外源乙烯对草莓果实微粒体Ca2+- ATPase活性和膜脂过氧化的调节

 以乳白期‘春星’草莓(Fragaria ananassa Duch．‘Chunxing’)果实为试材，研究了经钙调素(CaM)拮抗剂三氟拉嗪(TFP)、氯丙嗪(CPZ)各100 µmol·L^-1 和钙通道阻塞剂异博定(Verapamil)100 µmol·L^-1 预处理后再用乙烯(50 µL·L^-1 )处理的草莓果实中微粒体“Ca²⁺-ATP酶活性、O₂ 产生速率和MDA含量的变化。结果表明，外源乙烯对微粒体膜O₂ 产生速率无显著影响，处理早期提高微粒体膜Ca²⁺- ATPase总活性，对MDA含量影响不大；后期加速微粒体膜Ca²⁺- ATPase总活性下降，但仍保持较高的MDA含量和线粒体膜Ca²⁺-ATPase活性。CPZ、TFP和Verapamil预处理降低了上述乙烯处理下Ca²⁺- ATPase活性和MDA含量，但对微粒体膜O 产生速率亦无显著影响，这说明细胞内Ca²⁺-和CaM 可能参与了乙烯诱导的膜Ca²⁺-ATP酶活性与膜脂过氧化水平的调节。     

苹果矮化砧木‘辽砧2号’选育

从1135系的助列涅特与M9杂交实生苗中选出优良苹果矮化砧木—辽砧2号(原代号80-1-6),经过22年田间观查和区试鉴定,其矮化性、早果性、丰产性、生根性与M26相近,适应性强于M26,以其为中间砧的富士树栽后6年进入盛果期,平均株产为23.5kg,同龄M26为17.2kg;8年生树冠积为4.07m3,同龄M26为4.58m3,树体存活率较M26高55%,平均每666.7m2产量1000kg以上;与基砧山定子和生产上主栽的富士系、元帅系、国光系等品种亲和性良好,以自根砧和中间砧方式利用均可。     

Human interleukin-1β induces A375-S2 human melanoma apoptosis through caspase pathway

AIM: To study the molecular biological mechanism and signal transduction pathway of interleukin-1β (IL-1β)-induced apoptosis in A375-S2 melanoma cells. METHODS: Photomicrocropy showed typical apoptotic changes. The cytotoxic effect of IL-1β in vitro and influences of caspases in this effect were measured by MTT assay. The cytotoxicity of cells was assessed by LDH-based assay. Degradation of DNA was detected by agarose gel electrophoresis. RESULTS: The inhibitory effect of IL-1β on A375-S2 cell growth was in a dose and time-dependent manner, and cell death rate reached more than 90% at 72 h after treatment with 10^-9mol/L IL-1β. The inhibitors of caspase-family, -1, -3, -8, -9, and -10, partially blocked cell death at early stage. LDH assay showed that major IL-1β-induced cell death was apoptosis, and in a dose and time-dependent manner. Typical apoptotic DNA ladder was observed in agarose gel electrophoresis. CONCLUSION: IL-1β induced apoptosis in melanoma A375-S2 cells by activating caspase pathway.     

Changes of Ca2＋ activated potassium channels and cellular-proliferation in autogenous vein grafts

AIM: To investigate changes of Ca^2＋ activated potassium channels (K_Ca) in autogenous vein grafts. METHODS: The contraction of venous ring was measured by means of perfusion in vitro. The intimal proliferation and proliferation of cultured smooth muscle cells (vascular smooth muscle cells, VSMCs) were observed by the means of computerised image analysis and MTT method, respectively. Furthermore, whole cell mode of patch clamp was used to record K_Ca of VSMCs isolated from autogenous vein grafts. RESULTS: 1 week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P＜0.05, n=8 vs control), and they were more enhanced 4 weeks after vein transplantation (P＜0.01, n=8 vs control). TEA (blocker of Ca^2＋ activated potassium channels) increased MTT A₄₉₀ value of VSMCs from femoral vein in a dose-dependent manner (P＜0.05, n=8). K_Ca current density was significantly attenuated in VSMCs from autogenous vein grafts 1-4 weeks after transplantation (P＜0.05, n=5). CONCLUSION: K_Ca was inhibited in autogenous vein graft, which accounted for vasospasm and intimal proliferation.     

Effect of indomethacin on tumor invasion in human laryngeal cancer Hep-2 cells

AIM: To assess the effect of indomethacin on tumor invasion in a human laryngeal cancer Hep-2 cell line in vitro. METHODS: Hep-2 cells were exposed to indomethacin at different concentrations for 48 h. Then cell growth rate, the colony formation in soft agar medium and cell mobility were examined, and monolayer invasion assay was performed to assess cell invasion index. RESULTS: Preteatment with indomethacin inhibited the colony formation of Hep-2 cells and the cell mobility, and decreased the invasion index. CONCLUSION: Indomethacin can inhibit the invasion of Hep-2 cells.     

Effects of NF-κB decoy oligonucleotides on apoptosis in lung cancer cell A549

AIM: To investigate the effects of NF-κB decoy oligodeoxynucleotides (ODNs) on apoptosis in lung cancer cell A549. METHODS: The treatments of lung cancer cells (A549) were divided into three groups: group A (control group); group B (decoy ODN group) and group C (scramble decoy ODN group). FITC-labeled NF-κB decoy ODNs was transfected into A549 with LipofectAMINE^TM2000. The activation was observed by electrophoretic mobility shift assays (EMSA). The proliferation was observed by growth curve. The apoptosis of cells were observed by flow cytometry and TdT mediated dUTP-biotin Nick End Labeling (TUNEL). The expression of Bcl-2 and Fas were observed by Western blot. RESULTS: After FITC-labeled decoy ODNs was transfected for 1 hour, the decoy ODNs was detected in the nuclei of A549 cells. EMSA performed the depression of the NF-κB binding to the nucleus. The growth curve showed the inhibition of the A549 cell growth and the percentage of apoptosis was increased compare with control group by flow cytometry and TUNEL. The amount of apoptosis inhibitor (Bcl-2) in group A and group C were 2.0 times and 2.1 times more than that in group B, respectively. The level of apoptosis accelerator (Fas) in group B were 2.6 times and 2.3 times more than that in group A and group C, respectively via Western blot. CONCLUSION: The NF-κB decoy ODNs accelerate the apoptosis of lung cancer cell A549 and the mechanism may be due to its inhibiting the expression of Bcl-2 and increasing the level of Fas.     

鹅细小病毒VP2基因在大肠杆菌中的表达及纯化

将鹅细小病毒 (GPV) VP2基因插入原核表达载体 p PROEX- HTb,获得重组表达质粒 p PROEX- HTb- VP2。将其转化大肠杆菌 DH5α,用 IPTG诱导表达 ,表达菌体蛋白中可产生与预期大小相符的约 72 0 0 0的蛋白。以光密度扫描对该表达产物进行定量分析 ,表达产物约占菌体总蛋白的 14 %。经 His- tag金属螯合层析纯化 ,获得纯度较高的 6 His- VP2融合蛋白。 Western- blot结果表明 ,所得蛋白与鹅抗 GPV高免血清有较好的免疫反应性 ,说明在 VP2的 N端融合 6个组氨酸不影响其与特异抗体结合的活性。     

表达犬瘟热病毒H基因的重组犬2型腺病毒的构建与鉴定

将犬瘟热病毒 ( canine distemper virus,CDV ) H基因表达盒克隆入转移质粒 p VAXΔE3,构建含 CDV H基因表达盒的转移质粒 p VAXΔ E3L PH,用 Nru 和 Sal 分别酶切转移质粒 p VAXΔ E3L PH和犬 2型腺病毒全基因组质粒p Poly2 - CAV- 2 ,将 CDV H基因表达盒定向克隆入 p Poly2 - CAV- 2质粒中 ,构建 E3区部分缺失的包含 CDV H基因表达盒的犬 2型腺病毒重组质粒 p CAV- 2 / CDVL PH。 Asc 和 Cla 对 p CAV- 2 / CDVL PH进行双酶切 ,释放 CAV- 2 /CDVL PH重组基因组 ,利用脂质体将 CAV- 2 / CDVL PH重组基因组与去除 Sal 和 Nru 片段的 CAV- 2基因组两端片段共转染 DK细胞 ,盲传和重复转染 3次 ,4 d后出现典型 CAV- 2病变 ,获得重组病毒 CAV- 2 / CDVL PH。用 CDV H基因特异性引物经 RT- PCR扩增重组病毒 DK细胞培养物 ,能够扩增出相应的核酸片段 ,表明 CAV- 2 / CDVL PH在DK细胞繁殖的过程中能够转录 CDVL P H的 m RNA,重组病毒免疫犬 ,可以诱导犬产生特异的抗 CAV- 2 HI抗体和抗 CDV中和抗体     

IBDV地方变异株VP2基因的克隆与特性鉴定

应用 RT- PCR技术对 1株分离于地方免疫鸡群中暴发的传染性法氏囊病病例的传染性法氏囊病病毒 ( infec-tious bursal disease virus,IBDV) HN0 2 6株的 VP2基因进行了克隆与序列分析 ,并与相应毒株的 VP2高变区核苷酸及氨基酸序列进行了比较研究。结果表明 ,HN0 2 6株的核苷酸和氨基酸序列与变异株 Var- A的同源性最高 ,分别达97.3%和 97.6 % ;其次为超强毒株 UK6 6 1 ,分别为 95 .7%和 95 .2 % ;而和弱毒株 PBG98的同源率仅有 92 .8%和90 .3%。其中 ,在第一、二亲水区 ,HN0 2 6株均有 1个氨基酸发生了变化 ,即第 2 2 2位转变为 E、第 31 8位转变为 D。更重要的是 ,其 2 4 9位和 2 5 4位上的氨基酸分别为 K和 S,这些均为变异株的特性。另外 ,HN0 2 6株的七肽区保持SWSASGS不变 ,且第 2 79位和 2 84位氨基酸分别为 D和 A,又完全具备强毒株的特性。因此 ,初步确定所分离的HN0 2 6为有较强致病力的变异株 IBDV。     

猪瘟病毒E2(gp55)基因在昆虫细胞中的表达

将除去3'端跨膜区的猪瘟病毒E2基因亚克隆到杆状病毒转移载体pFastBacHTb,获得重组质粒pFBHT-E2,转化进含穿梭载体Bacmid的感受态细胞DH10Bac,发生转座作用,经抗性及蓝白斑筛选得到含E2基因的重组载体质粒rBacmid-E2,以脂质体介导的方法将此重组载体质粒转染sf9昆虫细胞,获得重组病毒,命名为rvBac-E2.经SDS-PAGE和Western-blot及ELISA等方法检测,结果表明,此E2基因在昆虫细胞中正确表达,表达的蛋白能与猪瘟阳性血清发生特异性反应.