Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G1-chalone, a tissue-specific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G1-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentration-independent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G1-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase alpha, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G1-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

H2O2对蛇足石杉离体培养物诱变效应的研究

为了获得高产石杉碱甲(Huperzine A,Hup A)的蛇足石杉[Huperzia serrata(Thunb.)Trev.]叶状体,对H_2O_2诱变后的叶状体进行了研究。结果表明,诱变后叶状体株系的Hup A含量显著提高,并获得高产株系SH42,其相对生长率和Hup A含量分别达到4499.28%和261.17μg g~(–1) DW,比起始叶状体分别提高了2.35倍和2.43倍;且株系间可溶性蛋白质谱带和SOD同工酶谱均存在差异,经过连续9代培养,变异叶状体可以稳定遗传。因此,H_2O_2对叶状体细胞具有良好的诱变效应,可以用于筛选高产Hup A株系。     

淀粉样肽Aβ导致线粒体功能紊乱的体内和体外研究

为探索阿尔茨海默症(AD)中β淀粉样肽(Aβ)对线粒体功能的影响,比较了稳定表达人野生型淀粉样前体蛋白(APP)的细胞和同时转入人Swedish突变APP及ΔE9突变PS1的双转细胞(swe.Δ9)的线粒体功能.结果发现,swe.Δ9细胞的线粒体膜电位、细胞色素c氧化酶活性、线粒体膜流动性、ATP含量均明显低于APP细胞,而APP细胞又明显低于对照的转入空质粒的细胞.在转基因小鼠上也得到类似结果:同时转入人Swedish突变APP和人PS1 M146V敲入的双转小鼠的细胞色素c氧化酶活性和ATP含量比只转入Swedish突变APP的Tg2576小鼠更低.结果证明了AD模型中线粒体功能损害程度与Aβ产量的正相关关系.     

Induction of Cell Death by Cytokines in Cell Cycle-Synchronous Tumor Cell Populations Restricted to G1and G2

In the present work, cytokine-mediated induction of cell death was investigated by flow cytometry in cell cycle-synchronous human tumor cell populations gained by centrifugal elutriation or by cell cycle blockade with mimosine and aphidicolin. Attention was payed to the question of whether the effector phase of cell death takes place in the same phase of the cell cycle in which the death signal is received. Another point of interest was the question whether synchronization of cell populations with respect to the cell cycle leads to increased synchronicity of the death phase. The results demonstrate that supernatants from monocyte/tumor cell interaction cultures containing tumor necrosis factor-α, interferons, and interleukins-1 and -6 or appropriate combinations of pure cytokines cause cell cycle arrest predominantly in G₁and to a lesser extent in G₂. Cell death is initiated from both arrest points. Cytokine-treated G₁cells do not enter S phase. They die within the same G₁phase in which they receive the death signal. In contrast, a high proportion of cytokine-treated G₂cells pass through mitosis and are arrested and die in the subsequent G₁phase, whereas only a smaller proportion of cells are arrested and die in G₂. The synchronicity of the death phase cannot be increased by the diverse methods of cell cycle synchronization applied. Interestingly, aurin-tricarboxylic acid, an agent known for inhibitory effects on nucleolytic activities and other protein/nucleic acid interactions, not only prevents cell death, but also cell cycle arrest.     

光皮桦花粉离体萌发试验

设置若干温度的培养环境以及各种浓度蔗糖、蔗糖+硼酸、蔗糖+硝酸钙、蔗糖+硼酸+硝酸钙培养基,研究温度以及蔗糖、硼、钙对光皮桦花粉离体萌发的影响,探明光皮桦花粉萌发的适宜温度和最佳培养基。结果表明:合适浓度的蔗糖、硼酸和硝酸钙均能显著促进光皮桦花粉萌发,而高浓度的蔗糖、硼和钙均抑制其花粉萌发;光皮桦花粉离体萌发的合适培养基为10%蔗糖+200mg.L-1硼酸+200mg.L-1硝酸钙,适宜培养温度为30℃;在此条件下培养4h后花粉萌发率基本稳定,培养6h后花粉管长度亦趋于稳定。     

离体条件下卡德丽亚兰的形态建成

采用光学显微镜和电子显微镜技术对离体培养条件下卡德丽亚兰原球茎产生及形态建成进行了系统的细胞学观察。结果表明,离体培养条件下卡德丽亚兰原球茎起源于母原球茎表皮、亚表皮或中央薄壁细胞,这些细胞形成胚性细胞后,通过不断的细胞分裂形成胚性细胞团,胚性细胞团保持细胞分裂的同时伴随着胚性细胞的液泡化过程,形成了由上百个薄壁细胞组成的原球茎;原球茎通过芽端分生组织区形成,进一步分化成芽,其下方有输导组织形成。这一过程几乎重演了其合子胚发育及分化的过程。原球茎生长过程中其薄壁细胞通过叶绿体储存大量淀粉粒,而在以后的形态建成中淀粉粒逐渐降解。     

The effect of fibrin on endothelial cell migration in vitro

Endothelial cells are known to migrate and come into contact with fibrin during numerous physiological processes, such as in wound healing and in tumor growth. The present study was initiated to investigate the effect of fibrin on endothelial cell migration in vitro. Endothelial cell migration was assayed by wounding confluent monolayers of bovine aortic endothelial cells with a razor blade and counting the number of cells crossing the wound per unit time. Wound-induced proliferation of endothelial cells was inhibited by mitomycin C-treatment without affecting endothelial cell migration, indicating that in this assay migration could be measured independent of proliferation. Migration of endothelial cells in vitro was inhibited by fibrin in a concentration dependent manner. Endothelial cell migration under fibrin was further reduced by plasminogen depletion of the serum, and fibrin still inhibited the migration of mitomycin C-treated endothelial cells. Kadish et al. (Tissue and Cell, 11, 99, 1979) previously reported that fibrin did not affect EC migration in vitro. The inability to inhibit EC migration with fibrin appears to be due to their assay system which employed agarose, since pre-treating the wounded monolayer with agarose eliminated the inhibition of EC migration by fibrin. The present results indicate that EC migration in vitro can be used as a model system for studying the interaction of fibrin with EC.     

Interfering transbody-mediated Her2 gene silencing induces apoptosis by G0/G1 cell cycle arrest in Her2-overexpressing SK-BR-3 breast cancer cells

We previously isolated an interfering transbody, 4MH2, which penetrated the cytosol of living cells and preferentially hydrolyzed the target Her2 (ErbB2) mRNA, resulting in Her2 gene silencing followed by apoptotic cell death in Her2-overexpressing breast cancer cells. Here, we report the apoptotic cell death mechanism mediated by 4MH2-induced Her2 gene silencing in Her2-overexpressing SK-BR-3 breast cancer cells, in comparison with a small interfering RNA (siRNA) targeting Her2 mRNA (Her2₁₈-siRNA). 4MH2 induced G₀/G₁ cell cycle arrest to cause apoptotic cell death in SK-BR-3 cells by triggering specific signaling pathways associated with Her2 knockdown, including upregulation of G₀/G₁ cell cycle arrest-associated p21^Cip1 and p27^Kip1, downregulation of cyclin D1, inhibition of Akt phosphorylation, and downregulation of antiapoptotic Bcl-xL, which are comparable to those mediated by Her2₁₈-siRNA. Our results suggest that 4MH2-mediated Her2 gene silencing can trigger the downstream signaling pathways caused by Her2 downregulation, comparable to those mediated by the corresponding siRNA.     

PDGF-BB, IGF-I and mechanical load stimulate DNA synthesis in avian tendon fibroblasts in vitro

Resident cells in the surface epitenon and internal compartment of flexor tendons are subjected to cyclic mechanical load as muscle contracts to move limbs or digits. Tendons are largely tensile load bearing tissues and are highly matrix intensive with nondividing cells providing maintenance functions. However, when an injury occurs, tendon cells are stimulated to divide by activated endogenous growth factors and those from platelets and plasma. We hypothesize that tendon cells detect mechanical load signals but do not interpret such signals as mitogenic unless an active growth factor is present. We have used an in vitro mechanical load model, application of cyclic strain to cells cultured on flexible bottomed culture plates, to test the hypothesis that tendon cells require platelet-derived growth factor (PDGF-BB) and insulin-like growth factor-I (IGF-I) in addition to mechanical load to stimulate DNA synthesis. In addition, we demonstrate that in avian tendon cells, load and growth factors stimulate phosphorylation of tyrosine residues in multiple proteins, including pp₆₀src, a protein kinase that phosphorylates receptor protein tyrosine kinases. A lack of mitogenic responsiveness to mechanical load alone by tendon cells may be a characteristic of a regulatory pathway that modulates cell division.     

Stepwise effects of cytokinin activity and DNA synthesis upon mitotic cycle events in partially synchronized tobacco cells

Cytokinin addition to tobacco cell suspensions induced synchronous cell division after an 18 h lag period. Although continuous presence of the cytokinin in the culture medium during this lag period was essential to division, cytokinin was not required during mitosis itself. For each cell generation, cytokinin-dependent events are thus completed before mitosis occurs.Two experiments suggested that these cytokinin-dependent events are independent of DNA synthesis:

1. (i) With or without cytokinin, DNA synthesis proceeded normally in the presence of auxin, for at least the time required for one cell generation in complete medium.

2. (ii) In the presence of cytokinin, when DNA synthesis in the lag period was inhibited by FUdR, one normal cell division occurred when cytokinin was withdrawn and DNA synthesis restored by thymidine addition.

In cytokinin-starved cells, metaphase was greatly prolonged although prophase was unaffected.     

Actions of prostacyclin (PGI2) and its product, 6-oxo-PGF1α on the rat gastric mucosa in vivo and in vitro

The effects of prostacyclin (PGI₂) and its breakdown product 6-oxo-PGF_1α on various aspects of gastric function were investigated in the rat. PGI₂ increased mucosal blood flow when infused intravenously. PGI₂ was a more potent inhibitor of gastric acid secretion in vivo than PGE₂. Like PGE₂, PGI₂ inhibited acid secretion from the rat stomach in vitro. PGI₂ had comparable activity to PGE₂ in inhibiting indomethacin-induced gastric erosions. Thus prostacyclin shares several of the activities of PGE₂, and may be involved in the regulation of gastric mucosal function.     

Pasteur effect in the in vitro vascularly perfused rat small intestine

Isolated small intestine perfused in vitro with media with low oxygen concentration was found to contain low levels of ATP when compared with rat small intestine in vivo. The addition of fluorocarbon FC 75 to an erythrocyte-free perfusion medium was found to result in a high phosphate potential and a low rate of lactate production from glucose in isolated perfused small intestine, resembling the in vivo condition. This allowed the demonstration of a Pasteur effect in that replacement of oxygen by nitrogen (or the addition of 2,4-dinitrophenol) led to a rapid increase of the rate of glycolysis, and a decrease of the ATP concentration in the tissue     

Deoxyribonucleoside triphosphate and DNA synthesis in synchronized cultures of Chlamydomonas

Pool sizes of dATP, dTTP, dGTP and dCTP were determined during the life cycle of Chlamydomonas using light-dark synchronized cultures. The pools of all four nucleotides were small until the start of the DNA synthesis, when they all increased in close time relationship with the increase in rate of DNA synthesis. The dTTP and dATP pools increased more than 200-fold while the pools of dCTP and dGTP expanded approx. 10 times.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle

Cyclin-dependent kinases are the key regulators of cell-cycle transitions. In mammalian cells, Cdk2, Cdk4, Cdk6 and associated cyclins control the G₁ to S phase transition. Because proper regulation of this transition is critical for an organism's survival, these protein kinases are exquisitely regulated at different mechanistic levels and in response to a large variety of intrinsic and extrinsic signals.     

The concurrent in vitro and in vivo release of PGE2 from controlled-release hydrogel polymer pessary for cervical ripening

Twenty five patients booked for induction of labour, at 38 weeks or more gestation, were administered a controlled release vaginal polymer pressary containing 10mg prostaglandin E₂(PGE₂), designed to release 0.6mg per hour in vivo. The release profile from the polymer was linear throughout the eight hour observation period with a correlation coefficient of 0.81, and regression slope of 0.93 mg/hr. with 95% confidence intervals of 0.63mg/hr. to 1.23 mg/hr. This compared with a concomitatnt release profile in vitro which was uniform with time for the first five hours, but then continued at a decreasing rate with a correlation coefficient of 0.98. The relationship between PGE₂ release and cervical score change was linear, with a correlation coefficient of 0.65. The results show that PGE₂ release from the pessary in vivo is predictable, and suggest that the controlled release pessary offers the advantages of greater control of cervical ripening than alternative vehicles currently available.     

Methylcobalamin corrects the deleterious in vitro effect of nitrous oxide on thymidylate synthetase

Summary In this study, cobalamin deficiency was produced in vitro by the use of nitrous oxide, known to inactivate the vitamin. In 14 sets of experiments, normal human lymphocytes stimulated with phytohemagglutinin on day 0 were exposed to nitrous oxide and oxygen on day 2. McCbl was delivered later to half of the cells. Untreated cells served as a control. On day 3, the cells were harvested, the lymphocytes were lysed, and the obtained extracts were assayed for thymidylate synthetase. In 16 other experiments the same procedure was performed, and the incorporation of radioactive thymidine or deoxyuridine by the intact cells was measured. In additional experiments, a deoxyuridine suppression test of treated and untreated stimulated lymphocytes was also performed. The results indicate that nitrous oxide significantly reduces the activity of thymidylate synthetase and that this reduction is significantly corrected by McCbl, suggesting a causative relation between the vitamin and the enzyme. However, there was no statistically significant effect of nitrous oxide demonstrated on the nucleoside incorporation nor on the deoxyuridine suppression test.An abstract of this article appeared in Blood 62: Suppl 1 37a, 1983.