Effects of FK506 on anti-glomerular basement membrane nephritis in rats

AIM:To investigate the effects of FK506 on anti-glomerular basement membrane(GBM) nephritis in rats. METHODS: Anti-GBM nephritis model was elaborated by rabbit anti-rat GBM antibody injection in SD rats in this study. The rats were divided into three groups: FK506 treated group(0.5 mg·kg^-1·d^-1, sc), untreated nephritis control group and normal control group. FK506 was administered daily six hours after injection of anti-GBM IgG. All the rats were observed urinary protein at the 4th day, the 14th day and the 21st day. At the same time, the kidney specimens were collected, and T cell transforming function was also monitored.RESULTS:Rats injected with rabbit anti-GBM Ab developed heavy proteinuria by 4 days, and serum creatinine and serum urea appeared which kept on the rising. Glmerular hypercellularity, crescents, and protein casts were observed in nephritic rats. By electron microscopy, the thickening of GBM and loss of foot processes were seen. T cell transforming function was higher than normal. But, all pathological changes obviously turned for the better in FK506 treated group. CONCLUSION:FK506 could inhibit the progression of rat anti-GBM nephritis.     

组氨醇脱氢酶抑制剂的研究动态

组氨醇脱氢酶(HDH)是组氨酸生物合成过程中最后两步反应的催化酶。对组氨醇脱氢酶的结构、催化机理、组氨醇脱氢酶抑制剂等方面的研究动态进行了较为详细的综述。     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Changes of intracellular potassium activity and membrane potential in guinea pig ventricular myocardium during superfusion with low sodium solutions and effects of low pH

AIM:To investigate the changes in intracellular potassium activity(aⁱK) and membrane potential(V_m) induced by low external sodium infusion (Low [Na]_o) and to detect the mechanisms involved and the relationship between aⁱK and V_m. METHODS:aⁱK and V_m were measured in infusion with different sodium concentrations using methods of convenient and ion selective microelectrodes in guinea pig ventricular myocardium. RESULTS:Low [Na]_o resulted in a decrease in aⁱK and an increase in V_m in a Na⁺ concentration-dependent manner.At the same time,contraction and resting potential increased, and action potential duration decreased markedly,but action potential amplitude was not affected. A change of the pH from 7.4 to 7.0 in low [Na]_o solution reduced the decrease in aⁱK, but did not affect the increase in V_m.CONCLUSION:A better linear relationship appeared between the changes in aⁱk and [Na]_o or in V_m and [Na]_o,while during each low [Na]_o the change in both aⁱK or V_m may reach a new balance level.     

奶牛MTT比色法的最佳条件探讨

为建立奶牛淋巴细胞增殖反应MTT比色法,对试验条件进行了研究。应用L16(45)正交试验,对影响MTT比色法的四个主要因素,包括ConA浓度、细胞浓度、培养液小牛血清浓度及培养时间进行了比较和探索。试验表明几个因素对其增殖反应都有显著影响(P<0.05),且最佳反应条件为:15μg/mL的ConA、1×106/mL细胞浓度、10%小牛血清及60h的培养时间;影响增殖反应的先后顺序为细胞浓度、ConA浓度、培养时间及血清浓度。     

利用远缘杂交创造核果类果树新种质的研究

以桃、杏、李、樱桃、杏梅等果树的 1 4个品种为亲本 ,3年来共进行了 80余个组合的远缘杂交试验 ,探讨了核果类果树远缘杂交的亲和性 ,并对远缘杂种幼胚进行了胚抢救。结果表明 ,铃铛花期授粉的坐果率显著高于初花期授粉 ;同一杂交组合 ,正反交坐果率差异显著 ,母本对远缘杂交亲和性影响很大 ,选择自交亲和或自然坐果率高的种或品种做母本容易克服远缘杂交的不亲和性 ;适宜场强的静电场、He -Ne激光处理及60 Coγ射线与He -Ne激光联合处理花粉 ,均能显著提高花粉离体萌芽率 ,用上述处理的花粉进行的远缘杂交坐果率也明显高于对照 ;60 Coγ射线单独处理则降低了花粉离体萌芽率 ,远缘杂交的坐果率也低于对照 ;对远缘杂种幼胚及时地进行胚抢救 ,并诱导形成多丛芽 ,是克服核果类果树远缘杂种不育性的有效方法。研究并筛选出了李、樱桃胚萌发与生长、多丛芽诱导与增殖以及生根培养等最佳培养基配方。目前已将欧洲甜樱桃×中国樱桃、大石早生李×泰安巴旦水杏及凯特杏×总统李等一批核果类果树远缘杂种定植于露地 ,其中欧洲甜樱桃与中国樱桃的种间杂种是国内外首次获得     

Effects of β-ME and RA on expression of GFAP in mesenchymal cells derived from mouse fetal liver

AIM: To investigate the effects of β-mercaptoethanol (β-ME) and all-trans rentinal acid (RA) on glial fibrillary acidic protein (GFAP) expression in mesenchymal cells derived from mouse fetal liver in vitro. METHODS: Cells suspension from 14.5-days-old mouse fetal liver were cultured in DMEM/HEPES/F12 supplemented with 20％ FCS and mesenchymal cells were acquired after discarding nonadherent cells. The 5th passage cells were induced by β-ME and RA. The characteristics of treated cells were assayed by immunocytochemistry staining at 5 hours and 5 days after induction. β-actin as an internal control, GFAP gene expression of mesenchyal cells was detected with semi-quantitative RT-PCR. RESULTS: After being inducted by β-ME and RA, 80％ approximately of the cells exhibited typical neural morphology and about 85％ expressed GFAP phenotype. Semi-quantitative RT-PCR showed that mRNA expression of GFAP increased in treated cells versus untreated cells (P<0.01). CONCLUSION: GFAP expression in mesenchymal cells derived from mouse fetal liver in vitro increases after being treated with β-ME and RA.     

Meiotic competence of mouse oocytes reconstructed by replacing the germinal vesicle with a male pronucleus and somatic nucleus

The present study examines the contribution of the nucleus to meiotic competence in mouse oocytes that were reconstructed using nuclear transfer. Three types of reconstructed oocytes were produced: MP‐GV, by transplanting the male pronucleus (MP) into germinal vesicle (GV) stage oocytes; 3T3‐GV, by transplanting the nucleus of a National Institute of Health (NIH) 3T3 cell into a GV stage oocyte; and 3T3‐MII, by transplanting the nucleus of an NIH 3T3 cell into a metaphase II (MII) stage oocyte. The fusion rates differed, but not significantly, in the MP‐GV, 3T3‐GV, and 3T3‐MII groups (77, 63, 56%, respectively). Then, meiotic competence was compared in MP‐GV, 3T3‐GV and non‐manipulated GV stage oocytes as a control. Nuclear envelope breakdown occurred in all the reconstructed oocytes, as well as the control ones. The percentage of first polar body extrusion differed between the MP‐GV (100%), 3T3‐GV (72%), and control (67%) groups. DNA staining with Hoechst 33342 revealed that in the MP‐GV‐group oocytes that had reached MII stage, the chromosomes were condensed and aligned in a regular array similar to the normal metaphase plate. By contrast, in 3T3‐GV group oocytes, the condensed chromosomes were irregularly scattered in the cytoplasm. These results suggest that the donor nucleus affects meiotic competence in reconstructed oocytes.     

猪胸膜肺炎放线杆菌外膜蛋白(OMP)5′末端保守区基因的克隆、表达及其免疫活性研究

以猪胸膜肺炎放线杆菌血清7型25-4株基因组DNA为模板,PCR方法扩增外膜蛋白(OMP)5′末端保守区基因片段(OMPc),酶切及核苷酸序列分析鉴定后,与原核表达载体质粒pGEX-6P-1进行连接,构建成重组表达载体pGEX-OM-Pc,转入大肠杆菌BL21中,以IPTG进行诱导,SDS-PAGE电泳分析发现,转化了重组质粒的菌株所表达的融合蛋白相对分子量为34 kD,与实际预测相符,命名为GST-OMPc.GST亲和层析柱进行纯化,ELISA方法对纯化蛋白进行检测.结果表明:纯化蛋白GST-OMPc能够与兔抗猪胸膜肺炎放线杆菌血清7型的阳性血清反应.OMPc蛋白的成功表达为其功能的研究打下基础.