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81.
Paul Salers L'Houcine Ouafik Pierre Giraud Anne Dutour Jean-Yves Maltese Charles Oliver 《Molecular and cellular biochemistry》1991,106(1):15-24
Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/106 cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN2) at 5 × 10–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH
Thyrotropin-Releasing Hormone or Thyroliberin
- His-Pro-DKP
Histidyl-ProlineDiketopiperazine
- TRH-OH
acid TRH or deamidated TRH
- LH-RH
Luteinizing Hormone-Releasing Hormone
- Z-Gly-Pro-CHN2
N-benzyloxycarboxyl-Gly-Pro-diazomethylketone
- PGP
Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-)
- PE
Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26) 相似文献
82.
Characterization of a frequent polymorphism in the coding sequence of the Tp53 gene in colonic cancer patients and a control population 总被引:2,自引:0,他引:2
Sylviane Olschwang Pierre Laurent-Puig Anne Vassal Rémy-J. Salmon Gilles Thomas 《Human genetics》1991,86(4):369-370
Summary We describe a simple method for characterizing a frequent polymorphism (that subsitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population. We could find no evidence that this polymorphism is associated with a marked predisposition to colorectal cancer. 相似文献
83.
84.
Ronald J. Hill Margaret R. Mott Fujiko Watt Theodora Fifis P. Anne Underwood 《Chromosoma》1986,94(6):441-448
An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci. 相似文献
85.
Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker 总被引:10,自引:0,他引:10
Lynne V. Mayne Anne Priestley Michael R. James Julian F. Burke 《Experimental cell research》1986,162(2):530-538
Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line. 相似文献
86.
Transposon Mutagenesis and Excision of R′ Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains
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Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10−7 to 10−9. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hgr transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with 32P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hgr both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hgr transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R′ plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome. 相似文献
87.
The O-methyl substituents of aromatic compounds constitute a C1 growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C1 substrate was determined by 14C radiotracer techniques. O-[methyl-14C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C1 substrate. The data showed that for every O-methyl carbon converted to [14C]acetate, two were oxidized to 14CO2. Quantitation of the carbon recovered in the two products, acetate and CO2, indicated that acetate was formed in part by the fixation of unlabeled CO2. The specific activity of 14C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO2 and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-14C]vanillate by strain TH-001 can be described as follows: 314CH3OC7H5O3 + CO2 + 4H2O → 14CH3COOH + 214CO2 + 10H+ + 10e- + 3HOC7H5O3. 相似文献
88.
Christian Boucher Anne Martinel Patrick Barberis Genevieve Alloing Claudine Zischek 《Molecular & general genetics : MGG》1986,205(2):270-275
Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acrr), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acrr mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne. 相似文献
89.
M N Fukuda B Bothner K O Lloyd W J Rettig P R Tiller A Dell 《The Journal of biological chemistry》1986,261(11):5145-5153
Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas. 相似文献
90.
Phorbol 12,13-Dibutyrate Increases Tyrosine Hydroxylase Activity in the Superior Cervical Ganglion of the Rat 总被引:3,自引:3,他引:0
Phorbol 12,13-dibutyrate (PDBu) increased the production of 3,4-dihydroxyphenylalanine (DOPA) in the superior cervical ganglion of the rat. This effect occurred without a detectable lag and persisted for at least 90 min of incubation. The action of PDBu was half-maximal at a concentration of approximately 0.1 microM; at high concentrations, PDBu produced about a twofold increase in DOPA accumulation. PDBu increased DOPA production in decentralized ganglia and in ganglia incubated in a Ca2+-free medium. The action of PDBu was additive with the actions of dimethylphenylpiperazinium, muscarine, and 8-Br-cyclic AMP, all of which also increase DOPA accumulation, and was not inhibited by the cholinergic antagonists hexamethonium (3 mM) and atropine (6 microM). Finally, PDBu did not increase the content of cyclic AMP in the ganglion. Thus, the action of PDBu does not appear to be mediated by the release of neurotransmitters from preganglionic nerve terminals, by the stimulation of cholinergic receptors in the ganglion, or by an increase in ganglionic cyclic AMP. PDBu also increased the incorporation of 32Pi into tyrosine hydroxylase. PDBu activates protein kinase C, which in turn may phosphorylate tyrosine hydroxylase and increase the rate of DOPA synthesis in the ganglion. 相似文献