Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.     

Millimeter wave exposure reverses TPA suppression of gap junction intercellular communication in HaCaT human keratinocytes

The effect of 30.16 GHz millimeter wave (MMW) exposure at 1.0 and 3.5 mW/cm2 on gap junction intercellular communication (GJIC) was studied in cultured HaCaT keratinocytes, using the fluorescence recovery after photobleaching (FRAP) technique and laser confocal scanning microscopy to follow the intracellular movement of 5,6-carboxyfluorescein diacetate dye. While MMW exposure alone for 1 h at either 1.0 or 3.5 mW/cm2 did not affect GJIC, MMW exposure in combination with 5 ng/ml TPA treatment reversed TPA induced suppression of GJIC. Exposure at 1.0 mW/cm2 resulted in a partial reversal, and exposure at 3.5 mW/cm2 resulted in essentially full reversal of the TPA suppression.     

Generating gene knockout rats by homologous recombination in embryonic stem cells

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell-based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ～1 year to complete, from derivation of ES cells to generation of knockout rats.     

香烟烟雾提取物抑制肺泡上皮细胞的增殖并诱导其凋亡

香烟烟雾提取物（cigarette smoke extract,CSE）中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.     

大鼠多囊卵巢颗粒细胞凋亡时MDA含量及SOD活性的变化

目的和方法以大鼠多囊卵巢（PCO）为动物模型,观察了PCO大鼠卵泡颗粒细胞凋亡的发生率,并检测了颗粒细胞凋亡时丙二醛（MDA）的含量及超氧化物歧化酶（SOD）活性的变化。结果①PCO颗粒细胞凋亡发生率明显高于正常（P<0．001）;②PCO颗粒细胞发生凋亡时,细胞内MDA含量增加而SOD活性降低。结论大鼠PCO颗粒细胞的凋亡可能与细胞内MDA含量增加和SOD活性降低有关。     

Mutant plant viruses with cell binding motifs provide differential adhesion strengths and morphologies

The ability of Tobacco mosaic virus (TMV) to tolerate various amino acid insertions near its carboxy terminus is well-known. Typically these inserts are based on antigenic sequences for vaccine development with plant viruses as carriers. However, we determined that the structural symmetries and the size range of the viruses could also be modeled to mimic the extracellular matrix proteins by inserting cell-binding sequences to the virus coat protein. The extracellular matrix proteins play important roles in guiding cell adhesion, migration, proliferation, and stem cell differentiation. Previous studies with TMV demonstrated that the native and phosphate-modified virus particles enhanced stem cell differentiation toward bone-like tissues. Based on these studies, we sought to design and screen multiple genetically modified TMV mutants with reported cell adhesion sequences to expand the virus-based tools for cell studies. Here, we report the design of these mutants with cell binding amino acid motifs derived from several proteins, the stabilities of the mutants against proteases during purification and storage, and a simple and rapid functional assay to quantitatively determine adhesion strengths by centrifugal adhesion assay. Among the mutants, we found that cells on TMV expressing RGD motifs formed filopodial extensions with weaker attachment profiles, whereas the cells on TMV expressing collagen I mimetic sequence displayed little spreading but higher attachment strengths.     

Cell origins and diagnostic accuracy of interleukin 27 in pleural effusions

The objective of the present study was to investigate the presence of interleukin (IL)-27 in pleural effusions and to evaluate the diagnostic significance of pleural IL-27. The concentrations of IL-27 were determined in pleural fluids and sera from 68 patients with tuberculous pleural effusion, 63 malignant pleural effusion, 22 infectious pleural effusion, and 21 transudative pleural effusion. Flow cytometry was used to identify which pleural cell types expressed IL-27. It was found that the concentrations of pleural IL-27 in tuberculous group were significantly higher than those in malignant, infectious, and transudative groups, respectively. Pleural CD4(+) T cells, CD8(+) T cells, NK cells, NKT cells, B cells, monocytes, macrophages, and mesothelial cells might be the cell sources for IL-27. IL-27 levels could be used for diagnostic purpose for tuberculous pleural effusion, with the cut off value of 1,007 ng/L, IL-27 had a sensitivity of 92.7% and specificity of 99.1% for differential diagnosing tuberculous pleural effusion from non-tuberculous pleural effusions. Therefore, compared to non-tuberculous pleural effusions, IL-27 appeared to be increased in tuberculous pleural effusion. IL-27 in pleural fluid is a sensitive and specific biomarker for the differential diagnosing tuberculous pleural effusion from pleural effusions with the other causes.