Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.     

人外周血淋巴细胞C-有丝分裂效应的研究

目的 研究秋水仙素（Ｃｅ）和秋水仙碱（Ｃｄ）对人外周血淋巴细胞有丝分裂效应的影响。方法 应用外周血淋巴细胞培养法,分析有丝分裂指数（ＭＩ）和Ｃ－有丝分裂效应。结果 Ｃｅ和Ｃｄ的ＭＩ与相应对照组相比,差异有显著性（Ｐ〈０．０１）,Ｃ－有丝分裂效应Ｃｅ和Ｃｄ处理组与相应对照组之间差异亦有显著性（Ｐ〈０．０１）。ＭＩ和Ｃ－有丝分裂效应,Ｃｄ的作用均较Ｃｅ强。并对Ｃ－有丝分裂效应与非整倍体诱导活性之间的关     

Induction of polyploidy in Adenovirus E1-transformed cells by the mitotic inhibitor colcemid

Adenovirus-transformed cells were tested for their ability to synthesize DNA in the presence of cell cycle inhibitory drugs. We show that transformed cells are completely resistant to the mitotic inhibitor colcemid, partly resistant to lovastatin, mimosine, aphidicolin and genistein but not to hydroxyurea or thymidine. When treated with colcemid, AdE1-transformed cells continue to synthesize DNA but do not divide and, therefore, become highly polyploid. This effect is dependent on the presence of both E1A and E1B.     

Growth in the pre-fusion murine allantois

Prior to fusion with the chorion, the extraembryonic mesoderm of the murine (Mus musculus) allantois differentiates with distal-to-proximal polarity into at least two cell lineages: a chorio-adhesive cell lineage called mesothelium, and the endothelium of the umbilical vasculature. How the allantois grows is less clear, but cell proliferation and addition of mesoderm from the underlying primitive streak appear to play important roles. The aim of this study was to analyze growth in the murine allantois. Techniques of histology and microsurgery were used to examine pre-fusion allantoises at nine developmental timepoints that differed by approximately 2 h. Cell counts revealed that allantoic size increased over time. Two hours of exposure to colcemid enhanced mitotic figures, which were used to calculate the relative number of proliferating cells (mitotic index, MI) in pre-fusion allantoises at each developmental timepoint. Cell proliferation was highest in nascent allantoises and showed signs of slowing by two somite pairs. By five to six-somite pairs, when most allantoises are attaching to the chorion, the overall MI decreased significantly. No regional differences in the mitotic index were observed at any developmental stage. Total cell numbers and the mitotic index were then used to discover the extent of streak contribution to pre-fusion allantoises. Cell proliferation and streak activity were highest in nascent allantoises, after which growth occurred predominantly by cell proliferation. Formation of allantoic regenerates by microsurgical removal and culture in intact conceptuses provided independent confirmation that, as the allantois matured, the primitive streak ceased to be a major contributor to its growth. Thus, the allantois grows by both mitosis and addition of mesoderm from the streak. That the periods of highest cell proliferation and streak activity coincided raises intriguing questions concerning their interplay in the control of growth in the murine allantois. Accepted: 26 May 2000