A Cathepsin B Inhibitor,E-64, Improves the Preimplantation Development of Bovine Somatic Cell Nuclear Transfer Embryos

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.     

Comparison of two methods for culturing neonatal rat cardiac fibroblasts

AIM:To establish and compare the methods for culturing neonatal rat cardiac fibroblasts culture. METHODS:Neonatal rat hearts were isolated by collagenase+trypsin or trypsin digestion. The cardiomyocytes and cardiac fibroblasts were isolated by different attachment techniques. The cellular morphology, purity and reactions to the reagent were tested. RESULTS:For morphology and purity, no difference between the 2 methods was observed, though more myocytes were harvested by the method of collagenase+trypsin digestion. For the cellular responses to reagent, the cardiac fibroblasts harvested with trypsin digestion had more potent proliferative ability than those with collagenase+trypsin digestion. CONCLUSION:There is no difference of cellular morphology and purity in the cardiac fibroblasts isolated by collagenase+trypsin digestion and trypsin digestion, but the fibroblasts with trypsin digestion have more potent proliferative ability.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

钙信号抑制剂加剧低钾胁迫对烟草幼苗光合特性及钾吸收的影响

  【目的】  研究低钾胁迫抑制钙信号传导对植株幼苗叶片光合特性及钾吸收的影响,为缓解作物的低钾胁迫提供理论依据。  【方法】  以烟草品种K326为试验材料进行了室内水培试验。烟草幼苗先在营养液正常钾(K+ 5 mmol/L)和缺钾(K+ 0.15 mmol/L)营养液中生长8 天,然后在营养液中分别施加钙通道有机阻断剂Vp、钙通道无机阻断剂LaCl₃和钙离子螯合剂(EGTA)钙信号抑制剂,继续生长4天后取样,测定植株干鲜重、电导率、活性氧含量、叶片抗氧化酶活性、叶片光合特性等指标和植株地上、地下部钾素浓度及钾离子吸收相关基因表达量。  【结果】  1)不添加钙通道抑制剂或螯合剂条件下,低钾胁迫的烟株地上部和根系干重较常钾水平显著降低了59.29%、39.82%,鲜重显著降低了51.58%、48.19%,除水汽压饱和亏外的其余5项光合特性指标显著降低,植株地上部和根系钾含量分别显著降低了39.31%和77.05%,而叶中可溶性蛋白、相对电导率、过氧化氢含量则显著增加,NKT2、NtKC1、NtHAK1、NtHAK5基因的相对表达量分别增加了2.09、3.32、3.23、12.34 倍。2)常钾水平下,3个钙抑制剂处理未明显影响地上部和根系干鲜重,而低钾水平下,钙抑制处理的地上部鲜重和干重显著高于无钙抑制剂对照。两个钾水平下,与各自对照相比,3个钙抑制剂处理的叶片相对电导率、活性氧积累量均显著增加,抗氧化酶SOD和POD活性、叶绿素含量显著减少,光合特性指标(除水汽压饱和亏外)明显降低。3)两个钾水平下,3个钙信号抑制剂处理的烟株地上部和根系钾含量均显著低于其对照,低钾胁迫下平均降低幅度分别达到25.54%和53.36%。低钾胁迫下,钾离子通道基因NKT2、NtKC1和转运蛋白基因NtHAK1、NtHAK5在根系中的相对表达量分别平均降低了60.60%、50.62%、38.61%、69.79%,钙通道相关基因NtCNGC1和NtCNGC11在根中的相对表达量分别平均降低了95.36%和87.04%。  【结论】  低钾胁迫明显影响烟株幼苗生长、光合作用及对钾素的吸收和积累。低钾水平下,外施钙通道抑制剂或螯合剂可进一步降低烟草叶片抗氧化酶活性,减弱烟株叶片光合作用,并抑制根中钾吸收相关基因的表达和烟株对钾素的吸收。     

HBV衣壳蛋白装配抑制剂的研究进展

乙型肝炎病毒(HBV)是一种常见的嗜肝性病原体,全球约有4亿人感染,带来严重的社会问题和经济负担。虽然目前已有预防和治疗HBV的药物,但由于耐药性等问题不能彻底清除病毒感染,新药开发依然迫在眉睫。介绍了HBV的病毒结构、复制和感染过程,对现有治疗药物的优缺点进行了总结。对以衣壳蛋白装配为靶点的新药研究进展进行综述,旨在为今后开发抗HBV的新药提供参考。     

脲酶测定法判定豆浆抗营养因子热失活情况研究

大豆抗营养因子的主要成分是胰蛋白酶抑制剂,在大豆制品加工过程中,通常采用高温加热钝化的方法使胰蛋白酶抑制剂失去活性。豆浆是人们日常生活中喜欢的饮品之一,因为脲酶测定方法比较简单,目前多以加热钝化后豆浆脲酶活性来表示抗营养因子的去除情况。本研究通过测定豆浆中胰蛋白酶抑制剂和脲酶的活性研究豆浆加热过程中二者失活是否具有一致性,判定以脲酶活性来体现豆浆安全的科学性,同时通过豆浆加热程度与胰蛋白酶抑制剂失活情况的关系,对家庭豆浆制作提供一种安全的加热模式。     

硝化抑制剂对小白菜生长和土壤性质的影响

施用硝化抑制剂可有效控制蔬菜硝酸盐积累,施用硝化抑制剂DMPP、土地精和双氰胺可提高小白菜的经济产量、叶面积、净光合速率,降低硝酸盐含量,并对栽培土壤有明显的改良作用。硝化抑制剂对小白菜的硝酸盐积累抑制作用为DMPP双氰胺土地精,3种硝化抑制剂在经济产量方面表现为DMPPDCDTC,比对照分别提高产量19.32%、14.83%、9.46%。对叶茎鲜重比的影响结果为DCD(84.7%)DMPP(84.4%)TC(83.8%)CK(78.1%)。通过对栽培土壤的研究发现硝化抑制剂能显著改良土壤的盐渍化,降低小白菜生长过程中土壤盐离子的含量,提高铵离子在土壤中的含量,抑制土壤耕层硝酸盐的生成。     

包膜及其与生化抑制剂结合型尿素在草甸棕壤中尿素态氮溶出特征

在室内恒温培养条件下,以日本70 d聚烯烃包膜尿素和大颗粒尿素为对照,研究4种丙烯酸树脂（MMA）包膜及其与生化抑制剂结合尿素肥料在土壤中尿素态氮溶出与水解特征,以确定不同种MMA包膜尿素在土壤中缓/控释效果与尿素转化机理。结果表明,4种MMA包膜尿素在土壤中尿素累积溶出量在48 d后达到最大值,接近100%,尿素态氮累积溶出80％的时间分别在第24和28 d,时段平均溶出率最高峰值均出现在9～12 d,为7.73%/d～8.04%/d。4种MMA包膜肥料尿素态氮在土壤中累积残留量高峰值分别出现在20和24 d。MMA膜和NBPT共同作用对抑制尿素释放作用效果十分显著, 其结合型肥料对抑制尿素释放作用最强。