目的：研究冬凌草甲素促进U937人淋巴瘤细胞分化的蔬噬细胞吞噬调亡小体的机制。方法：光学显微镜下计数检测吞噬率，PKC活力检测盒测定PKC活力，吖啶橙染色，Hoechst 33258染色及Western blot法。结果：酪氨酸蛋白激酶（PTK）抑制剂genistein和蛋白激酶C（PKC）广泛的抑制剂stauroporine均不同程度地抑制了冬凌草甲素诱导U937分化的蔬噬细胞对调亡小体的吞噬增强效果。2.7umol·L-1的冬凌草甲素处理U937细胞后，时间依赖性地增加了PKC活力。ERK磷酸化抑制剂PD98059阻断了冬凌草甲素的吞噬增强作用。免疫印迹结果显示冬凌草甲素作用U937细胞后，ERK磷酸化程度增加，而PD98059逆转了ERK磷酸化。结论：冬凌草甲素增加U937细胞对凋亡小体的吞噬作用，其吞噬机制是通过激活PTK和PKC激酶，导致下游ERK途径活化，从而增强吞噬过程。

目的：研究科凌草甲素诱导人组织淋巴瘤U937细胞凋亡的机制及ERK激酶在凋亡过程中的作用。方法：噻唑蓝（MTT）法，Hoechst 33258 梁色法，DNA凝胶电泳及Western blot检测法。结果：冬凌草甲素对U937细胞生长抑制作用呈时间剂量依赖性。27umol·L-1冬凌草甲素作用细胞12h后，Hoechst 33258 梁色细胞，出现明显凋亡小体，并诱导ERK发生磷酸化。ERK磷酸化抑制剂PD98059阻断了冬凌草甲素诱导的细胞生长抑制及DNA片段化。细胞内抑制调亡蛋白Bc-XL表达量时间依赖性减少，促调亡蛋白Bax表达量增加，而ERK抑制剂可逆转这种作用。结论：冬凌草甲素（27umol·L-1）诱导U937细胞调亡，这种作用是通过活佛ERK激酶，改变其下游Bax/Bcl-XL的表达比率，从而促进U937的细胞发生调亡

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) α and interleukin (IL)-1β release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity timedependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB /Akt) and the increased expression of PLCγ2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNFα and anti-IL-1β antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of ERK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNFα or IL-1β only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and ERK-dependent pathways.

A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4£102 CFU/ g DNA at 12.5 kV/cm and 1000, resulting in a time constant of approximately 5ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the Wrst report of transformation of bacteria A. hydrophila.

We previously reported that oridonin, a major component isolated from the plant Rabdosia rubescens HEMSL, induced apoptosis in human melanoma A375-S2 and cervical cancer HeLa cells. In the present study, oridonin was first evaluated for its effect on phagocytosis of apoptotic cells by macrophages. Preincubation of human histocytic lymphoma U937 cell-derived macrophages with 2.7mM oridonin significantly augmented phagocytosis of UV-irradiated (2.4 J/cm2, 4 min) U937 cells undergoing apoptosis in a dose-and time-dependent manner. However, less effect on synthetic fluoresbrite microspheres indicated that enhancement of apoptotic U937 cell uptake by oridonin was a selective effect. The oridonin-augmented phagocytosis was attenuated by anti-human TNFa and IL-1b antisera, suggesting that TNFa and IL-1b participate in the phagocytosis by oridonin-treated U937 cell-derived macrophages. In addition, the similar effect of phagocytosis was observed in oridonin-treated human monocyte-derived macrophages at 4 d maturation. Taken together, oridonin facilitates the phagocytic activity against apoptotic cells through TNFa and IL-1b release, which may be contribute to its antitumor activities.

Background: SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied. Methods: Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase α-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [3H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA). Results: We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase α-primase is one of its main targets. Conclusion: Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.

Cortical neuronal cultures were exposed to total ginkgolides (TG) in order to find out whether TG could rescue cultured cortical neurons from neurotoxicity damages. The cellular injuries were induced in the cells after 10 days in vitro by exposure to 1 mM L-glutamate (Glu) for 6–12 hr in serum-free medium, to 0.2 mM hydrogen peroxide (H2O2) for 6-12 hr, and then to free-glucose and hypoxia medium (an ischaemia model in vitro) for 4 hr. TG (0.01 to 100 ug/ml) was added to the growth medium 12 hr prior to or simultaneously the damage protected cortical neurons from toxic damages. Neuronal viability was confirmed by the assay of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium (MTT). The lactate dehydrogenase (LDH) release from the cultured medium was also detected. The results of present study demonstrated that TG protected cortical neurons from toxic damages induced by Glu, H2O2 or free-glucose and hypoxia.

目的酿酒酵母（Saccharomyces cerevisiae）丙酮酸脱羧酶（PDC）基因的克隆和表达。方法利用PCR技术从酿酒酵母的总cDNA中钓取出PDCsc基因，构建其高效表达质粒，利用Ion和ompT蛋白酶缺陷株Escherichia coli BL21（DE3）进行表达。结果扩增出长约1.7kb的PDCsc基因，成功构建其表达质粒pSC-22b，对转化菌株的SDS-PAGE分析结果显示：该基因获得高效表达，表达蛋白占细胞总蛋白的18.6%。结论成功构建高效表达PDCsc基因的工程菌株，为实现利用PDCsc进行的生物转化奠定基础。

主要介绍了酿酒酵母和移动单孢中丙酮酸脱羧酶的结构、性质及其在手性合成中的应用，包括反应的机理、底物范围和影响反应的因素。

用BamH I和Xba I两种限制酶酶切质粒pKG27，获得了编码F26G（F26G：furostanol gly-coside 26-O-β-glucosidase）的cDNA，然后将期重组到表达载体pET-22b上而得到pET-22b-CSF26G，利用大肠杆菌E.coli BL21进行表达，用胞内可溶蛋白进行酶反应，通过TLC、HPLC分析证明重组菌表达出高活性的F26G酶，并实现了呋甾皂苷的体外生物转化。