Activation of renal tubular epithelial cells by monocytes/macrophages and the relative mechanisms

AIM: To observe the direct effects of peripheral blood monocytes/macrophages (MO/MAC) on renal tubular epithelial cells (RTEC),and further probe into the possible mechanisms. METHODS: Conditioned medium(M-CM) of human peripheral blood MO/MAC was collected and added to HK-2,a human renal proximal tubular cell line.After incubation with M-CM for 24 hours,HK-2 cells were detected for DNA synthesis by [³H]-TdR incorporation,osteopontin (OPN) and α-smooth muscle actin (α-SMA) expression by Western blot,and fibronectin(FN) secretion by ELISA.Furthermore,anti-TGFβ₁ neutralizing antibody and interlukin-10(IL-10) were used separately to antagonize the effects of M-CM on HK-2 cells. RESULTS: ①DNA synthesis,α-SMA expression and FN secretion were all increased in HK-2 cells when incubated with M-CM.②When adding with anti-TGFβ₁ neutralizing antibody (5 mg/L) in the M-CM,the degree of upregulation of α-SMA and FN in HK-2 cells was much lower than that stimulated by M-CM alone.③M-CM added with IL-10 (20 μg/L) had a weaker ability to induce the increasing in α-SMA expression and FN excretion in HK-2 cells, compared with M-CM itself alone.M-CM from MO/MAC preincubated with IL-10 caused a lower upregulation of α-SMA expression in HK-2 cells than M-CM from non-preincubated MO/MAC. CONCLUSION: MO/MAC can directly induce proliferation,transdifferentiation and extracellular matrix secretion in RTEC.TGFβ₁ and proinflammatory cytokines secreted by MO/MAC might be involved in the aboveeffects.     

蛹虫草CmKexin基因克隆和菌丝体中表达检测

为了探讨蛹虫草类枯草杆菌蛋白酶(Subtilisin-like protease)的表达特性,以真菌蛹虫草菌丝体为研究对象,通过RT-PCR获得蛹虫草CmKexin基因的ORF序列,并进行序列分析。应用Northern杂交和Real-time PCR方法,检测蓝光照射后蛹虫草菌丝体中CmKexin基因的转录情况。结果获得蛹虫草CmKexin基因的ORF序列。对翻译蛋白质产物CmKexin进行生物信息学分析,表明该蛋白质含1个属蛋白转化酶的肽酶S8家族功能域(143~429)和1个前体蛋白转化酶P功能域(514~600),符合真菌类枯草杆菌蛋白酶的特征。蛹虫草菌丝体在黑暗预培养4 d后再蓝光照射,Northern Blotting和Real-time PCR检测均可得到相同的结果,即在持续的蓝光照射48~50 h时,出现CmKexin基因的瞬时大量转录。而其他时间内均未检测到该基因的大量转录。试验结果将为蛹虫草类枯草杆菌蛋白酶的利用提供理论依据。     

副溶血弧菌的外膜蛋白及其抗原性研究↑（*）

本文对１３株不同来源的副溶血弧菌的外膜蛋白及其抗原性进行了比较研究。１３株副溶血弧菌的外膜蛋白有相似的ＳＤＳ－ＰＡＧＥ图谱。大多数菌株有五条主要的蛋白质，其大致分子量分别为：ａ、６９ｋＤａ；ｂ、６３ｋＤａ；ｃ、４０ｋＤａ；ｄ、３０ｋＤａ；ｅ、２８ｋＤａ。其中蛋白质ｂ是所有菌株共有的。与吸附后的兔抗副溶血弧菌血清Ｗｅｓｔｅｒｎ印迹法结果显示抗血清与多数副溶血弧菌菌株的外膜蛋白有一条共同的免疫反应带，其大致分子量为４４ｋＤａ。     

Activation of JNK/SAPK pathway in vero cells induced by N-methyl-N'-nitro-N-nitrosoguanidine

AIM and METHODES: To evaluate the possible signal transduction mechanism of nontargeted mutagenesis in vero cells induced by DNA damaging agent N-methyl-N'-nitro-N-nitrosoguanidine(MNNG),the activation of c-Jun NH₂-terminal kinase/stress activated protein kinase(SAPK/JNK) pathway in vero cells induced by MNNG was studied. Western Blot analysis and Solid-phase kinase assay were used to measure the phosphorylation of JNK1 and kinase activity of JNKs, respectively. RESULTS: After 0.2 μmol/L, 2.5 h MNNG or 1 mg/L, 1 h cycloheximide (CHM) treatment, the proportion of phosphorylated JNK1 in cell extract increased significantly, simultaneously the kinase activity of JNKs increased dramatically(6.7 and 3.0 folds respectively), as measured by the phosphorylation of c-Jun, a substrate of JNKs. CONCLUSION: Both 0.2 μmol/L 2.5 h MNNG and 1 mg/L 1 h CHM treatment can induce the activation of JNK/SAPK pathway, one of the stress signal transduction pathways, in vero cells.     

Expression and activation of NF-κB and carcinogenesis of cervical cancer

AIM:To evaluate the significance of NF-κB p65 protein expression in the development of human cervical squmous cell cancer.METHODS:Immunohistochemical analysis was done in 125 casas of paraffin-embedded cervical tissue specimens of different histological grades (32 LSILs,33 HSILs,38 SCCs and 22 normal) to evaluate the expression of RelA.Western blotting was used to analyze the level of NF-κB p65 protein.RESULTS:① By using immunohistochemical analysis,RelA was mainly localized in cytosol in normal cervical tissue and low-grade squamous intraepithelial lesions,whereas in high-grade lesions and squamous cell carcinomas,RelA translocated into the nucleus.② By Western blotting analysis,RelA was detected in the cytosolic extracts in normal or LSILs.In cancer tissues,the expression of RelA increased in nuclear extracts while their expression in the cytosolic extracts was relatively less.CONCLUSIONS:Constitutive activation of NF-κB p65 may lead to oncogenesis.NF-κB p65 may be a new target for the treatment of human cervical squmous cancer.     

Effects of carbon monoxide on hypoxia inducible factor-1 expression in hypoxia preconditioned mice

AIM:To study the effect of carbon monoxide (CO) on hypoxia inducible factor-1α(HIF-1α) expression and on hypoxic tolerance duration in hypoxia preconditioned mice. METHODS:Mice were injected with normal saline (NS) or hemin intraperitoneally. Western blot was used to detect HIF-1α in hippocampus. The tolerant duration of each hypoxic run was recorded. RESULTS:After 1 and 2 runs of hypoxia exposure, there were no significant differences in HIF-1α expression and hypoxic tolerance duration between N and hemin injection groups. After 3 and 4 runs of hypoxia exposure, the content of HIF-1α was lower and hypoxic tolerance duration was shorter (P＜0.05) in hemin injection groups than that in NS injection groups. CONCLUSION:CO could decrease the expression of HIF-1α, and decrease hypoxic tolerance duration in hypoxia preconditioned mice. The mechanism may be that CO binds to hemoprotein and inhibits hemoprotein oxygen sensor.     

小鼠骨形成蛋白BMP3的原核表达及多克隆抗体制备与鉴定

为构建小鼠BMP3蛋白原核表达载体,纯化BMP3蛋白,制备并鉴定BMP3蛋白小鼠多克隆抗体,以及对BMP3进行系统进化关系研究。采用RT-PCR方法从小鼠肝脏中克隆获得BMP3蛋白的表达菌株;利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得BMP3蛋白,免疫小鼠制备BMP3蛋白多克隆抗体;采用ELISA法检测抗体滴度达1∶3 200倍,Western Blotting证实抗血清具有很好的特异性。通过DNAMAN软件对BMP3序列同源性分析发现其C-端在不同动物间存在较高同源性;MEGA软件对BMP3序列的系统发生分析证实BMP3在动物间具有明显的进化趋势。为BMP3蛋白调控骨代谢及与肿瘤的关系研究奠定基础。     

Study on chlamydia pneumoniae infection in the patients with spontaneous brain bleeding

AIM: To study the relationship between Chlamydia pneumoniae (Cpn) and spontaneous brain bleeding. METHODS: The IgG and IgM antibody titers to Cpn in peripheral blood from 9 patients of spontaneous brain bleeding were detected by microimmunofluorescence antibody technique (MIF). The Cpn DNA of arteries in brain bleeding region from these cases was detected by polymerase chain reaction (PCR). The Cpn major outer membrane protein (MOMP) expression of arteries in brain bleeding region from these cases were tested by Western blot. RESULTS: All the geometric average titer to IgG antibody (190.3±3.2) and the positive rate of IgG antibody (85.2%) and IgM antibody (30.8%) were significantly higher in spontaneous brain bleeding patients than those (48.1±2.0, 48.1% and 4.8%) in control (P<0.01,P<0.05,P<0.01). The positive rate of the Cpn DNA was 88.9%(8/9) and the Cpn MOMP expression was found in the arteries of brain bleeding region from all 9 cases. CONCLUSION: Spontaneous brain bleeding may be associated with the chlamydia pneumoniae infection.     

鸡毒支原体株间结构蛋白及其抗原性变异的比较研究

本研究以ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ Ｂｌｏｔ技术,应用鸡毒支原体国外强毒株Ｓ６、标准株ＰＧ３１,疫苗株Ｆ,北京分离 ＢＧ４４Ｔ、ＮＢ７２特异性多克隆抗血清对以上五株及疫苗株Ｖ、北京分离株Ｃ的结构及其抗原性进行了比较结果表明ＭＧ结构蛋白及其抗原性存在着株间的多样性和一定相似性,其中以Ｆ与Ｓ６、ＢＧ４４Ｔ与ＰＧ３１、ＮＢ７２与Ｃ更为接近,可能具有同源性。ＳＤＳ－ＰＡＧＥ显示出了ＭＧ株间结构蛋白微     

Influence of lanthanum nitrate on the expression of aquaporin 7 in the testis of rats

AIM: To find out whether different dosage of rare earth element-lanthanum can influence the expression of aquaporin 7(AQP 7) in the testis of rats. METHODS:Rats were fed with lanthanum nitrate[La(NO₃)₃]and killed 6 months later.Testes were then removed immediately to extract total RNA.Northern blot analysis is performed finally. RESULTS:0.1 mg/kg La(NO₃)₃ depressed the expression of AQP 7 in rat testis, while 20 mg/kg La(NO₃)₃ had no significant effect on it. CONCLUSION: AQP 7 expession is found in the rat testis; La(NO₃)₃ can depress the expression of AQP 7 in the rat testis.