinated in the erythrocytes (RBC) from patients with chronic renal failure. The study included healthy subjects (n = 7), patients on hemodialysis (HD) using polyacrylonitrile-type dialysis membrane (before and after HD) (n = 10), patients on HD using cuprophane-type dialysis membrane (before and after HD) (n = 6), and patients on continuous ambulatory peritoneal dialysis (CAPD) (n = 11). A significant decrease in SOD activity was found in HD groups using polyacrylonitrile- or cuprophane-type dialysis membrane. SOD activity was found to increase in patients undergoing CAPD. We have found that CAT activity is higher in all the CRF groups in respect to the control: with polyacrylonitrile-type dialysis membrane, with cuprophane-type dialysis membrane, and in CAPD treatment.