Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Comparative use of fructose and glucose in human liver and fibroblastic cell cultures

Summary The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5, mmol·1⁻¹ and 27.5 mmol·I⁻¹ glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [³H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5, mmol·1⁻¹ glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol·1⁻¹. Several possible explanation for the stimulation of cell growth in fructose medium were discussed. This work was supported by grants for the Institut National de la Santé et de la Recherche Médicale (ATP 82-79-114) and the Unité d'Enseignement et de Recherche, Le Kremlin-Bicêtre, Université Paris-Sud (C. R. 848).     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Synthesis of a cytosine nucleoside of 2-amino-2-deoxy-beta-D-xylofuranose.

1-(2-Amino-2-deoxy-beta-D-xylofuranosyl)cytosine (13) was synthesized by three routes: (a) coupling of 2-deoxy-3,5-di-O-p-nitrobenzoyl-2-(trifluoroacetamido)-D-xylofuranosyl chloride (5) with 2,4-dimethoxypyrimidine and subsequent treatment with methanolic ammonia, (b) coupling of 5 with 4-N-acetyl-2-O,4-N-bis(trimethylsilyl)cytosine followed by treatment with methanolic ammonia, and (c) thiation of 1-[3,5-di-O-acetyl-2-deoxy-2-(trifluoroacetamido)-beta-D-xylofuranosyl]uracil (6) by treatment with phosphorus pentasulfide in pyridine followed by amination of the resulting 4-thionucleoside 12 with metanolic ammonia. The best yield was obtained via route (a).     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。