活性氧参与一氧化氮诱导的神经细胞凋亡

采用激光共聚焦成像技术，用氧化还原敏感的特异性荧光探针(DCFH-DA和DHR123)直接研究了一氧化氮供体S-亚硝基-N-乙酰基青霉胺(SNAP)诱导未成熟大鼠小脑颗粒神经元凋亡过程中的细胞胞浆、线粒体中活性氧水平的变化，发现神经细胞经0.5 mmol/L SNAP处理1 h后，细胞胞浆及线粒体中活性氧水平大大增加.一氧化氮清除剂血红蛋白能够有效抑制细胞胞浆、线粒体中活性氧的产生，防止细胞凋亡.外源性谷胱甘肽对细胞也具有良好的保护作用，而当细胞中谷胱甘肽的合成被抑制后，一氧化氮的神经毒性大大增强.实验结果表明一氧化氮通过促进神经细胞产生内源性活性氧而启动细胞凋亡程序，而谷胱甘肽可能是重要的防止一氧化氮引发神经损伤的内源性抗氧化剂.     

新疆维吾尔族妇女宫颈癌组织中乳头瘤病毒16型E6基因的克隆和序列分析

为了分析新疆南部地区维吾尔族妇女宫颈癌组织中HPV16型E6基因结构特点，从中国新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取DNA，以宫颈癌活检组织标本DNA为模板进行PCR扩增，获得HPV16 E6基因，将其克隆到pUCm-T载体上，并对其进行基因全序列分析.PCR检测结果显示宫颈癌组织中HPV16 E6阳性率为82.35%（14/17）；测序结果显示，新疆株HPV16 E6基因全长456 bp，大小与德国标准株一致.E6基因的第247位碱基发生T→G突变，并由此引起所编码的氨基酸亦发生改变.上述结果表明，中国新疆南部地区维吾尔族妇女宫颈癌患者组织中HPV16 E6的基因结构与德国标准株HPV16 E6基因之间存在差异.     

Epidermal structures and stomatal parameters of Chinese endemic Glyptostrobus pensilis (Taxodiaceae)

Glyptostrobus pensilis K. Koch, the only living species, is endemic to southern China. Epidermal structures of G .  pensilis have been studied on leaves collected from Guangzhou, southern China, the native locality of the species, and from Hangzhou, eastern China, the cultivated locality. Leaves are linear, linear-subulate and scale-like. Epidermal cells are rectangular and elongate parallel to the mid-vein on areas lacking stomata, and short, with rounded corners, on intrastomatal areas. Stomatal bands lie parallel to the mid-vein on both surfaces of leaves. Commonly the stomata have five or six subsidiary cells. Stomatal parameters (density and index) of the same surfaces of linear leaves from Guangzhou and Hangzhou show no statistically significant differences ( P  > 0.05). Considering the stomatal parameters of the same surfaces of linear-subulate leaves between the two localities, the stomatal index of the abaxial surfaces reveals no significant differences ( P  > 0.05), while the stomatal index of the adaxial surfaces and the stomatal density of both surfaces exhibit significant differences ( P  < 0.05). Intra-individual variation in stomatal index is smaller than that in stomatal density based on the coefficient of variability of stomatal parameters of the same areas of leaves. When studying the correlation between stomatal parameters of G. pensilis and atmospheric CO₂ concentrations, the stomatal parameters of linear leaves are mostly significant, and stomatal index is more useful than stomatal density.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 153–162.     

LABORATORY SUSCEPTIBILITY OF PLUTELLA XYLOSTELLA TO METARHIZIUM ANISOPLIAE AND NOMURAEA RILEYI*

Abstract Five isolates of Metarhizium anisopliae and one isolate of Nomuraea rileyi were bioassayed against larvae of Plutella xylostella. Larvae were treated by exposing cabbage leaf discs previously immersed in conidial suspension, and were then incubated at 25^oC and observed daily. One of M. anisopliae isolates, F11248, originally from Mastotermes darwiniensis was found being most virulent against P. xylostella. The LC₅₀ was estimated as 2.03 times 10⁴conidia/ml with 9 d mortality data, and the LT₅₀ was 4.97 d at concentration of 10⁷conidia/ml. Isolate F163 (N. rileyi) showed virulence to P. xylostella in both tests, but no cadavers sporulated.     

Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A recombinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down-stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous recombination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by genome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowlpox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation.Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were assayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T)were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cytokines (IFN-Y and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice.We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.     

蛋白质组学中新蛋白质鉴定的研究方法和策略

当前,基于生物质谱进行蛋白质鉴定的技术已经成为蛋白质组学研究的支撑技术之一.产生的数据主要使用数据库搜索的方法进行处理,这种方法的一大缺陷是不能鉴定数据库中未包含的蛋白质,因此如何充分利用质谱数据对蛋白质组研究的意义很大,而新蛋白质鉴定更是其中一个重要的内容.新蛋白质鉴定是蛋白质鉴定的一个方面,新蛋白质的定义按照序列和功能的已知程度分为3个层次;以蛋白质鉴定的方法为基础,目前新蛋白质鉴定的方法可分为denovo测序和相似序列搜索结合的方法以及搜索EST、基因组等核酸数据库的方法2大类;两者各有利弊.存在各自的问题和相应处理的策略.不同的研究者可以根据具体目的应用和发展不同的鉴定方法,同时新蛋白质的鉴定也将随着蛋白质组学研究的发展而更加完善.     

Adrenal cells in tissue culture. The effect of papaverine and amytal on steroidogenesis, respiration and replication

The smooth-muscle relaxant papaverine has been shown to be a potent inhibitor of cyclic AMP phosphodiesterase activity (Kukovetz, W. R., and Poch, G. (1970) Naunyn Schmiedebergs Arch. Pharmakol.267, 189). Because of this, papaverine was tested in monolayer cultures of functional mouse adrenal cortex tumor cells for possible stimulatory effects on Steroidogenesis. At 10^?5m, papaverine was found to inhibit ACTH-stimulated steroidogenesis 50% and at 10^?4m, > 95%. This was associated with a > 10-fold increase in [¹⁴C]lactate production from [¹⁴C]glucose and a 50% reduction in ³²P_i, incorporation into macromolecules. These findings were similar to those observed with the barbiturate amytal, an inhibitor of the mitochondrial electron-transport chain at the level of oxidation of NADH (Site I). Papaverine was 100 times more effective than amytal in inhibiting steroidogenesis and 1000 times more effective in initiating an increase in glycolysis. In intact tumor cells and mitochondria isolated from normal rat adrenals, papaverine (10^?4m) completely inhibits oxygen uptake supported by malate or α-ketoglutarate. Oxygen uptake is restored by the addition of succinate, suggesting that, like amytal, papaverine inhibits respiration at Site I.Papaverine does not inhibit NADPH-supported cholesterol side-chain cleavage in bovine adrenal acetone powders or 11β-hydroxylation in normal rat adrenal cortex mitochondria. By contrast, amytal inhibits both these activities at concentrations comparable to that effective in intact adrenal cells, suggesting a direct interaction of amytal with cytochrome P-450. Both papaverine and amytal inhibit incorporation of thymidine into nuclear DNA to an extent far greater than that observed with either maximally stimulating levels of cyclic AMP or high concentrations of ACTH. Succinate does not reverse the inhibitory effects of either papaverine or amytal on thymidine incorporation into DNA. Papaverine increases intracellular cyclic AMP in both resting and ACTH-treated cells. However, the effects of papaverine on steroidogenesis, glycolysis, ATP-P_i exchange, and DNA synthesis in adrenocortical cells are not directly attributable to this action.     

基于目标颜色基及梯度方向匹配的菌落分割计数算法

【目的】通过菌落测试片提取菌落并计数,在农业、食品业、医疗卫生等领域中是一项常用且重要的工作。目前,菌落自动计数算法大都是以菌落培养皿为主要工作对象,对菌落测试片适用性较差。另外,目前相关技术在常规的粘连物体分割中有着较好的效果,但在菌落分割计数中,由于菌落本身的形态特征,对粘连菌落分割计数的效果尚不够精准。【方法】为解决此类问题,本文提出一种基于目标颜色基及梯度方向匹配的菌落分割计数算法。首先利用图像中菌落的颜色特征作为基,将图像转换到基空间内,以增强菌落与背景之间的差异,其次利用菌落图像的梯度幅值特征对梯度方向进行滤波,然后通过梯度方向进行匹配,进而将粘连的菌落分割,最后利用非极大值抑制的方法筛选出菌落并计数。【结果】经试验,本研究算法的计数精度可达98.00%,能够满足实际需求。【结论】在针对菌落的目标分割计数中,本研究算法不仅计数精度高,而且具有较好的鲁棒性,在对不同厂家的菌落总数测试片菌落分割计数中均有优异效果;然而在对大面积目标的检测分割中算法的准确率会有所下降,因此,该算法更适合于菌落等小目标的检测分割。     

铜绿假单胞菌fur基因缺失突变株的构建及其表型分析

铁摄取调节蛋白(ferric uptake regulator, Fur)是控制铜绿假单胞菌铁代谢和毒力的关键调节因子。许多课题组尝试构建铜绿假单胞菌fur的缺失突变株均失败,因此铜绿假单胞菌的fur一直被认为是必需基因,这导致其生物学功能一直未得到全面的解析。【目的】构建铜绿假单胞菌fur的缺失突变株,并对该突变株的表型进行分析。【方法】以铜绿假单胞菌PAO1为亲本菌株,通过同源重组的方法构建fur缺失突变株,研究该基因对铜绿假单胞菌生长、铁载体生物合成、抗氧胁迫能力、鞭毛形成、生物被膜形成和毒力等的影响。同时,通过遗传分析对fur缺失突变株生长缺陷表型的原因进行探究。【结果】本研究成功构建了铜绿假单胞菌fur基因的缺失突变株,发现缺失突变fur极大地限制了铜绿假单胞菌的生长能力,并降低了该菌对限铁环境的生长适应性,但不影响该菌对高铁环境的生长适应性。铜绿假单胞菌Δfur的这种生长缺陷表型是细胞生长增殖变慢造成的,而不是诱导细胞死亡引起的。然而,其他异源的fur基因能完全互补Δfur的这种生长缺陷表型,暗示铜绿假单胞菌的Fur蛋白在功能上不存在独特性。尽管Fur与毒素-抗毒素系统PacTA存在功能关联性,但是铜绿假单胞菌Δfur的这种生长缺陷表型却与PacT毒素无关。除了影响铜绿假单胞菌的生长表型,缺失突变fur还使铜绿假单胞菌丧失了对铁载体生物合成的抑制作用,导致该菌对H₂O₂更敏感并丧失了鞭毛的形成能力,同时降低了该菌对大蜡螟幼虫的毒力。此外,缺失突变fur还显著提升了铜绿假单胞菌的胞内环二鸟苷酸(cyclic diguanylate, c-di-GMP)水平,从而诱导pelF和pslA基因的表达,进而促进铜绿假单胞菌生物被膜的形成。【结论】fur是可以缺失的非必需基因,在铜绿假单胞菌的正常生长、铁载体生物合成、抗氧胁迫能力、鞭毛形成、生物被膜形成和毒力等方面都发挥着十分重要的作用,这为针对铜绿假单胞菌的疫苗和抗菌药物开发奠定了基础。