Protective effect of retinoic acid on interleukin-1 beta-induced cytotoxicity of pancreatic beta-cells

Cytokines produced by immune cells in pancreatic islets infiltrating are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of retinoic acid (RA) on cytokine-induced beta-cell dysfunction were examined. RA significantly protected interleukin-1 beta (IL-1) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of rat insulinoma cell (RINm5F), and also reduced in IL-1 and IFN-gamma-induced nitric oxide (NO) production, which correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism, by which RA inhibited iNOS gene expression, appeared to involve the inhibition of NF-kappa B activation. Our results suggest possible therapeutic value of RA for the prevention of diabetes mellitus progression.     

缺氧诱导因子-1在缺氧预处理心肌保护中的作用及其机制研究

目的:研究缺氧诱导因子-1(HIF-1)在缺氧预处理(HPC)心肌细胞保护中的作用及其机制。方法:在培养的SD乳鼠心肌细胞缺氧/复氧(H/R)模型上,观察HPC对于24h后心肌细胞H/R损伤的影响,以MTT法测定心肌细胞存活率,试剂盒测定培养液中乳酸脱氢酶(LDH)活性。制备心肌细胞蛋白提取物,以磷酸化的细胞外信号调节激酶(ERK1/2)抗体测定HPC后不同时间ERK1/2活性,以聚丙烯酰胺电泳迁移实验观察HIF-1α磷酸化,并观察蛋白磷酸酶激动剂BDM和ERKs的上游激酶(MEK1/2)抑制剂PD98059对于HPC诱导的HIF-1α磷酸化以及心肌细胞保护作用的影响。结果:HPC可以提高心肌细胞H/R后存活率、减少LDH漏出,并激活ERK1/2,使HIF-1α发生磷酸化;蛋白磷酸酶激动剂BDM和ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的HIF-1α磷酸化和心肌细胞保护作用。结论:HPC可以提高乳鼠心肌细胞对于H/R的耐受性,其机制涉及ERKs介导的HIF-1α磷酸化。     

Characterization of a galactose-1-phosphate uridylyltransferase gene from the marine red alga Gracilaria gracilis

The metabolism of D-galactose is a major feature of red-algal physiology. We have cloned and sequenced a gene from the red alga Gracilaria gracilis that encodes a key enzyme of D-galactose metabolism, galactose-1-phosphate uridylyltransferase (GALT). This gene, designated GgGALT1, is apparently devoid of introns. A potential TATA box, four potential CAAT boxes, and a repeated sequence occur in the 5′-flanking region. The predicted 369-aa peptide shares significant sequence similarity with GALTs from other organisms (human, 47%; Saccharomyces cerevisiae, 49%; Solanum tuberosum, 49%). Southern-hybridization analysis reveals two related, but apparently not identical, GALT genes in the nuclear genome of G. gracilis. Sequence analysis indicates that the GgGALT1 enzyme lacks a rubredoxin “knuckle” motif, which in bacterial and fungal GALTs is involved in binding zinc. An open reading frame encoding a potential peptidyl tRNA hydrolase occurs 179 bp downstream from the GgGALT1 gene. Received: 6 April / 2 June 1998     

胰岛素样生长因子-1对骨骼肌源性干细胞的促增殖效应

目的 观察胰岛素样生长因子-1(IGF-1)对骨骼肌源性干细胞(MDSCs)生长的影响.方法 采用连续预贴壁法从新生小鼠后肢肌分离培养MDSCs;用含2%胎牛血清的DMEM培养基促进其向骨骼肌细胞分化.免疫细胞化学SP法检测于细胞标志Sca-1和骨骼肌细胞标志肌节(α-sarcomeric)肌动蛋白的表达情况;采用四甲基偶氮唑盐(MTT)比色法检测IGF-1对MDSCs增殖的影响,并分析IGF-1效应与培养时间以及与IGF-1浓度之间的关系.结果 从新生小鼠后肢肌成功分离培养MDSCs,90%以上的MDSCs呈Sca-1阳性;在分化培养中MDSCs能够产生α-sarcomeric 肌动蛋白阳性的肌管;IGF-1对MDSCs促增殖作用随细胞培养时间的延长逐渐明显;随IGF-1浓度的增加而增加,并逐渐趋于饱和.结论 IGF-1对体外培养的MDSCs有促进增殖的作用.     

Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

Previous results indicated that the herpes simplex virus 1 (HSV-1) U(L)31 gene is necessary and sufficient for localization of the U(L)34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U(L)31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U(L)31 gene. The replication of the U(L)31 deletion virus was restored on U(L)31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U(L)34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U(L)34 protein localized at the nuclear membrane in rabbit skin cells, and U(L)31 complementing CV1 cells infected with the U(L)31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U(L)34 protein to the nuclear membrane. We speculate that this function partially complements that of U(L)31 and may explain why U(L)31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.     

编码N端截短的IκBα突变体重组腺病毒的构建与鉴定

目的：通过去除N端丝氨酸32/36磷酸化位点，获得人胎盘组织IκBα突变体（IκBαM）基因，构建其复制缺陷型重组腺病毒（AdIκBαM），并进行体外表达和活性检测。方法:PCR定点克隆IκBαM基因（203-1 003 bp），亚克隆至pShuttle和pGEM-T，进行PCR、双酶切、DNA测序和同源性分析。将重组质粒pShuttle-IκBαM中含CMV启动子、IκBαM cDNA和PolyA信号的表达单元定向插入Ad5腺病毒载体，构建成重组腺病毒AdIκBαM，再经脂质体介导共转染293细胞进行包装。Western blotting检测AdIκBαM在293细胞中蛋白表达情况，电泳迁移率实验观察AdIκBαM抑制佛波酯诱导的ECV304细胞核因子κB（NF-κB）激活的作用。结果:成功克隆长801 bp的新型IκBαM基因，与GenBank中登陆的IκBα基因（接受号M69043）相应核苷酸序列一致。所制备的AdIκBαM滴度为4.0×1012 pfu/L。AdIκBαM介导IκBαM基因在293细胞中表达，并以剂量依赖性方式显著抑制佛波酯诱导的ECV304细胞NF-κB活化 。结论：AdIκBαM有效介导IκBαM基因表达并特异性抑制NF-κB活性，有望应用于哮喘的基因治疗。     

Type 1 and type 2 tumor infiltrating effector cell subpopulations in progressive breast cancer

Effector T cells fall into two subpopulations based on cytokine-secretion. Type 1 cells secrete IFN-gamma, whereas type 2 cells secrete IL-4, IL-10, and GM-CSF. NKT cells represent a third subpopulation that secretes similar cytokines and have been associated with immunoregulation. Using the TS/A adenocarcinoma, we assessed the phenotype and kinetics of tumor-infiltrating lymphocytes (TIL) in mice challenged subcutaneously in the mammary region. Flow cytometric analysis shows that T cells do not infiltrate the primary tumor site until days 7-14 following tumor challenge. Both CD4 and CD8 TILs were predominantly CD44(High) and expressed CD25, CD69, and CD95 cell surface activation markers. Activated CD4/CD44(High) TIL numbers reached peak levels at day 21 that precipitously decreased by day 28 whereas corresponding CD8 cell numbers progressively increased, however, at lower levels and with later kinetics. Intracellular cytokine staining showed that greater numbers of IL-4-producing Th2 cells were elicited and with earlier kinetics than that of IFN-gamma-producing Th1 cells. T cells co-expressing DX5 (CD3(+)/DX5(+)) emerged (>21 days), suggesting a recruitment of NK-like T cells at later stages of tumor progression. Moreover, tumors selectively up-regulated TGF-beta, MIF, and IP-10 gene expression at times as early as day 4, with peak levels at day 7 in vivo. Such gene expression remained elevated and correlated with a continued progression in tumor growth suggesting that preferential effector cell recruitment and production of select factors during different stages of tumor maturation may aid in regulating effective endogenous antitumor responses in progressive breast cancer.     

针刺对多囊卵巢综合征大鼠卵巢TGF-α、EGFR表达的影响

目的:研究针刺对多囊卵巢综合征(PCOS)大鼠卵巢转化生长因子α(TGF-α)、表皮生长因子受体(EGFR)表达的影响,探讨针刺促排卵的作用机制。方法:24日龄雌性大鼠颈背部皮下注射脱氢表雄酮(DHEA)的油溶液制作(PCOS)模型,对照组同期皮下注射油剂。PCOS大鼠随机分为模型组和针刺组。模型组不作处理,针刺组大鼠从80日龄起针刺关元、中极、双侧三阴交、双侧子宫穴,1次/天,15min/次,连续6周。治疗结束后各组大鼠断头处死,迅速取血并分离血清,-20℃冰箱保存,待测性激素水平。摘取双侧卵巢,称重,4%多聚甲醛固定,作HE染色和免疫组织化学染色。结果:与模型组相比,针刺组卵巢湿重、卵巢TGF-α、EGFR表达及血清睾酮(T)、雌二醇(E2)水平均显著降低(P<0.01),而卵泡刺激素(FSH)、黄体生成素(LH)、孕酮(P4)水平差异无统计学意义(P>0.05)。结论:针刺能显著降低PCOS大鼠卵巢TGF-α及EGFR的表达,抑制TGF-α对卵巢和激素合成的作用,改善PCOS大鼠多囊样变和高雄激素血症,促进排卵。     

Pentoxifylline attenuates the development of hyperalgesia in a rat model of neuropathic pain

Pentoxifylline, a non-specific cytokine inhibitor, has shown to be beneficial in inflammatory pain in both experimental and clinical studies. The present study demonstrates for the first time, to our knowledge, the antihyperalgesic effect of pentoxifylline in the neuropathic pain using L5 spinal nerve transection rat model. In a preventive paradigm, pentoxifylline (12.5, 25, 50, or 100 mg/kg intraperitoneally) was administered systemically daily, beginning 1 h prior to nerve transection. Pentoxifylline (50, or 100 mg/kg i.p.) produced significant decrease in the mechanical and thermal hyperalgesia. However, pentoxifylline (100 mg/kg i.p.) did not influence the paw pressure thresholds and paw withdrawal latency in sham-operated rats. In order to understand the possible antinocicieptive effect of pentoxifylline in neuropathic pain, we examined the level of TNFα, IL-1β, IL-6 and IL-10 protein in the contralateral brain on day 7 post-transection. Pentoxifylline administration resulted in a dose-dependent reduction of the production of proinflammatory cytokines like TNFα, IL-1β and IL-6, and enhancement of IL-10. Furthermore, we investigated the activity of nuclear factor kappa B (NF-κB) in the contralateral brain on days 7 after surgery. In accordance with the change of proinflammatory cytokines, Pentoxifylline (50 or 100 mg/kg) significantly inhibited the activation of NF-κB in the brain. This research supports a growing body of literature emphasizing the importance of neuroinflammation and neuroimmune activation in the development of neuropathic pain states, and the potential preventive value of pentoxifylline in the treatment of neuropathic pain.