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Query: UNIPROT:P06889 (Mol)
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Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.
Mol Gen Genet 1982
PMID:pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors. 628 16

A 7.5 kb KpnI-generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. serotype O9,12; Vi+; H-d), S. typhi Vinegative strain H400 (i.e. serotype O9,12; Vi-; H-d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906-O4 demonstrated that these strains express the O4 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi+; H-d and the serotype of H404 is O4,12; Vi-; H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906-O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Microbiol 1994 Aug
PMID:Construction and characterization of isogenic O-antigen variants of Salmonella typhi. 752 93

Most bacteriophages undergo a dramatic expansion of their capsids during morphogenesis. In phages lambda, T3, T7 and P22, it has been shown that expansion occurs during the packaging of DNA into the capsid. The terminase-DNA complex docks with the portal vertex of an unexpanded prohead and begins packaging. After some of the DNA has entered, the major head protein undergoes a conformational change that increases both the volume and stability of the capsid. In phage T4, the link between packaging and expansion has not been established. We explored the possibility of such a connection using a pulse-chase protocol and high resolution sucrose gradient analysis of capsid intermediates isolated from wild-type T4-infected cells. We show that the first particle appearing after the pulse is an unexpanded prohead, which can be isolated in vitro as the ESP (empty small particle). The next intermediate to appear is also unexpanded, but contains DNA. This new intermediate, the ISP (initiated small particle), can also be isolated on agarose gels, permitting confirmation of both its expansion state and DNA content ( approximately 10 kbp). It appears, therefore, that >/=8% of the T4 genome enters the head shell prior to expansion. Following packaging of an undetermined amount of DNA, the capsid expands, producing the ILP (initiated large particle), which is finally converted to a full head upon the completion of packaging. An expanded, empty prohead, the ELP (empty large particle), was also observed during 37 degrees C infections, but failed to mature to phage during the chase. Thus the ELP is unlikely to be an intermediate in normal head assembly. We conclude by suggesting that studies on assembly benefit from an emphasis on the processes involved, rather than on the structural intermediates which accumulate if these processes are interrupted.
J Mol Biol 1998 Dec 04
PMID:Capsid expansion follows the initiation of DNA packaging in bacteriophage T4. 982 6

ISP-1 is a new type of immunosuppressant, the structure of which is homologous to that of sphingosine. In a previous study, ISP-1 was found to inhibit mammalian serine palmitoyltransferase, the primary enzyme involved in sphingolipid biosynthesis, and to reduce the intracellular pool of sphingolipids. ISP-1 induces the apoptosis of cytotoxic T cells, which is triggered by decreases in the intracellular levels of sphingolipids. In this study, the inhibition of yeast (Saccharomyces cerevisiae) proliferation by ISP-1 was observed. This ISP-1-induced growth inhibition was also triggered by decreases in the intracellular levels of sphingolipids. In addition, DNA duplication without cytokinesis was detected in ISP-1-treated yeast cells on flow cytometry analysis. We have cloned multicopy suppressor genes of yeast which overcome the lethal sphingolipid depletion induced by ISP-1. One of these genes, SLI2, is synonymous with YPK1, which encodes a serine/threonine kinase. Kinase-dead mutants of YPK1 did not show any resistance to ISP-1, leading us to predict that the kinase activity of the Ypk1 protein should be essential for this resistance to ISP-1. Ypk1 protein overexpression had no effect on sphingolipid biosynthesis by the yeast. Furthermore, both the phosphorylation and intracellular localization of the Ypk1 protein were regulated by the intracellular sphingolipid levels. These data suggest that the Ypk1 protein is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. The Ypk1 protein was reported to be a functional homologue of the mammalian protein kinase SGK, which is a downstream kinase of 3-phosphoinositide-dependent kinase 1 (PDK1). PDK1 phosphotidylinositol (PI) is regulated by PI-3,4,5-triphosphate and PI-3,4-bisphosphate through the pleckstrin homology (PH) domain. Overexpression of mammalian SGK also overcomes the sphingolipid depletion in yeast. Taking both the inability to produce PI-3,4, 5-triphosphate and PI-3,4-bisphosphate and the lack of a PH domain in the yeast homologue of PDK1, the Pkh1 protein, into account, these findings further suggest that yeast may use sphingolipids instead of inositol phospholipids as lipid mediators.
Mol Cell Biol 2000 Jun
PMID:Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. 1082 4

Cannabinoids have recently been shown to induce the expression of the cyclooxygenase-2 (COX-2) isoenzyme in H4 human neuroglioma cells. Using this cell line, the present study investigates the contribution of the second messenger ceramide to this signaling pathway. Incubation of cells with the endocannabinoid analog R(+)-methanandamide (R(+)-MA) was associated with an increase of intracellular ceramide levels. Enhancement of ceramide formation by R(+)-MA was abolished by fumonisin B1, a ceramide synthase inhibitor, whereas inhibitors of neutral sphingomyelinase (spiroepoxide, glutathione) and serine palmitoyltransferase (l-cycloserine, ISP-1) were inactive in this respect. R(+)-MA caused a biphasic activation of the p38 and p42/44 mitogen-activated protein kinases (MAPKs), with phosphorylation peaks occurring after 15-min and 4- to 8-h treatments, respectively. Inhibition of ceramide synthesis with fumonisin B1 was associated with a suppression of R(+)-MA-induced delayed phosphorylations of p38 and p42/44 MAPKs and subsequent COX-2 expression. The involvement of ceramide in COX-2 expression was corroborated by findings showing that C2-ceramide and neutral sphingomyelinase from Bacillus cereus caused concentration-dependent increases of COX-2 expression that were suppressed in the presence of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazol (SB203580, a p38 MAPK inhibitor) or 2'-amino-3'-methoxyflavone (PD98059, a p42/44 MAPK activation inhibitor). In contrast, dihydro-C2-ceramide being used as a negative control did not induce MAPK phosphorylation and COX-2 expression. Collectively, our results demonstrate that R(+)-MA induces COX-2 expression in human neuroglioma cells via synthesis of ceramide and subsequent activation of p38 and p42/44 MAPK pathways. Induction of COX-2 expression via ceramide represents a hitherto unknown mechanism by which cannabinoids mediate biological effects within the central nervous system.
Mol Pharmacol 2003 Nov
PMID:Ceramide is involved in r(+)-methanandamide-induced cyclooxygenase-2 expression in human neuroglioma cells. 1457 69

The S1 serine protease family is one of the largest gene families known. Within this family there are several subfamilies that have been grouped together as a result of sequence comparisons and substrate identification. The grouping of related genes allows for the speculation of function for newly found members by comparison and for novel subfamilies by contrast. Analysis of the evolutionary patterns of genes indicates whether or not orthologs are likely to be identified in other species as well as potentially indicating that hypothesized orthologs are in fact not. Looking at subtle differences between subfamily members can reveal intricacies about function and expression. Previously, we have described genes encoding two novel serine proteinases, ISP1 and ISP2, which are most closely related to tryptases. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching and invasion in vitro. Additionally both ISP1 and ISP2 are co-expressed in the endometrial gland during the time of hatching, suggesting that they may also both participate in zona lysis from within the uterine lumen. Here, we demonstrate that the ISPs are tandemly linked within the tryptase cluster on 17A3.3. We suggest that remarkable similarities within the 5'-untranslated and first intron regions of ISP1 and ISP2 may explain their intimate co-regulation in uterus. We also suggest that ISP genes have evolved through gene duplication and that the ISP1 gene has also begun to adopt an additional new function in the murine preimplantation embryo.
Mol Reprod Dev 2004 Oct
PMID:Origin of the murine implantation serine proteinase subfamily. 1529 13

The 3.0-3.1A X-ray structures of the cytochrome b(6)f complex from Mastigocladus laminosus and Chlamydomonas reinhardtii obtained in the presence of the p-side quinone-analogue inhibitor tridecyl-stigmatellin (TDS) are very similar. A difference occurs in the p-side binding position of TDS. In C.reinhardtii, TDS binds in the ring-in mode, as previously found for stigmatellin in X-ray structures of the cytochrome bc(1) complex. In this mode, the H-bonding chromone ring moiety of the TDS bound in the Q(p) niche is proximal to the ISP [2Fe-2S] cluster, and its 13 carbon tail extends through a portal to the large inter-monomer quinone-exchange cavity. However, in M.laminosus, TDS binds in an oppositely oriented ring-out mode, with the tail inserted toward the Q(p) niche through the portal and the ring caught in the quinone-exchange cavity that is 20A away from the [2Fe-2S] cluster. Site-directed mutagenesis of residues that might determine TDS binding was performed with the related transformable cyanobacterium Synechococcus sp. PCC 7002. The following changes in the sensitivity of electron transport activity to TDS and stigmatellin were observed: (a) little effect of mutation L193A in cytochrome b(6), which is proximal to the chromone of the ring-out TDS; (b) almost complete loss of sensitivity by mutation L111A in the ISP cluster binding region, which is close to the chromone of the ring-in TDS; (c) a ten and 60-fold increase associated with the mutation L81F in subunit IV. It was inferred that only the ring-in binding mode, in which the ring interacts with residues near the ISP, is inhibitory, and that residue 81 of subunit IV, which resides at the immediate entrance to the Q(p) niche, controls the relative binding affinity of inhibitor at the two different binding sites.
J Mol Biol 2004 Nov 19
PMID:Molecular control of a bimodal distribution of quinone-analogue inhibitor binding sites in the cytochrome b(6)f complex. 1552

We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals.
Hum Mol Genet 2005 Dec 15
PMID:Targeted disruption of hepatic frataxin expression causes impaired mitochondrial function, decreased life span and tumor growth in mice. 1627 35

The sphingolipid ceramide has been recognized as an important mediator in the apoptotic machinery, and its efficient conversion to glucosylceramide has been associated with multidrug resistance. Therefore, inhibitors of glucosylceramide synthase are explored as tools for treatment of cancer. In this study, we used D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol to sensitize Neuro-2a murine neuroblastoma cells to the microtubule-stabilizing agent paclitaxel. This treatment resulted in a synergistic inhibition of viable cell number increase, which was based on a novel mechanism: (a) After a transient mitotic arrest, cells proceeded through an aberrant cell cycle resulting in hyperploidy. Apoptosis also occurred but to a very limited extent. (b) Hyperploidy was not abrogated by blocking de novo sphingolipid biosynthesis using ISP-1, ruling out involvement of ceramide as a mediator. (c) Cyclin-dependent kinase 1 and 2 activities were synergistically decreased on treatment. In conclusion, instead of inducing apoptosis through ceramide accumulation, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol by itself affects cell cycle-related proteins in paclitaxel-arrested Neuro-2a cells resulting in aberrant cell cycle progression leading to hyperploidy.
Mol Cancer Ther 2006 Mar
PMID:PDMP sensitizes neuroblastoma to paclitaxel by inducing aberrant cell cycle progression leading to hyperploidy. 1654 73

Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays a central role in the immune system. Homing of lymphocytes from blood into secondary lymphoid tissues beyond high endothelial venules is highly dependent on the interaction between the chemokines CCL19, CCL21, CXCL12, and CXCL13, and their receptors CCR7, CXCR4 and CXCR5. However, the molecular mechanism(s) of lymphocyte egress from secondary lymphoid tissues to lymph remained unclear. We have found a new class of immunomodulator, FTY720 by chemical modification of vegetative wasp-derived natural product, ISP-I (myriocin). FTY720 has been shown to be highly effective in experimental allograft and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating activity in these models. The reduction of circulating lymphocytes by FTY720 is caused by sequestration of lymphocytes into secondary lymphoid tissues and thymus. FTY720 is rapidly converted to (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P] by sphingosine kinase 2 in vivo. (S)-FTY720-P acting as a potent agonist of S1P receptor type 1 (S1P1), induces long-term down-regulation of S1P1 on lymphocytes, and thereby inhibits the migration of lymphocytes toward S1P. Thus, it is presumed that FTY720-induced lymphocyte sequestration is due to the inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymus by its active metabolite (S)-FTY720-P. Throughout the analysis of the mechanism of action of FTY720, it is clarified that S1P/S1P1 interaction plays an important role for lymphocyte egress from secondary lymphoid tissues and thymus.
Cell Mol Immunol 2006 Feb
PMID:Role of sphingosine 1-phosphate receptor type 1 in lymphocyte egress from secondary lymphoid tissues and thymus. 1654 44


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