Detection of Penaeus monodon-type baculovirus (MBV) infection in Penaeus monodon Fabricius by in situ hybridization

Abstract. A digoxigenin-labelled DNA probe was used for in situ detection of the Penaeus monodon -type baculovirus (MBV) derived from cloned MBV polyhedrin genome in cultured Penaeus monodon Fabricius. First, the specificity of the probe against MBV DNA with dot blot hybridization analysis was verified. This probe indicated that cloned MBV polyhedrin fragment can be used as an MBV-specific probe. This was then used to microscopically examine sections of MBV-infected tissues for a blue-purple precipitate indicative of a positive reaction for MBV. MBV-positive cells were located only in the epithelium of the hepatopancreatic tubules and of the midgut. Furthermore, comparison of the susceptibility to MBV infection among several life-stages of the shrimp showed that the MBV genome was found in the zoea, mysis, post-larva, and adult stages, whereas MBV DNA was not detected in either eggs or nauplii. The results were quantified from in situ hybridization with an image analyser to compare the degree of cell infection among groups of cultured P. monodon collected from various farms in Taiwan.     

大白菜突变体库的构建及M2叶片表型变异的研究

 利用不同浓度甲基磺酸乙酯（EMS）诱变大白菜自交系‘A03’种子,根据M₁代植株发芽率、成活率、育性及M₂代成活率,筛选出适宜的EMS诱变浓度为0.4%。采取EMS种子诱变1次、EMS种子诱变2次及EMS花蕾诱变结合小孢子培养的方法构建了含有4 253个家系及相应自交M₂代种子的大白菜突变体库。在该突变体库M₂群体中分别对苗期6 404株幼苗及莲座期7 756个单株的叶片性状进行了形态学调查,总的表型变异频率高达37.62%和26.33%。     

Effects of maternal limb ischemic preconditioning on fetal hippocampal neuron apoptosis induced by intrauterine distress-reoxygenation in rats

AIM: To evaluate the influence of maternal limb ischemic preconditioning (LIP) on the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats. METHODS: Intrauterine ischemia was induced by clamping the uterine and uterine branch of the ovarian blood vessels with aneurysm clamps for a period of 15 min followed by removal of the clamps to permit reperfusion. Sprague-Dawley (SD) rats (n=12) were randomly divided into 4 groups on the 19th pregnant day: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. The cesarean birth occurred on embryonic day 21 to obtain 12 fetal rats alive in each group. The fetal rats were decapitated and the pyramidal cells in CA1 hippocampus were observed under light microscope. The neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and the apoptotic rate was calculated. The expression of Bcl-2 and Bax were detected by immunohistochemical method and Western blotting. RESULTS: The rates of neuronal apoptosis in FD group and LIP+FD group were significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The expression of Bcl-2 and Bax in FD group and LIP+FD group was significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The ratio of Bcl-2/Bax was lower in FD group than that in S group. Compared with FD group, the rate of neuronal apoptosis was significantly lower (P<0.05), while the ratio of Bcl-2/Bax was significantly higher (P<0.05) in LIP+FD group. CONCLUSION: Maternal limb ischemic preconditioning attenuates the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats, which may be associated with the up-regulation of Bcl-2 expression.     

Expression of Cdc25a in cross-species hepatocellular carcinoma tissues and its significance

AIM：To study the expression of cell division cycle 25a protein (Cdc25a) in hepatocellular carcinoma (HCC) tissues of different species, including human, rat and tree shrew, and to verify the feasibility of the strategy of cross-species oncogenomics screening. METHODS：Real-time fluorescence quantitative PCR and Western blotting were applied to detect the expression of Cdc25a at mRNA and protein levels in the HCC tissues, corresponding HCC-adjacent liver tissues and normal liver tissues collected from humans, rats and tree shrews. RESULTS：The mRNA expression of Cdc25a in the HCC tissues of humans, rats and tree shrews was higher than that in normal liver tissues (P<0.05). The mRNA levels of Cdc25a in the HCC tissues of humans and rats were higher than those in the corresponding HCC-adjacent liver tissues (P<0.05). The mRNA expression of Cdc25a in the HCC tissues was significantly correlated with the portal vein tumor thrombus, the extrahepatic metastasis and the clinical stage, but was not correlated with the recurrence of tumor, the diameter of tumor, the number of tumor, the level of serum alpha-fetoprotein and the differentiation of tumor. The protein levels of Cdc25a in the HCC tissues of humans, rats and tree shrews were higher than those in the corresponding HCC-adjacent liver tissues and the normal liver tissues. CONCLUSION：Cdc25a may be a particularly crucial molecule for hepatocarcinogenesis. The cross-species oncogenomics screening may represent a feasible and convenient way for identifying key molecules of human HCC.     

Simple and effective method for monitoring ambulatory electrocardiogram in toads

AIM：To screen out a suitable lead for monitoring the ambulatory electrocardiogram (ECG) in unrestrained toad, and to investigate its practicability. METHODS：After subcutaneously implanting the electrodes in toads under anaesthesia, the ambulatory ECG of 5 leads were monitored with BL-420S data acquisition and analysis system, and the leads which could well express the waveform in ECG were screened out. The recovery process of the toads from the artificial hibernation within 6 h, the day-to-day stability of the heart rate (HR) and the heart rate variability (HRV) in 5 successive days of hibernation, and the HR and HRV after freeze-thawing process were monitored to determine its practicability. RESULTS：Two out of 5 leads showed better ECG waveforms. Compared with 6 h post hibernation, lowered HR at 0 h and 1 h was observed, and the standard deviation of normal R-R intervals (SDNN) was significantly increased (P<0.05 or P<0.01), but the HR and SDNN from 2 to 5 h showed no significant difference, suggesting that the cardiac function reached the steady state after 2 h recovery. The HR at 2 h and 4 h on day 4 and day 5 decreased significantly compared with that on day 1 (P<0.05 or P<0.01), followed with a significant increase in SDNN (P<0.05 or P<0.01), suggesting that the ECG remained stable within 3 d. The HR increased, while SDNN decreased significantly at 1 h and 12 h post-thawing compared with that at pre-freeze (P<0.05), indicating the damaged cardiac function after freeze-thawing process. CONCLUSION：The method of subcutaneously implanting electrodes is suitable for effectively monitoring the ambulatory ECG in toads.     

Attenuated Salmonella carrying pGRIM-19-si-survivin co-expression plasmid inhibits growth of prostate cancer subcutaneous xenografts in vivo

AIM：To explore the effects of pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella on prostate cancer subcutaneous xenograft growth in nude mice. METHODS：Prostate cancer xenograft model was established in nude mice. Co-expression plasmids carried by attenuated Salmonella were introduced by intraperitoneal injection. The xenograft volumes were monitored timely. Immunohistochemical staining, RT-PCR and TUNEL assay were applied to investigate the related mechanisms that pGRIM-19-si-survivin inhibited tumor growth in vivo. RESULTS：Compared with psi-survivin and pGRIM-19 carried by attenuated Salmonella (control groups), the tumor volumes were reduced markedly in pGRIM-19-si-survivin plasmid group. The mean shrinkage rates were 2.36 and 3.02 times. pGRIM-19-si-survivin co-expression plasmid carried by attenuated Salmonella inhibited survivin expression but strengthened GRIM-19 expression obviously (P<0.05). The mRNA expression of apoptosis-related proteins such as Bcl-xL, Stat3, cyclin D1 and c-Myc was inhibited, and the vascular endothelial growth factor (VEGF) mRNA and Ki67 protein were also inhibited, but the caspase-3 mRNA expression was up-regulated (P<0.05) with significant cell apoptosis. CONCLUSION： pGRIM-19-si-survivin co-expression plasmid carried by human attenuated Salmonella inhibits the growth of prostate cancer subcutaneous xenografts in nude mice by promoting cell apoptosis and inhibiting prostatic cancer proliferation.     

High concentration of extracellular ATP causes autophagy and apoptosis of SH-SY5Y cells

AIM：To examine the effects of high concentration of extracellular ATP on human neuroblastoma SH-SY5Y cell injury. METHODS：Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time. The cell viability was detected by CCK-8 assay. The variation of autophagic vacuoles was observed with monodansylcadaverine staining. The cell apoptosis was analyzed by Hoechst 33258 staining. Meanwhile, apoptotic rate was detected by flow cytometry. The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) were determined by Western blotting. RESULTS：Compared with control group, the survival rate of SH-SY5Y cells was significantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h). The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment, trended to decrease over time and returned to control level at 6 h. The protein expression of LC3-Ⅱ was significantly increased at 1 h with ATP treatment, which was consistent with the time points of increasing autophagic vacuoles. LC3-Ⅱ expression level gradually decreased at 2~3 h with ATP treatment, and returned to control level at 6 h. Compared with control group, the apoptotic rate and the expression level of caspase-3 were enhanced synchronously. The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION：High concentration of extracellular ATP induces the autophagy and apoptosis of SH-SY5Y cells. The increased autophagy shows up, followed by the climax of apoptosis until 6 h. With the prolonged duration of ATP, apoptosis is the main process in the cells.     

小株采种阳畦中白菜幼苗症状的识别

目前,小株采种是白菜杂交一代种子生产的主要方式,而播种和育苗环节是小株采种的两个关键技术环节,阳畦育苗过程中如何识别幼苗受害症状至关重要。     

溧阳白芹铅污染原因探析

为寻找溧阳白芹铅超标原因,探索生态修复和控制措施,溧阳市蔬菜办公室在溧阳城郊溧阳白芹基地进行了水、土、肥、产品等四大类样品的铅含量检测,对照相关标准,通过对试验区生态环境的调研和铅污染评价,认为溧阳市城郊溧阳白芹主产区的土壤、基肥、培土与否及铅富集等对溧阳白芹重金属铅污染影响不明显,灌溉水则是重金属铅污染的主因。     

浅水芹夏秋轻简栽培技术研究

为探索浅水芹夏秋轻简栽培技术,研究了不同稻草覆盖量和覆盖方式对水芹生长的影响。试验结果表明,浅水芹夏秋栽培宜采用免催芽直接排种;覆盖方式以遮阳网大棚覆盖为最佳,其次为稻草覆盖,最适宜的稻草覆盖量为200~250 kg/667 m2。