SummaryPseudomonas aeruginosa produces three types of bacteriocins: R-, F-and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the Ftype from l phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.
A new compound with an immunosuppressive property was purified from culture filtrates of Isaria sinclairii and was chemically modified to FTY720. Rat spleen cells incubated with FTY720 demonstrated features characteristic of apoptosis--such as the absence of surface microvilli, chromatin condensation, and the formation of apoptotic bodies--by electron microscopy, and genemic DNA fragmentation by agarose gel electrophoresis. When FTY720 was administered in liver-allografted rats at a dose of 0.5 mg/kg from day 1 to day 14 after transplantation, the recipients survived significantly longer than the control group. Pretransplant treatment with 5 mg/kg of FTY720 one day before and on the day of grafting induced a remarkable prolongation of recipient survival, and three of 10 recipients survived for longer than 50 days. Furthermore, administration of FTY720 at 5 mg/kg on days 3 and day 4 after grafting also prolonged survival. In canine kidney allografting, a pretransplant 2-day course of FTY720 at 5 mg/kg prolonged graft survival. Daily administration of FTY720 in combination with CsA resulted in a significant prolongation of graft survival in a synergistic manner. In addition, FTY720 appeared to be nontoxic in canine recipients. These results demonstrated that FTY720, having a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation.
1 Our previous studies revealed that the immunosuppressive agent, FTY720, mainly induces mitochondria-involved apoptosis in some types of cancer cells, since Bcl-2 overexpression prevents the FTY720-induction of apoptotic stimuli. Furthermore, FTY720 induces G0/G1 cell cycle arrest. The present study further examines the correlation between intracellular signaling kinases with FTY720-induced mitochondria-involved apoptosis. 2 Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time-and concentration-dependent manner. FTY720 also induced Bad (Ser 136 ) and ribosomal p70S6 kinase (p70 S6k ) (Thr 389 ) dephosphorylation. 3 FTY720-induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3 0 -kinase (PI 3-kinase) pathway inhibition. 4 FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL-1. BALL-1 cells were resistant to FTY720-induced apoptosis. 5 Okadaic acid (OA) inhibited the FTY720-induced dephosphorylation of Akt and p70 S6k , suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. 6 OA partially inhibited FTY720-induced caspase-3 activation. 7 PP2A or PP2A-like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 8 Overall, the results suggest that FTY720 activated PP2A or PP2A-like phosphatase and dephosphorylated Akt pathway factors resulting in the enhancement of apoptosis via mitochondria. British Journal of Pharmacology (2003) 138, 1303 -1312. doi:10.1038/sj.bjp.0705182 Keywords: FTY720; Akt; PKB; P70 S6k ; Bad; PP2A; apoptosis; leukemia cells Abbreviations: CDK, cyclin-dependent kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; OA, okadaic acid; p70 S6k , ribosomal p70 S6 kinase; PDGF, platelet-derived growth factor; PDK, phosphoinositide-dependent kinase; PDK1, phosphoinositide-dependent kinase 1; phospho-, phosphorylated; PI(3,4)P 2 , phosphatidylinositol 3,4-diphosphate; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-triphosphate; PI 3-kinase, phosphatidylinositol 3 0 -kinase; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PP, protein phosphatase; pRb, retinoblastoma protein; SGK, serum and glucocorticoid-inducible kinase; DC m , mitochondrial membrane potential
Background: A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements.
Most strains of Pseudomonas aeruginosa produce various types of bacteriocins (pyocins), namely, R-, F-, and S-type pyocins. The production of all types of pyocins was shown to be regulated by positive (prtN) and negative (prtR) regulatory genes. The prtN gene activates the expression of various pyocin genes, probably by the interaction of its product with the DNA sequences conserved in the 5' noncoding regions of the pyocin genes. The prtR gene represses the expression of the prtN gene, and its product, predicted from the nucleotide sequence, has a structure characteristic of phage repressors and seems to be inactivated by the RecA protein activated by DNA damage. A model for the regulation of the pyocin genes is proposed.
In the early stage of Drosophila embryogenesis, DNA replication initiates at unspecified sites in the chromosome. In contrast, DNA replication initiates in specified regions in cultured cells. We investigated when and where the initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In the DNA polymerase ␣ gene (DNApol␣) locus, where an initiation region, oriD␣, had been identified in cultured Kc cells, repression of origin activity in the coding region was detected after formation of cellular blastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation regions between oriD␣ and the Drosophila E2F gene (dE2F) downstream of DNApol␣. At least four initiation regions showing replication bubbles were identified in the 65-kb DNApol␣-dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specification levels of origin usage in 5-h embryos are in the intermediate state compared to those in more differentiated cells. Further, we found a spatial correlation between the active promoter regions for dE2F and the active initiation zones of replication. In 5-h embryos, two known transcripts differing in their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. In Kc cells, only one transcript was expressed and functional replication origins were observed only in the region including the promoter region for this transcript.Many observations indicate that DNA replication is not initiated at random sites but rather within specified chromosomal regions in metazoan somatic cells or cultured cells, although the DNA structures essential for initiation of DNA replication in higher eukaryotes remain to be clarified (see reference 8 for a review). In contrast, initiation sites are not specified to the same degree in early embryos of Drosophila melanogaster (36) and Xenopus laevis (23).We previously identified an initiation zone of replication, oriD␣, located downstream of the gene encoding the 180-kDa subunit of DNA polymerase ␣ (DNApol␣) in cultured Kc cells from D. melanogaster (38). We were unable to find other initiation regions within the 40-kb region from oriD␣ to DNApol␣ and its upstream region (35,38). While cultured Kc cells duplicate in approximately 24 h and their S phase lasts for about 8 h, nuclei in early embryos of Drosophila duplicate their DNA in 10 min. Embryogenesis of Drosophila is started by fertilization that occurs at the time of oviposition. The first 2 h of embryogenesis consists of 13 cycles of rapid and almost synchronous nuclear divisions that have only M and S phases (32) (see Fig.
Chromosomal DNA replication units in early embryos of D.melanogaster were studied using two-dimensional gel replicon mapping techniques. DNA was prepared from nuclei encapsulated into agarose beads. This method substantially improved preservation of replication intermediates more than standard DNA preparation methods, and allowed us to detect replication intermediates for even single-copy chromosomal regions without their selective enrichment. Analysis with tandem repeats of histone genes indicated that DNA replication initiates at multiple locations on the repeating unit. The initiation sites were not localized to a defined site, but rather distributed throughout the repeating unit. DNA replication on a single-copy chromosomal region was also suggested to initiate at numerous sites, probably with little regard for the specific DNA sequences.
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