A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-³H]-2′-deoxyuridine or [5-³H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-³H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.     

Smurf2 is a TRAF2 binding protein that triggers TNF-R2 ubiquitination and TNF-R2-induced JNK activation

TRAF2 plays a central role in TNF-induced signalling to NF-κB and JNK/p38 MAPK. To better understand the molecular mechanisms that mediate this dual function of TRAF2, we performed a yeast two-hybrid screening for TRAF2 interacting proteins using the Sos recruitment system. This resulted in the identification of the E3 ubiquitin ligase Smurf2 as a TRAF2 binding protein. TRAF2 overexpression was shown to trigger Smurf2 ubiquitination and the formation of a TNF-R2/Smurf2 complex. Smurf2 on its turn promoted TNF-R2 ubiquitination and the relocalization of TNF-R2 as well as TRAF2 to a detergent-insoluble cell fraction. This was associated with enhanced TNF-R2-induced JNK activation, whereas TNF-R2-induced NF-κB activation remained unaffected. These results suggest an important role for Smurf2 binding to TRAF2 in determining specific signalling outputs of TNF-R2.     

人乳头瘤病毒2(HPV-2)变异株的突变E2蛋白转录抑制作用的研究

皮肤寻常疣的发生与多种基因型别HPV的感染密切相关.本研究利用PCR方法对1例临床罕见的寻常疣患者感染的HPV-2毒株LCR及E2基因序列进行扩增、测序,分别构建含HPV-2变异株及原毒株LCR的重组CAT基因报导质粒pBLCAT-LCR和表达突变及野生型E2蛋白的重组真核表达质粒pcDNA3.1-E2,通过瞬时转染HeLa细胞,研究变异株启动子活性及突变E2蛋白的转录抑制作用.结果显示,患者感染的HPV-2变异株LCR及E2基因均存在多处点突变.变异株早期启动子活性明显高于原毒株;突变的E2蛋白转录抑制作用较野生型E2蛋白显著降低;变异株LCR上E2结合位点核苷酸的突变明显降低E2蛋白对病毒早期启动子的抑制作用.提示HPV-2变异株启动子活性增强及突变E2蛋白转录抑制作用的降低与这一罕见巨大寻常疣临床表型之间存在着重要的联系.     

过氧化氢和氯化钙对香蕉幼苗抗寒性的影响

用H₂O₂和CaCl₂单独或混合使用的方法喷洒香蕉幼苗,并置于低温培养箱中进行冷胁迫处理,发现它们可提高香蕉幼苗冷胁迫期间叶片POD活性,降低细胞质泄漏,增加可溶性糖含量及减缓叶绿素降解,从而减轻冷伤害程度。H₂O₂和CaCl₂混合处理的效果优于单独处理,二者有协同效应。     

PPP2R2A在乳腺癌细胞中结合GFPT2并导致其去磷酸化

PPP2R2A是PP2A磷酸酶的调控亚基之一,以往的研究报道显示,PPP2R2A可促进肿瘤细胞生存和生长。本研究通过串联亲和纯化联合HPLC-Chip-ESI/MS/MS筛选PPP2R2A的相互作用蛋白质,分析结果显示,L-谷氨酰胺-D-果糖-6-磷酸转氨酶1(Glutamine-fructose-6-phosphate transaminase 1,GFPT1)和L-谷氨酰胺-D-果糖-6-磷酸转氨酶2(Glutamine-fructose-6-phosphate transaminase 2,GFPT2)是PPP2R2A可能的结合蛋白。通过免疫荧光共定位、GST Pull-down和免疫共沉淀等方法,进一步确认了PPP2R2A和GFPT1及GFPT2的相互结合。通过shRNA下调PPP2R2A后,GFPT2的磷酸化水平显著增加,但GFPT1的磷酸化水平改变不明显。GFPT2是O-GlcNAC糖基化修饰通路中的一个限速酶,在乳腺癌细胞MDA-MB-231中下调PPP2R2A后,蛋白质O-GlcNAC糖基化修饰水平增加。这些结果表明,PPP2R2A可直接结合GFPT2,并导致其去磷酸化,进而影响细胞内O-GlcNAC糖基化修饰。     

TXA2/PGI2与心血管疾病

血栓素(Thromboxane,TXA2)和前列环素(Prostacyclin,PGI2)均为花生四烯酸的代谢物,是前列腺素(Prostaglandins,PGs)中生物活性最强的一对。在正常情况下,二者在体内保持一定的平衡,相互拮抗、相互协调,共同维持血液循环畅通,与心血管疾病关系密切。本文即就其生物特性及与心血管病的关系等进行综述,对人们全面认识TXA2/PGI2具有一定的参考价值。     

Adaptor Protein2 (AP2) Orchestrates CXCR2‐Mediated Cell Migration

The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, β2, μ2 and σ2 subunits, facilitates clathrin‐mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl‐terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand‐mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand‐mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2‐μ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, μ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2‐μ2, is replaced with μ2 mutated in Patch 1 and/or Patch 2 domains, ligand‐mediated receptor binding and internalization are not lost. However, chemotaxis requires AP2‐μ2 Patch 1, but not Patch 2. AP2‐σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2‐σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2‐mediated receptor internalization can be dissociated from AP2‐mediated chemotaxis.      

不同污染负荷土壤中镉和铅的吸附－解吸行为

采用一次平衡法,对3种不同污染负荷土壤Cd2 和Pb2 的吸附-解吸行为进行了比较.结果表明,低污染负荷土壤对Cd2 和Pb2 的吸附能力高于高污染负荷土壤.3种土壤对Cd2 的吸附等温线与Freundlich方程有较好的拟合性,Pb2 的等温吸附过程可用Langmuir方程与Freundlich方程来描述.双常数方程是描述这3种不同污染负荷土壤中Cd2 和Pb2 吸附动力学行为的最优模型,其次为Elovich方程,最差模型是一级动力学方程.Pb2 的解吸滞后现象较Cd2 明显.高污染负荷土壤的吸附态Cd2 、Pb2 解吸率高于低污染负荷土壤,Cd2 、Pb2 解吸量与其初始吸附量之间的关系符合二次幂方程.3种土壤Cd2 、Pb2 的解吸速率随重金属初始浓度的增加而增加,随解吸时间的延长而降低.     

不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应*

目的:探讨不同氧浓度下小鼠骨骼肌卫星细胞系(C2C12细胞)对H₂O₂刺激反应的变化及其机制。方法:小鼠骨骼肌卫星细胞系(C2C12细胞),经培养复苏后,将细胞分为7组,每组设8个复孔,各组分别加入浓度为0.1 mmol/L、0.25 mmol/L、0.5 mmol/L、0.75 mmol/L、1 mmol/L、2 mmol/L的H₂O₂,分别作用1 h、2 h后测细胞活力,选择细胞H₂O₂刺激的最佳作用时间和浓度;C2C12细胞分为不同氧浓度组:21% O₂、12% O₂、8% O₂、5% O₂每组设8个复孔,12 h后,H₂O₂作用1 h,收集细胞;检测细胞Nrf2蛋白荧光和蛋白表达量,测定Nrf2和抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1 的mRNA表达量及细胞ROS水平。结果:选择H₂O₂作用时间相对较短的1 h和浓度0.5 mmol/L作为本实验的H₂O₂刺激条件。与21%O₂组相比,12%O₂组细胞Nrf2蛋白荧光增强,Nrf2 的mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的 mRNA表达均显著增加(P＜0.05或P＜0.01),细胞 ROS水平明显降低(P＜0.01);8%O₂组仅GPX-1 mRNA显著增加(P＜0.05),其他指标变化不大;5%O₂组细胞 Nrf2 mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、NQO-1、GPX-1的 mRNA表达均明显降低(P＜0.05或P＜0.01),细胞 ROS水平则明显升高(P＜0.01)。结论:不同氧浓度下C2C12细胞中Nrf2介导的抗氧化系统对H₂O₂刺激反应不同,12 h的12% O₂浓度可促进C2C12细胞Nrf2的抗氧化作用,而5% O₂浓度的严重低氧则作用相反。     

SNT-2 interacts with ERK2 and negatively regulates ERK2 signaling in response to EGF stimulation

The control of cellular responses with fibroblast growth factors and neurotrophins is mediated through membrane-linked docking proteins, SNT (suc1-binding neurotrophic target)-1/FRS2alpha and SNT-2/FRS2beta. ERK1/2 are members of the mitogen-activated protein kinase family that regulate diverse cellular activities in response to various stimuli. Here, we demonstrate that SNT-2 does not become tyrosine phosphorylated significantly in response to EGF but forms a complex with ERK2 via the region of 186-252 amino acid residues, and the complex formation is enhanced upon EGF stimulation. SNT-2 downregulates ERK2 phosphorylation, suppresses and delays ERK2 nuclear accumulation which occurs following EGF stimulation. In contrast, the mutant SNT-2 which carries deletion of 186-252 amino acids and lacks ERK2 binding does not have these effects. These observations suggest that SNT-2 negatively regulates ERK2 signaling activated via EGF stimulation through direct binding to ERK2.     

ERK1／2信号通路对黄芪苷Ⅳ抗H2O2诱导H9c2细胞氧化损伤的作用

目的：研究黄芪苷Ⅳ（AST）是否通过细胞外信号调节激酶1／2（ERK1／2）通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。方法：用200μmoL／L的H2O2处理细胞6h,采用MTT法检测细胞存活率,建立H2O2诱导的H9c2细胞氧化损伤模型;比色法测定细胞培养液中乳酸脱氢酶（LDH）活性、总超氧化物歧化酶（T—SOD）和锰超氧化物歧化酶（Mn—SOD）活力以及丙二醛（MDA）含量;Western blot检测H9c2细胞ERK1／2蛋白的磷酸化水平。结果：在H2O2浓度为200μmol／L作用6h条件下,细胞存活率降低程度适中,实验结果重复性好,确定后续实验采用200μmol／L H2O2作用6h建立模型。与H2O2组比较,10mg／L及20mg／L AST均显著提高细胞存活率（P〈0．01）,使细胞培养液中LDH活性显著降低（P〈0．01）,T—SOD及Mn—SOD活力显著提高（P〈0．01）,MDA含量显著降低（P〈0．01）。10mg/L及20mg/L AST均显著增加H2O2损伤的H9c2细胞p—ERK1／2蛋白的表达（P〈0．01）,当用PD98059（ERK1／2的抑制剂）预处理后,AST的作用则被取消。结论：黄芪苷Ⅳ可以通过ERK1／2通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。     

Protein kinase CK2 promotes cancer cell viability via up-regulation of cyclooxygenase-2 expression and enhanced prostaglandin E2 production

Augmented expression of protein kinase CK2 is associated with hyperproliferation and resistance to apoptosis in cancer cells. Effects of CK2 are at least partially linked to signaling via the Wnt/β-catenin pathway, which is dramatically enhanced in colon cancer. Cyclooxygenase-2 (COX-2), a Wnt/β-catenin target gene, has been associated with enhanced cancer progression and metastasis. However, the possibility that a connection may exist between CK2 and COX-2 has not been explored previously. Here we investigated changes in COX-2 expression and activity upon CK2 modulation and evaluated how these changes affected cell viability. COX-2 expression and cell viability decreased upon selective inhibition of COX-2 with SC-791 or CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), both in human colon (HT29-ATCC, HT29-US, DLD-1) and breast (ZR-75) cancer cells, as well as in human embryonic kidney (HEK-293T) cells. On the other hand, ectopic CK2α expression promoted up-regulation of COX-2 by activating the Wnt/β-catenin pathway in HEK-293T cells. Noteworthy, over-expression of either CK2α, β-catenin or COX-2, as well as supplementation of the medium with prostaglandin E2 (PGE2), all were individually sufficient to overcome limitations in cell viability triggered by CK2 inhibition either upon addition of DMAT or over-expression of a dominant negative CK2α variant. Altogether, these findings provide new insight to the role of CK2 in cancer by up-regulating COX-2 expression and thereby PGE2 production.     

ABA诱导玉米叶质外体H2O2积累的机制

通过组织化学染色和电镜观察并结合酶活性分析表明,ABA可通过诱导玉米（Zea mays L、）叶片质膜NADPH氧化酶、细胞壁POD及质外体PAO活性的升高,使其质外体产生H2O2;其中质膜NADPH氧化酶起主要作用。     

Degradation of 2-bromo-, 2-chloro- and 2-fluorobenzoate by Pseudomonas putida CLB 250

Abstract Pseudomonas putida strain CLB 250 (DSM 5232) utilized 2-bromo-, 2-chloro- and 2-fluorobenzoate as sole source of carbon and energy. Degradation is suggested to be initiated by a dioxygenase liberating halide in the first catabolic step. After decarboxylation and rearomatization catechol is produced as a central metabolite which is degraded via the ortho-pathway. After inhibition of ring cleavage activities with 3-chlorocatechol, 2-chlorobenzoate was transformed to catechol in nearly stoichiometric amounts. Other ortho -substituted benzoates like anthranilate and 2-methoxybenzoate seem to be metabolized via the same route.