Bacteriophage Mu-induced modification of DNA is dependent upon a host function.

The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.     

聚乙二醇对东北红豆杉培养细胞的

在改良的B     

超声波对红豆杉悬浮细胞生长及紫杉醇释放的研究

分析了超声波对中国红豆杉悬浮细胞培养的生长,紫杉醇合成及释放的影响,细胞对不同强度及作用时间的超声波反应不同,用38kHz,120s的超声强度处理悬浮细胞,紫杉醇胞外释放率由对照的10％左右提高到40－50％,总产量提高了47％,超声波处理植物细胞,提供了在保持细胞生长的前提下有效刺激胞内次生代谢物的简易操作方法。     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

芫菁斑蝥素对喉癌细胞和胃癌细胞的抑制作用

【目的】 研究提取自眼斑芫菁Mylabris cichorii (Linnaeus)体内的斑蝥素对人喉癌HEP-2细胞和人胃癌BGC-823细胞的抑制、以及对细胞周期分布的影响。【方法】 将斑蝥素作用于经体外培养的人喉癌HEP-2细胞和人胃癌BGC-823细胞, 采用MTT法进行体外细胞抑制实验, 测定斑蝥素对这2种癌细胞生长的抑制率与剂量效应;采用流式细胞术测定斑蝥素处理的人喉癌HEP-2细胞的细胞周期;并通过光学显微镜观察其细胞形态学改变。【结果】 斑蝥素浓度为1.28 μmol/L时, 对HEP-2细胞有显著抑制作用, 且随药物浓度升高其抑制作用增强, 呈剂量效应关系, 抑制中浓度为2.88 μmol/L;斑蝥素浓度为20.4 μmol/L时, 对BGC-823细胞有显著抑制作用, 且随药物浓度升高其抑制作用增强, 呈剂量效应关系, 抑制中浓度为54.85 μmol/L。用浓度1.44和2.88 μmol/L的斑蝥素处理HEP-2细胞24 h后, G2-M期分布从8.21%增加到22.29%, S期细胞分布从14.33%增加到21.61%, 且随药物浓度升高其阻滞作用增加, 呈剂量效应关系。G0-G1期细胞分布都有所降低, 从77.45%降低到56.10%, G0-G1期峰前无显著的亚二倍体峰出现, 说明斑蝥素未能够诱导HEP-2细胞发生凋亡。光镜检查显示：HEP-2细胞可出现细胞收缩、胞膜突出、核碎裂等现象。【结论】 斑蝥素对治疗喉癌的效果可能较为理想, 而对胃癌的作用则不明显。     

The induction of lymphokine synthesis and cell growth in IL 3-dependent cell lines using antigen-antibody complexes

Several IL 3-dependent murine bone marrow-derived cell lines can be stimulated to grow with antigen-antibody (Ag.Ab) complexes. The Ag.Ab complexes induced lymphokine gene expression and the synthesis of IL 2, GM-CSF, IL 3, and BSF-1 (IL 4). The lymphokines produced by these IL 3-dependent cells appeared to stimulate their own growth, as both IL 3 and BSF-1 (IL 4) stimulated the growth of IL 3-dependent cells. Ag.Ab complexes also stimulate the growth of primary cultures of bone marrow cells that have been previously activated with IL 3. Normal bone marrow, IL 2-, and GM-CSF-dependent bone marrow cell lines could bind Ag.Ab complexes, but binding did not result in the induction of lymphokine synthesis or cell growth. Hyperimmune serum from mice also stimulated lymphokine synthesis and cell growth in IL 3-dependent cells, and the stimulatory activity was removed by treatment with Staphylococcus aureus protein A, suggesting the presence of Ag.Ab complexes.     

ERK phosphorylation is dependent on cell adhesion in a subset of pediatric sarcoma cell lines

Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.     

Thiopyronine-sensitized photodynamic effect on cell growth, RNA and DNA synthesis of Chinese hamster ovary cells

After treatment of Chinese hamster ovary (CHO) cells with very low concentrations of thiopyronine (TP; 1 microgram/ml) and visible light, a delay in growth of cell cultures (prolongation of the lag phase] was observed. The lengthened lag phase, however, was followed by normal growth of the cells. The length of the lag period is dependent on the irradiation dose applied. A similar effect on DNA and RNA synthesis could be seen after photodynamic treatment with TP in CHO cells: the maxima of RNA and DNA synthesis occur later but are not significantly reduced after treatment with low concentrations of TP and irradiation with visible light. This result is further evidence that the photodynamic effect with TP does not involve attack on nuclear DNA in eukaryotic cells.     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth.

Hepatocyte growth factor (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates growth and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0-fold occurred with 10 ng/ml HGF. This stimulatory effect was additive with both acidic and basic fibroblast growth factors, while it was inhibited by transforming growth factor-beta 1. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of 22 pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes.     

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long-Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.     

Inhibitory effect of novel 5-O-acyl juglones on mammalian DNA polymerase activity, cancer cell growth and inflammatory response

We previously found that vitamin K(3) (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on juglone (5-hydroxy-1,4-naphthoquinone), which is a 1,4-naphthoquinone derivative, and chemically synthesized novel juglones conjugated with C2:0 to C22:6 fatty acid (5-O-acyl juglones). The chemically modified juglones enhanced mammalian pol inhibition and their cytotoxic and anti-inflammatory activities. The juglone conjugated with oleic acid (C18:1-acyl juglone) showed the strongest inhibition of DNA replicative pol α activity and human colon carcinoma (HCT116) cell growth in 10 synthesized 5-O-acyl juglones. C12:0-Acyl juglone was the strongest inhibitor of DNA repair-related pol λ, as well as the strongest suppression of the production of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS) in the compounds tested. Moreover, this compound caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ears. C12:0- and C18:1-Acyl juglones selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. These data indicate that the novel 5-O-acyl juglones target anti-cancer and/or anti-inflammatory agents based on mammalian pol inhibition. Moreover, the results suggest that acylation of juglone is an effective chemical modification to improve the anti-cancer and anti-inflammation of vitamin K(3) derivatives, such as juglone.     

The effect of abscisic acid on cell growth, cell division and DNA synthesis in the maize root meristem

Cis -abscisic acid (ABA), when applied to maize ( Zea mays L. cv. LG 11) roots, decreases the rates of cell growth and cell division in the meristem. It also decreases the rate at which nuclei become labelled with [³H]-thymidine and enter mitosis. Removing the root cap accelerates the entry of nuclei into the DNA synthetic phase of the mitotic cycle and enhances the rate of cell proliferation in the quescent centre. ABA diminishes these effects, but does not suppress them. Thus, ABA cannot wholely substitute for the presence of a cap. One of the primary effects of applied ABA is to retard cell enlargement which may in turn affect the rate of cell division; natural endogenous ABA may act similarly. ABA might in this way assist in maintaining the quiescent centre in intact roots, but cannot be the sole agent involved.     

Differential effects on growth, cell cycle arrest, and induction of apoptosis by resveratrol in human prostate cancer cell lines.

Epidemiologic studies have suggested that nutrition plays an important role in carcinogenesis and that 30% of cancer morbidity and mortality can potentially be prevented with proper adjustment of diets. Resveratrol, a polyphenol present in red wines and a variety of human foods, has recently been reported to exhibit chemopreventive properties when tested in a mouse skin cancer model system. In this study, we investigated the effects of resveratrol on growth, induction of apoptosis, and modulation of prostate-specific gene expression using cultured prostate cancer cells that mimic the initial (hormone-sensitive) and advanced (hormone-refractory) stages of prostate carcinoma. Androgen-responsive LNCaP and androgen-nonresponsive DU-145, PC-3, and JCA-1 human prostate cancer cells were cultured with different concentrations of resveratrol (2. 5 x 10(-5)-10(-7) M). Cell growth, cell cycle distribution, and apoptosis were determined. Addition of 2.5 x 10(-5) M resveratrol led to a substantial decrease in growth of LNCaP and in PC-3 and DU-145 cells, but only had a modest inhibitory effect on proliferation of JCA-1 cells. Flow cytometric analysis showed resveratrol to partially disrupt G1/S transition in all three androgen-nonresponsive cell lines, but had no effect in the androgen-responsive LNCaP cells. In difference to the androgen-nonresponsive prostate cancer cells however, resveratrol causes a significant percentage of LNCaP cells to undergo apoptosis and significantly lowers both intracellular and secreted prostate-specific antigen (PSA) levels without affecting the expression of the androgen receptor (AR). These results suggest that resveratrol negatively modulates prostate cancer cell growth, by affecting mitogenesis as well as inducing apoptosis, in a prostate cell-type-specific manner. Resveratrol also regulates PSA gene expression by an AR-independent mechanism.     

Solid-phase synthesis of 2'-hydroxychalcones. Effects on cell growth inhibition, cell cycle and apoptosis of human tumor cell lines

Thirty-one 2'-hydroxychalcones were prepared via solid-phase synthesis by base-catalyzed aldol condensation of substituted 2'-hydroxyacetophenones and benzaldehydes. Chalcones were tested for their growth inhibitory activity in three human tumor cell lines (MCF-7, NCI-H460 and A375-C5) using the SRB assay. Results revealed that several of the tested compounds caused a pronounced dose-dependent growth inhibitory effect on the tumor cell lines studied in the low micromolar range. To gain further insight on the cellular mechanism of action of this class of compounds, studies of their effect on cell cycle profile as well as on induction of cellular apoptosis were also carried out. Generally, the tested chalcones interfered with the cell cycle profile and increased the percentage of apoptotic MCF-7 cells. The results here presented may help to identify new chalcone-like structures with optimized cell growth inhibitory activity which may be further tested as potential antitumor agents.     

Aromatase activity and the effect of estradiol and testosterone on DNA synthesis in endometrial carcinoma cell lines

Human endometrial and breast carcinoma cell lines were examined for aromatase activity and the effects of sex steroids (estradiol and testosterone) on DNA synthesis. Aromatase activity was high (greater than 500 fmol/10⁷ cells/24 h) in the cell lines MCF-7 and OMC-2, moderate (100–499 fmol/10⁷ cells/24 h) in the cell lines HEC-59 and Ishikawa, and low (less than 100 fmol/10⁷ cells/24 h) in the HHUA cell line. A substantial stimulation of DNA synthesis by estradiol (10⁻⁹M) was observed in cell lines HEC-59, OMC-2, and MCF-7, with an increase in [³H]thymidine uptake of over 250%. The Ishikawa cell line was stimulated moderately (115–249%). No estradiol-induced increase in DNA synthesis was observed in HHUA. Responsiveness of DNA synthesis to testosterone was observed in cell lines that showed the greatest response to estradiol, namely HEC-59, OMC-2, and MCF-7. Otherwise, estrogen-responsiveness did not always correlate with a significant aromatase activity. These data suggest that some but not all endometrial carcinomas may possess an aromatase-dependent growth stimulating system.     

Inhibition of replicative DNA synthesis and induction of unscheduled DNA synthesis in permeable sarcoma cell by bleomycin.

Unscheduled DNA synthesis was induced by bleomycin in isolated rat liver nuclei and in permeable mouse ascites sarcoma cells. ATP significantly enhanced the bleomycin effect of inducing unscheduled DNA synthesis. Replicative DNA synthesis in permeable mouse ascites sarcoma cells was inhibited by bleomycin. The apparent inhibition or stimulation by bleomycin of in vitro DNA synthesis was thought to be determined by a balance between inhibited DNA replicase activity and induced unscheduled DNA synthesis.