Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways

Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

Analysis of tissue-specific methylation patterns of mouse mammary tumor virus DNA by two-dimensional Southern blotting.

We used a two-dimensional Southern blotting procedure to analyze the tissue-specific methylation patterns of the five endogenous mouse mammary tumor viruses in the GR/A mouse strain. Our findings suggest that in certain tissues (brain, kidney, and liver) all proviruses are extensively methylated. In other tissues (spleen, placenta, and testes) all proviruses are hypomethylated to some degree. In these tissues individual proviruses display both quantitative and qualitative differences in methylation. We interpret the general patterns of tissue-specific hypomethylation in terms of a "hitch-hiker" model: mouse mammary tumor virus proviral methylation patterns reflect the tissue-specific activity of neighboring sequences. The observation that certain sites on particular proviruses are differentially methylated in a tissue-specific fashion may reflect tissue-specific differences in the makeup or conformation, or both, of proviral-containing chromatin.     

辣椒四个品种的核型研究

本文对我国栽培辣椒Capsicum annuum L.的四个品种即汉川椒、华椒一号、牛角椒和二金条进行了核型分析,其染色体数目均为2n=2x=24,核型公式均为2n=24=20m+2sm+2st,但汉川椒和华椒一号具一对随体,牛角椒和二金条具两对随体。本文还对它们的进化关系进行了讨论。     

辣椒四个品种的核型研究

本文对我国栽培辣椒Capsicum annuum L.的四个品种即汉川椒、华椒一号、牛角椒和二金条进行了核型分析,其染色体数目均为2n=2x=24,核型公式均为2n=24=20m+2sm+2st,但汉川椒和华椒一号具一对随体,牛角椒和二金条具两对随体。本文还对它们的进化关系进行了讨论。     

Chemical and enzymatic biotin-labeling of oligodeoxyribonucleotides.

Biotin has been converted to 2-(biotinylamido)ethanol and condensed to phosphorylated oligonucleotides in a solid phase synthesis. The 5'-biotinylated oligonucleotides were enzymatically coupled to other DNA fragments by T4 DNA ligase or T4 RNA ligase. The hybridization properties of such biotin-labeled oligonucleotide probes were studied.     

四川成都市郊正常成年男性血清生殖激素水平及其频数分布

本文用放射免疫测定法(RIA),测定了200～290例中国四川成都市郊30～73岁的正常成年男性血清睾酮(T)促黄体激素(LH).卵泡刺激素(FSH)及泌乳激素(PRL)的水平及其分布,进行了不同年龄水平差异显著性的观察,见到睾酮与泌乳激素水平无显著差异,促黄体激素及卵泡刺激素水平,随年龄之增加而升高,差异有显著性,并测得它们在血清中水平的分布:LH、FSH及PRL均为对数正态公布,T既非正态又非对数正态,但其频率分布较对称,接近正态分布。