Hemorrhagic activity and mechanism of FⅡa′a fibrinolytic enzyme from Agkistrodon acutus venom

AIM: To study the local hemorrhagic activity of a fibrinolytic enzyme (FⅡa) from Agkistrodon acutus venom and its mechanism. METHODS: The local hemorrhagic activity was determined by subcutaneous injection on the back of mouse. The effects of FⅡa on factor X, prothrombin, gelatin, and collagen were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Platelet aggregation assays were performed in rat platelet-rich plasma (PRP). Human umbilical vein endothelial cells (HUVEC) were cultured and passaged in complete M 199 medium. Cell viability and nuclear morphology change were determined by fluorescein diacetate (FDA) staining and Hoechst 33258 staining, respectively. RESULTS: The minimum hemorrhagic dose (MHD) of FⅡa was 89 μg.In vitro, FⅡa (0.25 g/L) degraded factor X, prothrombin, collagen, and gelatin, and dose-dependently (0.25, 0.50,0.75, and 1.00 g/L) inhibited the platelet aggregation induced by ADP in rat PRP. When HUVEC in culture treated with FⅡa, HUVEC showed detachment in a dose-dependent manner, but no apoptosis sign was observed.CONCLUSION: FⅡa had local hemorrhagic activity, and the mechanism was related to the degradation of factor X,prothrombin, gelatin, and collagen, the inhibition of ADP-induced platelet aggregation, and inducement of HUVEC detachment.     

Detection of cyclin b1 expression in g(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis

Protein complex of cyclin B1 and cyclin-dependent protein kinase 1 induces phosphorylation of key substrates that mediate cell cycle transition during the G(2)-M phase. It is believed that cyclin B1 accumulates in the S phase of the cell cycle and reaches the maximal level at mitosis but is absent in G(1)-phase cells. In the present study, we demonstrated that cyclin B1 was expressed in the arrested G(1)-phase MOLT-4 lymphocyte leukemia cells and in G(1) phase T-7 transitional tumor cells, as determined by flow cytometry. In addition, we showed that cyclin B1 was detected in the G(1) phase in breast cancer cells from patient tissues and in lymphocytes from patients with leukemia. These findings were confirmed for the first time by postsorting Western blot analysis and by confocal microscopy. Furthermore, by using postsorting Western blotting, we found that cyclin B1 was expressed in different time-window sections of the G(1) phase under different conditions. For the asynchronously growing T-7 cells, cyclin B1 was detected in early G(1) phase, whereas in MOLT-4 cells arrested in G(1)-S phase, cyclin B1 was mainly detected in late G(1) phase. We propose that the cyclin B1 expressed in the G(1) phase may differ from that expressed in the G(2)-M phase, and that this unscheduled type of cyclin B1 may play an important role in tumorigenesis and apoptosis.     

中性粒细胞粘附分子CD11b表达对新生儿败血症早期诊断价值的研究

目的　观察新生儿败血症中性粒细胞粘附分子CD11b表达的规律 ,并评价其在新生儿败血症早期诊断中的价值。方法　将 5 1例临床疑似败血症的新生儿根据其临床表现及WBC、PLT、血浆CRP和未成熟中性粒细胞数与中性粒细胞总数比值 (I/T)四项指标 ,分为败血症和可疑败血症两组。采用全血流式细胞术检测患儿和 15例正常对照组新生儿中性粒细胞CD11b的平均荧光强度 (MFI)。结果　败血症组 2 3例 ,可疑败血症组 2 8例。两组中性粒细胞CD11b分别为 (32 0± 189)、(4 5 6± 2 13)MFI,均显著低于正常对照组的 (10 90± 338)MFI(t分别为 - 9 0 1、- 7 5 6 ,P均 <0 0 0 1) ,败血症组又低于可疑败血症组 ,差异有显著性 (t=- 2 39,P <0 0 5 )。高CRP组患儿CD11b为 (2 11± 16 4 )MFI,低于低CRP组的 (5 0 5± 2 6 5 )MFI,差异有显著性 (t=2 6 4 ,P <0 0 5 )。中性粒细胞CD11b≤ 6 0 0MFI对疑似败血症新生儿诊断的敏感性、特异性、阳性和阴性预测值分别为 86 3%、10 0 %、10 0 %、6 8 2 % ,CD11b检测阳性率为 86 3% ,高于血培养的阳性率 (17 6 % ) ,差异有显著性 (χ2 m=31 2 ,P <0 0 5 )。结论　新生儿败血症中性粒细胞粘附分子CD11b表达下调 ,其下调与感染严重程度有一定关系。中性粒细胞CD11b的动态检测对早期     

Lipolysis and apoptosis of adipocytes induced by neuropeptide Y—Y5 receptor antisense oligodeoxynucleotides in obese rats

目的:探讨神经肽Y-Y5受体反义基因治疗后饮食性肥胖大鼠减肥减重效应与外周白色脂肪细胞体积、数目变化的关系。方法:建立饮食性肥胖大鼠模型,侧脑室注射Y5受体编码起始区反义、正义、错义寡脱氧核糖核酸及生理盐水,采用MPLAS-500多媒体彩色病理图文分析系统计算平均脂肪细胞面积,基因组DNA提取物凝胶电泳检测脂肪细胞凋亡,RT-PCR分析凋亡相关基因bc1-2、bax表达的改变。结果:(1)Y5受体反义基因治疗后大鼠进食量与体重显著降低,外周白色脂肪组织湿重与平均脂肪细胞面积明显减少;(2)脂肪组织基因组DNA提取物凝胶电泳出现凋亡特征性梯状条带;(3)凋亡相关基因bc1-2表达下调、bax表达上调。结论:平均脂肪细胞面积减小、脂肪细胞凋亡增加可能是Y5受体反义基因治疗减肥减重的重要原因。     

Palmitic acid-induced apoptosis in pancreatic β-cells is increased by liver X receptor agonist and attenuated by eicosapentaenoate

Saturated fatty acids are implicated in the development of diabetes via the impairment of pancreatic islet β-cell viability and function. Liver X receptors (LXRs) and eicosapentaenoate (EPA) are known regulators of fatty acid metabolism. However, their roles in the pathogenesis of diabetes remain incompletely understood. The aim of this study was to determine the effects of EPA and the LXR agonist T0901317 on saturated fatty acid (palmitic acid)-induced apoptosis in the insulinoma β-cell line INS-1, a model for insulin-secreting β-cells. T0901317 significantly promoted palmitic acid-induced apoptotic cell death in the INS-1 cells. Consistent with these results, caspase-3 activity and BAX and sterol regulatory element binding protein-1c (SREBP-1c) mRNA levels were markedly increased in INS-1 cells co-administered palmitic acid and T0901317. The production of reactive oxygen species was considerably higher in the cells cultured concurrently with T0901317 and palmitic acid than in the cells incubated with either agent alone. EPA treatment attenuated the cellular death promoted by palmitic acid and T0901317 in the INS-1 cells, disclosing a possible mediating mechanism involving the inhibition of SREBP-1c. Finally, T0901317 up-regulated the palmitic acid-induced expression of p27(KIP1), transforming growth factor beta 1, and SMAD3 proteins in INS-1 cells. These results demonstrate that palmitic acid-induced apoptosis in β-cells is enhanced by T0901317 via the activation of LXRs and is blocked by EPA via the inhibition of SREBP-1c, suggesting that the regulation of lipogenesis and lipotoxicity affecting pancreatic β-cell viability and insulin production may be a unique strategy for diabetes therapy.     

Environmental inhibitors of 11β-hydroxysteroid dehydrogenase type 2

11β-Hydroxysteroid dehydrogenase (11β-HSD) regulates glucocorticoid action by catalyzing the interconversion of active cortisol and inactive cortisone in glucocorticoid and mineralocorticoid target tissues. Two distinct isoforms, 11β-HSD1 and 11β-HSD2, have been characterized. 11β-HSD1 is widely expressed, uses NADP+/NADPH as cofactors, and contributes to the metabolic syndrome and related conditions by increasing cortisol level. 11β-HSD2 is an NAD⁺-dependent oxidase, converting cortisol to cortisone to lower active glucocorticoid level. The inhibition of 11β-HSD2 activity is generally detrimental to health because the buildup of local cortisol concentration can cause symptoms of apparent mineralocorticoid excess, fetal developmental defect and lower testosterone level in males. In this review, we focus on many environmental inhibitors of 11β-HSD2 including licorice components, gossypol, phthalates, organotins, alkylphenols and perfluorinated substances.     

脓毒症患者外周血T淋巴细胞亚群变化及临床意义

目的 观察脓毒症患者外周血T淋巴细胞亚群水平变化.探讨脓毒症患者外周血T淋巴细胞亚群在发病中作用及预后意义.方法 以温州医学院附属第二医院急救中心及急诊ICU2007年8月～2009年7月收治的脓毒症患者64例为研究对象,采用流式细胞仪检测64例脓毒症患者(脓毒症组)入院后第1、7、14天外周血CD3+、CD4+、CD8+T淋巴细胞计数,并按病情好转与未好转分为两组,比较两组的异同.同时以同期20例健康体检者作为健康对照组.结果 2组脓毒症患者第1天CD3+、CD4+T淋巴细胞计数及CD4+/CD8+T淋巴细胞计数比值均明显低于健康对照组(P＜0.05),CD8+T淋巴细胞计数比较无明显统计学差异;2组脓毒症患者之间第1天CD3+、CD4+T淋巴细胞计数及CD4+/CD8+T淋巴细胞计数比值存在统计学差异,未好转组明显低于好转组(P＜0.05);好转组CD3+、CD4+T淋巴细胞计数及CD4+/CD8+T淋巴细胞计数比值随着治疗时间变化逐渐得到纠正,在第14天时接近健康对照组(P＞0.05).未好转组无明显改变.结论 脓毒症患者可以出现免疫失衡,表现为CD3+、CD4+、CD4+/CD8+比值的下降,且其和疾病严重程度存在一定相关性.CD3+、CD4+、CD4+/CD8+比值可以作为脓毒症治疗过程中的疾病严重程度的检测指标.