Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad

A pseudomonad has been isolated from sewage, which can utilize 3-chlorobenzoic acid as a sole carbon source. In cells grown on benzoate the enzymes of the -ketoadipic acid pathway are present. Considerable enzymic activities for chlorinated substrates were found in benzoate grown cells only for the oxygenation of 3-chlorobenzoate and the dehydrogenation of 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid. 3-Chlorobenzoate grown cells show additional high activities for the turnover of 3- and 4-chlorocatechols and chloromuconic acids.Abbreviations Used DHB (-)-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (derived from the trivial name, dihydrodihydroxybenzoate) - 3- and 5-Cl-DHB correspondingly 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid     

Isolation of ouabain-resistant human diploid fibroblasts

Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of 10^?7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 10⁸ unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2–5 × 10^?8 M, while resistant clones required 15–180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of ³H-ouabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition. Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.     

Activation of phosphorylating vesicles by net transfer of phosphatidyl choline by phospholipid transfer protein

Vesicles formed with phosphatidyl ethanolamine, phosphatidyl choline, cardiolipin, coupling factors and hydrophobic proteins from bovine heart mitochondria catalyzed a rapid³²P_i-ATP exchange. When phosphatidyl choline was deleted during the assembly of the vesicles, little³²P_i-ATP exchange was observed. Exchange activity was induced by incubating such deficient vesicles with phosphatidyl choline liposomes in the presence of a phosphatidyl choline transfer protein isolated from bovine heart. Transfer of [³²P] phosphatidyl choline was demonstrated by isolation of the activated vesicles by sucrose density centrifugation.     

Inhibition by 1-beta-D-arabinofuranosylcytosine of the incorporation of N-acetylneuraminic acid into glycolipids and glycoproteins of hamster embryo cells

1-β-D-Arabinofuranosylcytosine which interferes with DNA synthesis in bacteria and mammalian cells and brings about transformation of hamster embryo fibroblasts, has been found to inhibit the incorporation of N-Acetylneuraminic acid into glycolipids and glycoproteins of both normal and transformed hamster embryo cells in tissue culture. Three hours after commencement of treatment (10^?3M ara-C), incorporation of [¹⁴C] thymidine into DNA was inhibited by 95 per cent, while incorporation of [³H] D-glycosamine (precursor of sialic acid) into glycolipids and glycoproteins was inhibited by 85 per cent. At 24 hours, the inhibition of incorporation of the two labelled components was 83 and 80 per cent respectively. In homogenates of both cell types, incorporation of [¹⁴C] N-acetylneuraminic acid was competitively inhibited by ara-CMP. Ara-C was found to have no effect on the incorporation of [¹⁴C] choline into phospholipids of cells grown in tissue culture. These results suggest that interference with DNA synthesis by ara-C may not be the only factor involved in cell transformation by this substance.     

溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

猪-C反应蛋白的分离纯化及其性质的研究

以猪血清为材料,通过磷酸乙醇胺—琼脂糖亲和层析,Sepharose 4B柱层析和Sephacryl—S300凝胶过滤,获得了猪C—反应蛋白的结晶。猪C—反应蛋白可与肺炎球菌壁C多糖发生特异的沉淀反应,这种结合是依赖钙离子的。EDTA和一些磷脂代谢产物如磷酸胆碱,磷酸乙醇胺等,能抑制猪C—反应蛋白与C多糖的结合。在SDS—聚丙烯酰胺凝胶电泳及梯度聚丙烯酰胺凝胶电泳中,猪C—反应蛋白表现出与人C—反应蛋白相同的行为,亚基是一条分子量为23.5kD的肽链,全分子的表观分子量为150kD。猪C—反应蛋白与兔抗人C—反应的蛋白的抗血清能发生免疫交叉反应。     

The androgen-dependent rat prostate protein,probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1

Summary Rat prostate extracts contain an abundant 20–22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2–1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val-tyr-leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein “probasin”. This work was supported in part by NCI grant CA37589 (W. L. M., J. W. C.) and the Medical Research Council of Canada (R. J. M.).     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.