Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2

Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.     

Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry

A rapid gas chromatography-mass spectrometric method for the determination of nalmefene in human plasma is described. The procedure involves protein precipitation, extraction with ethanol-chloroform mixture and derivatization with pentafluropropionic anhydride. The deuterated analog of nalmefene, 6beta-naltrexol-d(7), was used as the internal standard. Quantitation was achieved on a HP-1 column (12 mx0.2 mm I.D.) with negative chemical ionization (NCI) using methane:ammonia (95:5) as the reagent gas. The standard curves were fitted using a quadratic equation with the curve encompassing a range of 0.5 to 200 ng/ml, and the intra- and inter-assay variations for three different nalmefene levels were less than 10% throughout. The limit of quantitation was found to be 0.5 ng/ml. The method described is highly specific and reproducible, and could also be applied for the determination of naltrexone and 6beta-naltrexol. Application of the method to actual human plasma samples is demonstrated.     

Isolation and characterization of a human novel RAB (RAB39B) gene

Rab proteins are small-molecular-weight GTPases that control vesicular trafficking in eukaryotic cells. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a novel Rab protein, which showed 74.2% identity with previously isolated Rab39A at the amino acid level. RAB39B was expressed in a variety of human tissues and located in human chromosome Xq28. It consisted of two exons spanning 3764 bp of human genomic DNA.     

吗啡对培养海马神经元钙离子作用的机制研究

目的：研究吗啡对海马神经元[Ca^2 ]i影响的机制,为探索吗啡成瘾的神经生物学机制与可能的治疗途径。方法：荧光探针Fluo-4标记细胞内游离钙后,用激光共聚焦显微镜检测吗啡对大鼠原代培养海马神经元[Ca^2 ]i的影响。结果：吗啡急性刺激引起海马神经元[Ca^2 ]i升高,CTOP不能阻断吗啡引起的细胞内[Ca^2 ]i增加,而naltrindole能阻断吗啡引起的细胞内[Ca^2 ]i反应;Thapsigargin预处理阻断吗啡诱导的细胞内[Ca^2 ]i增加,Verapamil预处理不能完全抑制吗啡引起的细胞内[Ca^2 ]i增加;吗啡长时程作用后,海马神经元[Ca^2 ]i升高,加入纳络酮急性戒断后,不能阻断吗啡引起的细胞内[Ca^2 ]i升高,反而引起[Ca^2 ]i异常升高。结论：吗啡急性刺激引起的海马神经元内游离钙增加主要来源于δ2阿片受体介导的IP3敏感的钙库释放。     

The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material. In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis. We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase. Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site. F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring. Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring. Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton. We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism. Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.     

Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan

A herpesvirus has been isolated for the first time from a population of European eels Anguilla anguilla cultured in a recirculated system in Taiwan. Syncytia formation was detected in EP-1 (eel epidermis) cell cultures inoculated with cell-free homogenates prepared from both integument and visceral organs of moribund fish. Inoculation of homogenates onto EK (eel kidney) cell cultures induced giant cell formation. Subsequent passages produced a consistent and progressive cytopathic effect (CPE) in cell cultures. In this study, EP-1 cell cultures infected with EEHV (European eel herpesvirus) were examined using an electron microscope. Numerous nucleocapsids of about 100 nm in diameter were found within the nucleus of infected cells, whereas enveloped particles were observed within the cytoplasm. The mature viral particle, about 235 nm in diameter, had an electron-dense core with a hexagonal nucleocapsid surrounded by a coarse capsule. Histopathological examination of moribund fish showed epithelial hyperplasia with intracytoplasmic metabolic inclusions in the skin. Macrophage aggregates were found in liver, spleen, and kidney. A pair of primers designed from channel catfish virus and salmonid herpesvirus 1 was used in a polymerase chain reaction. A 402 bp fragment was amplified and cloned from genomic DNA of EEHV. The nucleotide homology was 99% (298 of 300) with DNA polymerase of eel herpesvirus (anguillid herpesvirus). EEHV nucleic acids were detected within melanomacrophages in the skin, liver, spleen and kidney by in situ hybridization (ISH).