有氧运动上调Agtr1a基因甲基化改善高血压肠系膜动脉ACE1-AT1R收缩轴功能

血管紧张素Ⅱ 1A受体(angiotensin Ⅱ type 1A receptor, AT1aR)是Ang Ⅱ的主要受体亚型。AT1aR基因(Agtr1a)启动子区DNA甲基化水平的变化是调控AT1aR表观遗传的重要机制。为明确运动是否通过调节Agtr1a基因启动子区甲基化水平而减弱ACE1-AT1R收缩轴功能,从而起到改善高血压血管功能的作用,本研究选用3月龄自发性高血压大鼠(spontaneously hypertensive rat, SHR)和正常血压对照组大鼠(Wistar-Kyoto, WKY),随机分为正常血压安静组WKY-C、正常血压有氧运动组WKY-E、高血压安静组SHR-C、高血压有氧运动组SHR-E,各组n=24。12周跑台运动结束后,有氧运动显著减低运动组大鼠血压和体重(P0.05);采用微血管环张力测定技术测定肠系膜动脉对去甲肾上腺素(norepinephrine, NE)、血管紧张素Ⅱ(angiotensin Ⅱ, Ang Ⅱ)的反应性。结果显示,有氧运动显著减弱高血压大鼠肠系膜动脉对血管收缩因子NE、AngⅡ的收缩反应(P0.05);高效液相色谱法(high performance liquid chromatography, HPLC)测定血浆中ACE1-AT1R收缩轴主要活性肽血管紧张素原(angiotensinogen, AGT)、AngⅡ的水平。结果显示,有氧运动显著减弱高血压大鼠肠系膜动脉对血管收缩因子NE、AngⅡ的收缩反应(P0.05);免疫印迹法和q-PCR技术测定肠系膜动脉ACE1、AT1R蛋白质和AT1aR的mRNA水平相对含量。结果显示,有氧运动显著降低高血压大鼠肠系膜动脉ACE1、AT1R蛋白质和AT1aR mRNA水平(P0.05);亚硫酸氢盐测序BSP法测定Agtr1a基因的启动子区甲基化水平。结果显示,有氧运动显著上调高血压大鼠肠系膜动脉Agtr1a基因启动子区甲基化水平(P0.05)。本研究表明,有氧运动通过上调高血压肠系膜动脉Agtr1a基因启动子区甲基化水平,即而减弱RAS系统ACE1-AT1R收缩轴功能,从而抑制高血压血管张力增高,缓解血压增高。     

6aR,12aR,12a-羟基紫穗槐苷元上调Bim诱导肝癌细胞SMMC-7721发生凋亡的机制

肝细胞癌(HCC)是一种高度恶性的肿瘤。化疗是临床上一种重要的治疗方法,但由于化疗药物的毒副作用和耐药效应,现有的化疗药物或多或少有一定的局限性。因此,寻找毒副作用小且有效的化疗药物一直是肝癌患者的迫切需求。本研究从紫穗槐种子中分离纯化出一种天然产物6aR,12aR,12a-羟基紫穗槐苷元,采用CCK-8法检测了其对人肝癌SMMC-7721细胞的杀伤作用,应用流式细胞术检测了其对肝癌细胞的凋亡诱导情况,通过免疫印迹法检测了6aR,12aR,12a-羟基紫穗槐苷元上调Bim蛋白表达进而诱导肝癌细胞凋亡的作用机制。结果表明,6aR,12aR,12a-羟基紫穗槐苷元可呈浓度依赖性和时间依赖性抑制人肝癌SMMC-7721细胞的活力,24 h、48 h和72 h的半数抑制浓度(IC50)分别为21.77μmol/L、5.65μmol/L、5.0μmol/L;同时可浓度依赖性地提高细胞凋亡率;可通过下调抑凋亡蛋白Survivin、Mcl-1、Bcl-XL、Bcl-2,上调促凋亡蛋白Bak、Bim、Bax、Bid的表达,进而促进PARP和caspase-3的活化,从而诱导细胞发生凋亡;进一步地研究发现:6aR,12aR,12a-羟基紫穗槐苷元通过延长Bim的半衰期而增加Bim的积累,且具有和MG132类似的生物学功能,能够阻断蛋白的降解途径来抑制Bim降解。上述研究表明,6aR,12aR,12a-羟基紫穗槐苷元可通过抑制蛋白降解从而上调细胞中Bim蛋白的水平,进而诱导肝癌细胞SMMC-7721发生凋亡。     

Identification of serotonin 2A receptor as a novel HCV entry factor by a chemical biology strategy

Hepatitis C virus(HCV)is a leading cause of liver disease worldwide.Although several HCV protease/polymerase inhibitors were recently approved by U.S.FDA,the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistanee.Viral entry is an essential target step for antiviral development,but FDA-approved HCV entry inhibitor remains exclusive.Here we identify serotonin 2A receptor(5-HT2aR)is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy.The silencing of 5-HT2aR and clinically available 5-HT2aR antagonist suppress cell culture-derived HCV(HCVcc)in different liver cells and primary human hepatocytes at late endocytosis process.The mechanism is related to regulate the correct plasma membrane localization of claudin 1(CLDN1).Moreover,phenoxybenzamine(PBZ),an FDAapproved 5-HT2aR antagonist,inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs.The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice.Our results not only expand the understanding of HCV entry,but also present a promising target for the invention of HCV entry inhibitor.     

C3aR过度活化与糖尿病肾病肾组织损伤的关系

C3aR是补体C3裂解产物C3a的受体.最近的一些研究提示C3aR通路可能参与了糖尿病肾病(DN)的病理过程,但有关C3aR通路在DN中的确切病理作用及有关机制远未清楚.需要特别指出的是,现有的有关C3aR参与DN肾组织损伤的证据主要来自一些动物模型的研究,临床上尚缺乏较为系统全面的对DN患者肾组织C3aR通路与肾组织损伤关系的观察分析.为此,本文首次以较大的样本量分析了不同病理时期DN患者肾组织C3aR和C3a的表达变化情况及其与DN患者肾组织损伤的相关性.在此基础上,进而利用体外细胞模型,对高糖环境下C3aR活化致肾小球足细胞损伤的作用及机制进行了探讨.结果显示:a.与正常对照组相比,DN患者肾组织C3a和C3aR的表达水平随DN的进展而升高,C3aR在DN患者肾组织中的表达上调主要见于肾小管上皮细胞和肾小球足细胞;b.DN患者肾组织C3aR和C3a水平与患者肾组织损伤程度,特别是小管和小管间质损伤程度、肾小球足细胞损伤程度具有显著相关性;c.外加C3a激活C3aR可使高糖环境中的足细胞的细胞骨架发生明显改变、足细胞标记分子表达下调、足细胞通透性增加.这些结果说明:a.DN患者肾组织中确实存在C3a/C3aR轴过度活化的现象;b.C3a/C3aR轴的过度活化很可能在DN患者肾组织损伤,特别是小管和小管间质损伤、肾小球足细胞损伤中具有重要作用;c.可能通过破坏成熟足细胞特有的细胞骨架,改变足细胞标记分子表达,增加足细胞的通透性,C3a/C3aR轴过度活化参与DN足细胞损伤过程.本文不仅为C3a/C3aR通路参与DN病理过程提供了新的必不可少的临床证据,也增加了对C3a/C3aR通路过度活化致DN患者肾组织损伤机制,特别是肾小球足细胞损伤机制的了解,这对于拓展对DN病理机制的认识,发展DN防治新思路,无疑都是有益的.     

以田鼠作为动物模型来研究社会行为的进化:基因、脑与行为

田鼠属的一些近缘种间具有独特的社会行为多态性。例如Microtusochrogaster和M .pinetorum为一夫一妻制 ,而M .montanus和M .pennsylvanicus则为独居和一夫多妻制。无论是在野外还是人工饲养的条件下 ,单配制的田鼠其雌、雄成年个体一经交配即在两者之间形成长期的配偶关系并且双亲共同哺育后代。已证明神经多肽加压素 (Vasopressin)参与了田鼠单配制行为的神经调控。本篇综述了过去以及近期关于加压素调控田鼠配偶关系形成的研究结果和进展。首先 ,阐述了加压素V1a受体 (V1aR)在脑分布的种间差异 ,并以此来鉴别特定脑区在配偶关系形成中的功能 ;其次 ,探讨了运用V1aR拮抗物的药理学方法来决定究竟哪些脑区参与配偶关系的形成 ,还描述了田鼠种间V1aR基因结构和功能的不同 ,以及这些不同对V1aR在大脑的分布和行为调控潜在的作用机制 ;最后 ,讨论了最新的研究结果 ,即对一夫多妻制田鼠进行脑V1aR基因的改造 ,从而使之表现出一夫一妻制田鼠的行为。总之 ,了解复杂的社会性行为的遗传和神经机制可以加深我们对种间和种内行为分歧进化的理解     

缺氧通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR表达

C3aR是补体C3a的受体．在肾脏,已发现C3aR表达于包括肾小管上皮细胞在内的多种细胞．在特定的病理情况下,C3aR表达上调并参与多种肾脏疾病的病理过程,但有关C3aR在肾脏细胞中的表达调控机制仍不清楚．小管间质缺氧是肾脏疾病中的一种常见现象,也是一种重要致病因素．为了探讨缺氧对C3aR的表达调控作用,本文利用体外缺氧模型,对模型条件下C3aR在肾小管上皮细胞中的表达变化情况进行了分析,同时检测了HIF-1α和NF-κB的表达变化及活化情况,以及HIF-1α和NF-κB抑制剂对C3aR的表达影响情况．结果发现缺氧可诱导C3aR mRNA及蛋白质水平的表达上调、HIF-1α和NF-κB的核转移．HIF-1α和NF-κB抑制剂可阻断缺氧对C3aR的诱导作用,且HIF-1α抑制剂可抑制NF-κB的核转移．这些结果说明缺氧可通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR的表达．考虑到C3aR活化可促进肾小管的损伤,　我们推测C3aR通路可能参与了缺氧和NF-κB诱导的肾小管损伤过程,可能是防治缺氧和NF-κB诱导肾组织损伤的一个新靶标．     

过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析

利用半定量RT-PCR、免疫组化和Western blotting的方法,同时从mRNA水平和蛋白质水平对过敏毒素受体(C3aR)在不同病理阶段的2型糖尿病肾病模型小鼠——db/db小鼠肾脏中的表达情况进行了较为系统的分析．结果发现：a．在糖尿病前的db/db小鼠(4周龄的db/db小鼠),C3aR与作为正常对照的db/m小鼠相比没有明显差异．随着肥胖的加剧,高血糖、蛋白尿的发生和发展,C3aR在db/db小鼠肾脏中的表达显著升高．b．免疫组化分析显示,C3aR广泛地表达于db/m和db/db小鼠肾脏的皮质和髓质,分布于肾脏的上皮细胞中(包括肾小管上皮细胞、肾小球中的脏层上皮细胞(足细胞)和壁层上皮细胞)．从部位来看,皮髓交界处的肾小管中C3aR表达量明显要比其他部位的多．在肾小球,C3aR特异地存在于足细胞部位．在db/m小鼠,不同周龄小鼠肾脏中C3aR的表达量并没有明显变化,但在db/db小鼠,从8周龄开始,分布在db/db小鼠肾小管上皮细胞和小球足细胞中的C3aR均随小鼠周龄的增加而增加,至少在时间上,与小鼠糖尿病肾病的发生发展相关,其中尤以足细胞中和皮髓交界处肾小管上皮细胞中的变化最为明显． c．在糖尿病肾病小鼠中高表达C3aR的肾小管上皮细胞常有空泡变性的情况．上述工作印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了C3aR与糖尿病肾病的相关性,同时揭示了C3aR在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示C3aR的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制．     

缺氧通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR表达（英文）

C3aR是补体C3a的受体.在肾脏,已发现C3aR表达于包括肾小管上皮细胞在内的多种细胞.在特定的病理情况下,C3aR表达上调并参与多种肾脏疾病的病理过程,但有关C3aR在肾脏细胞中的表达调控机制仍不清楚.小管间质缺氧是肾脏疾病中的一种常见现象,也是一种重要致病因素.为了探讨缺氧对C3aR的表达调控作用,本文利用体外缺氧模型,对模型条件下C3aR在肾小管上皮细胞中的表达变化情况进行了分析,同时检测了HIF-1α和NF-κB的表达变化及活化情况,以及HIF-1α和NF-κB抑制剂对C3aR的表达影响情况.结果发现缺氧可诱导C3aR mRNA及蛋白质水平的表达上调、HIF-1α和NF-κB的核转移.HIF-1α和NF-κB抑制剂可阻断缺氧对C3aR的诱导作用,且HIF-1α抑制剂可抑制NF-κB的核转移.这些结果说明缺氧可通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR的表达.考虑到C3aR活化可促进肾小管的损伤,我们推测C3aR通路可能参与了缺氧和NF-κB诱导的肾小管损伤过程,可能是防治缺氧和NF-κB诱导肾组织损伤的一个新靶标.     

金黄色葡萄球菌CHIPS基因克隆及原核表达

趋化抑制蛋白(Chemotaxis Inhibitory Protein ofStaphylococcus aureus,CHIPS)是由金黄色葡萄球菌分泌到菌体外的一种蛋白。在感染早期,它能特异性地与中性粒细胞和单核细胞上的C5a受体(C5aR)和fMLP受体(FPR)结合,从而阻止中性粒细胞和单核细胞对C5a和fMLP的结合作用,导致对病原吞噬作用的延迟。人们可以利用CHIPS对C5a-C5aR的阻止作用来研制治疗由C5a诱发的炎症性疾病的药物。将CHIPS作为免疫原防治疾病也将成为新的研究课题。实验成功构建了CHIPS蛋白的原核表达系统,为CHIPS免疫原性研究及蛋白的其他功能研究奠定了基础。     

高表达C3aR的肥大细胞在糖尿病肾病患者肾组织中的分布及病理意义分析

选取糖尿病肾病(diabetic nephropathy,DN)早期、中期、晚期以及正常移植供肾各15例,分别作为DN早期组、中期组、晚期组和正常对照组,利用各个病例的肾穿刺组织标本进行C3aR免疫组化染色,分析各组中高表达C3aR浸润细胞的数量和分布特点,及其与DN发生、发展的关系,利用连续切片、免疫荧光双套色染色、免疫电镜、及甲苯胺蓝染色等方法对DN患者肾组织中高表达C3aR的浸润细胞进行细胞类型鉴定.结果表明:a.光镜和免疫电镜的形态学分析显示DN患者肾组织中高表达C3aR的浸润细胞在形态上具有肥大细胞的特点,免疫荧光双套色染色的分析显示,C3aR浸润细胞CD45阴性、CD68和类胰蛋白酶阳性,与肥大细胞的情况一致,甲苯胺蓝特殊染色进一步证实这是一种肥大细胞.b.正常移植供肾组织中虽有肥大细胞分布,但数量很少;从DN早期组到DN中期组,再到DN晚期组,肾组织中肥大细胞的数量有一种不断增加的趋势;DN患者肾组织肥大细胞的数量与24 h尿蛋白和血肌酐水平均具有很好的线性相关性.上述工作不仅揭示了在DN发生发展过程中肥大细胞在DN患者肾组织中的数量及分布情况,提示肥大细胞很可能参与了DN的发生发展过程,同时,DN患者肾组织肥大细胞高表达过敏毒素C3a受体C3aR的发现,提示C3a/C3aR通路很可能在DN患者肥大细胞的招募和激活过程中有重要作用.     

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.     

Synthesis and biological evaluation of 1,3-oxathiolane 5-azapyrimidine, 6-azapyrimidine, and fluorosubstituted 3-deazapyrimidine nucleosides

(2R,5S)-5-Amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,2,4-triazine-3(2H)-one (8) and (2R,5R)-5-amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2,4-tr iazine-3(2H)-one (9) have been synthesized via a multi-step procedure from 6-azauridine. (2R,5S)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,3, 5-triazine-2(1H)-one (11) and (2R,5R)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,3,5-triazine-2(1H)-one (12), and the fluorosubstituted 3-deazanucleosides (19-24) have been synthesized by the transglycosylation of (2R,5S)-1-[2-[[(tert-butyldiphenylsilyl) oxy]methyl]-1,3-oxathiolan-5-yl] cytosine (2) with silylated 5-azacytosine and the corresponding silylated fluorosubstituted 3-deazacytosines, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by deprotection of the blocking groups. These compounds were tested in vitro for cytotoxicity against L1210, B16F10, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1 and HBV.     

Indole alkoloids from Nauclea officinalis with weak antimalarial activity

Five indole alkaloids (naucleofficines A-E) were isolated from the stems (with bark) of Nauclea officinalis: (E)-2-(1-beta-d-glucopyranosyloxybut-2-en-2-yl)-3-(hydroxymethyl)-6,7-dihydro-indolo[2,3-a]quinolizin-4(12H)-one (1), (E)-1-propenyl-12-beta-d-glucopyranosyloxy-2,7,8-trihydro-indolo[2,3-a]pyran[3,4-g]quinolizin-4,5(13H)-dione (2), (E)-2-(1-hydroxybut-2-en-2-yl)-11-beta-d-glucopyranosyloxy-6,7-dihydro-indolo[2,3-a]quinolizin-4(12H)-one (3), (E)-1-propenyl-4-hydroxy-2,4a,7,8,13b,14,14a-hepthydro-(4alpha,4abeta,13balpha,14abeta)indolo[2,3-a]pyran[3,4-g]quinolizin-5(13H)-one (4) and 1-(1-hydroxyethyl)-10-hydroxy-7,8-dihydro-indolo[2,3-a]pirydine[3,4-g]quinolizin-5(13H)-one (10-hydroxyangustoline) (5), together with two known compounds, naucleidinal (6) and angustoline (7). All of the structures of the seven compounds above were elucidated by spectroscopic methods including use of 1D- and 2D-NMR spectroscopic analyses. Compounds 2 and 3 are rare examples of monoterpene indole alkaloids with a glucopyranosyloxy group attached to position C-12. In vitro activity screening of the above seven compounds showed weak to moderate inhibitory activity against Plasmodium falciparum.     

Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases

8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-(2)H]8-HODE and [10-(2)H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess-Martin periodinane followed by reduction to hydroxy fatty acids with NaB(2)H(4). The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-(2)H(2)]5,8- and [8,11-(2)H(2)]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC-MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.     

Two novel C29-5beta-sterols from the stems of Opuntia dillenii

Two novel C29-5beta-sterols, opuntisterol [(24R)-24-ethyl-5beta-cholest-9-ene-6beta,12alpha-diol] (1) and opuntisteroside [(24R)-24-ethyl-6beta-[(beta-d-glucopyranosyl)oxy]-5beta-cholest-9-ene-12alpha-ol] (2), together with nine known compounds, beta-sitosterol (3), taraxerol (4), friedelin (5), methyl linoleate (6), 7-oxositosterol (7), 6beta-hydroxystigmast-4-ene-3-one (8), daucosterol (9), methyl eucomate (10) and eucomic acid (11), were isolated from the stems of Opuntia dillenii collected in Guizhou Province, China. Their structures were elucidated mainly by spectroscopic analysis. The absolute configuration of 1 were deduced from comparative 1H NMR data of the (S)- and (R)-methoxyphenyl acetate derivatives. Compounds 6-8, 10 and 11 were isolated from O. dillenii for the first time.     

Non-glycosidic iridoids from Cymbaria mongolica

Six non-glycosidic iridoids, i.e. (1R,4S,4aS,7S,7aS)-7-hydroxyl-4-hydroxy- methyl-7-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta[e]pyran-3-one (1), (1S,4R,4aS,7S,7aS)-7-hydroxyl-4-hydroxymethyl-7-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta[e]pyran-3-one (2), (1R,4R,4aS,7S,7aS)-7-hydroxyl-4-hydroxy-methyl-7-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta[e]pyran-3-one (3), (1R, 4R, 4aS, 7aS)-4,7-dihydroxymethyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta-6-ene[e]pyran-3-one (4), (1R, 4R, 4aS, 7aS)-4,7-dihydroxymethyl-1-hydroxyl-1,4,4a, 7a-tetrahydrocyclopenta-6-ene[e]pyran-3-one (5), (1R, 4S, 4aS, 7aS)-4,7-dihydroxy-methyl-1-methoxyl-1,4,4a,7a-tetrahydrocyclopenta-6-ene[e]pyran-3-one (6), as well as five known non-glycosidic iridoids mussaenin A (7), gardendiol (8), isoboonein (9), 4-epi-alyxialactone (10) and rehmaglutin D (11) have been isolated from the Chinese medicinal plant Cymbaria mongolica. Their structures were elucidated by spectroscopic methods. These compounds exhibit significant antitumor and antibacterial activity.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

苏铁的化学成分研究

对苏铁(Cycas revoluta)的化学成分进行研究,采用多种色谱技术(硅胶、氧化铝和Sephadex LH-20等)从苏铁茎的乙酸乙酯部位分离得到16个化合物,其结构由HR-ESI-MS、1H和13CNMR等波谱学方法鉴定为5,6-去氢柳杉酚(1)、cunningine A (2)、6-羟基-5,6-去氢柳杉酚(3)、6α-羟基-7-氧代弥罗松酚(4)、ligballinol (5)、xanthoxyol (6)、callislignan A (7)、(2R,3R)-bis[(4-hydroxy-3-methoxyphenyl) methyl]-1,4-diacetate (8)、开环异落叶松脂醇(9)、二氢山柰酚(10)、4'-甲基木犀草素(11)、5-methoxypinosylvin (12)、N-benzoylphenyl alaninol(13)、(E,4R)-4-hydroxy-4,5,5-trimethyl-3-(3-oxobut-1-enyl) cyclohex-2-enone (14)、3-hydroxy-5α,6α-epoxy-β-io-none (15)和acuminantin (16)。以上化合物均为首次从苏铁属植物中分离得到,其中化合物13和16为首次以天然产物报道。本研究还首次发现了苏铁属植物中含有松香烷型二萜类(1～4)和芪类(12)化合物。化合物11具有中等的体外α-葡萄糖苷酶抑制活性。     

Mechanism of biosynthesis of 11R-and 12R-hydroxyeicosatetraenoic acids by eggs of the sea urchin Strongylocentrotus purpuratus

11(R)-Hydroxyeicosatetraenoic acid [11(R)-HETE] and 12(R)-HETE are biosynthesized by eggs of the sea urchin S. purpuratus. We report here the isolation of the 11(4)- and 12(R)-hydroperoxy-eicosanoids from incubations of the desalted 30-50%(NH4)2SO4 fraction of the egg homogenate; biosynthesis required the addition of calcium but not NADPH. Egg 11- and 12-HETE were formed from octadeuterated arachidonic acid without loss of geminal 2H from C11 or C12, thus revealing that 11- or 12-keto intermediates are not involved in the biosynthesis. The results support the conclusion that egg 11(R)- and 12(R)-HETE are synthesized by a lipoxygenase and not by an NADPH-dependent cytochrome P450 monooxygenase mechanism.     

Plant peptide hormone phytosulfokine (PSK-alpha): synthesis of new analogues and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In our studies on structure/activity relationship in PSK-alpha the synthesis of a series of analogues was performed: [H-D-Tyr(SO3H)1]- (9), [H-Phe(4-SO3H)1]- (10), [H-D-Phe(4-SO3H)1]- (11), [H-Phg(4-SO3H)1]- (12), [H-D-Phg(4-SO3H)1]- (13), H-Phe(4-NHSO2CH3)1]- (14), [H-D-Phe(4-NHSO2CH3)1]- (15), [H-Phe(4-NO2)1]- (16), [H-D-Phe(4-NO2)1]- (17), [H-Phg(4-NO2)1]- (18), [H-D-Phg(4-NO2)1]- (19), [H-Hph(4-NO2)1]- (20), [H-Phg(4-OSO3H)1]- (21), [Phe(4-NO2)3]- (22), [Phg(4-NO2)3]- (23), [Hph(4-NO2)3]- (24), [H-Phe(4-SO3H)1, Phe(4-SO3H)3]- (25) [H-Phe(4-NO2)1, Phe(4-NO2)3]- (26), [H-Phg(4-NO2)1, Phg(4-NO2)3]- (27), [H-Hph(4-NO2)1, Hph(4-NO2)3]- (28) and [Val3]- PSK-alpha (29). For modification of the PSK-alpha peptide chain the novel amino acids and their derivatives were synthesized, such as: H-L-Phg(4-SO3H)-OH (1), H-D-Phg(4-SO3H)-OH (2), Fmoc-Phg(4-SO3H)-OH (3), Fmoc-D-Phg(4-SO3H)-OH (4), Boc-Phg(4-NHSO2CH3)-OH (5), Boc-D-Phg(4-NHSO2CH3)-OH (6) Boc-Phe(4-NHSO2CH3)-OH (7), and Boc-D-Phe(4-NHSO2CH3)-OH (8). Peptides were synthesized by a solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK according to the Matsubayashi et al. test.