从绿藻礁膜（Monostroma nitidum Wittr）分离半乳糖专一的凝集素

礁膜（Monostroma nitidum Wittr）经 25%～80%硫酸铵分级、DEAE-纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到纯化礁膜凝集素（Monostroma nitidum lectin,MNL）,在SDS-PAGE上显示单一蛋白染色带. 用Sephadex G-200层析测得其分子质量为66.6 kD, 用SDS-PAGE测得其分子质量为66.2 kD．该凝集素可以凝集人A、B、AB、O型红细胞,且凝集活性相同. 在对人（A、B、AB、O）、兔、鲤、鲫、鼠、羊、鸡、狗的红细胞凝集作用中,兔凝集作用最强．该凝集素在pH 4.00～10.53范围内均有活性,但在pH 5.20～9.40范围内活性最大．经100 ℃热处理30 min后,该凝集素对兔红细胞血凝活性保留25%,活性最大的温度范围为25～55 ℃．MNL被EDTA抑制,最小抑制浓度为3.13 mmol/L,但对 Ca^２＋和Mg^２＋不敏感．该凝集素凝集兔红细胞的作用不被D -果糖、D-甘露糖、D-葡萄糖、蔗糖、麦芽糖、γ-球蛋白、牛甲状腺球蛋白所抑制,但被D- 半乳糖和乳糖抑制,最小抑制浓度分别为5 mmol/L和2.5 mmol/L．     

正红菇菌丝体凝集素的分离纯化及生化特性

正红菇菌丝体经磷酸缓冲液浸提、硫酸铵分级沉淀、DEAE-Sepharose FF离子交换层析和Sephadex G-100分子筛层析纯化得到红菇凝集素（Russula vinosa Lectin,RVL）。经SDS-PAGE检测为单一蛋白带,其亚基相对分子质量为55kDa,Sephadex G-100凝胶过滤测得相对分子质量为55.25kDa,提示RVL分子只有一个亚基。RVL中性糖含量为3.87%,经酸水解测定含15种氨基酸。温度在20–60℃、pH在5–9的范围内,凝集活性保持相对的稳定。RVL的凝血活性受Mn2+、Zn2+、Ca2+的影响。糖抑制实验表明,在供试的11种糖中,D-甘露糖强烈抑制RVL的凝血活性。抑菌实验显示,RVL对供试的细菌没有抑制作用,对稻瘟病菌、绿色木霉、红色链包霉、黑曲霉菌丝生长有显著的抑制作用。     

青霉属真菌Penicillium sp. CPCC 400786的抗病毒活性成分

采用抗艾滋病毒抑制剂筛选模型对一株青霉属真菌Penicillium sp. CPCC 400786发酵产物的乙酸乙酯提取物进行活性评价,结果显示,其对艾滋病毒有较强的抑制活性。采用正相硅胶柱、Sephadex LH-20凝胶柱和半制备HPLC等色谱技术对乙酸乙酯提取物进行分离纯化,从中分离得到8个化合物。通过波谱数据分析,分别鉴定为：oxalicine A（1）、oxalicine B（2）、cis-4,6-dihydroxymellein（3）、亚油酸（4）、十八烯酸（5）、肉豆蔻酸（6）、尿嘧啶（7）、胸腺嘧啶（8）。化合物1和2为杂萜类化合物。对化合物1-6进行了抗艾滋病毒（HIV-1）和抗甲型流感病毒（H1N1）的活性评价。结果显示,化合物1具有良好的抗H1N1活性,其IC₅₀值为38.5μmol/L,比阳性对照药利巴韦林稍弱（IC₅₀=20.5μmol/L）;化合物1和2具有抗HIV-1的活性,其IC₅₀值分别为22.4、67.8μmol/L;其他化合物未显示抗病毒活性。本研究为从青霉属中发现更多抗病毒活性杂萜分子提供了依据。     

雷丸凝集素的纯化及理化性质的研究

雷丸（OmphalialapidescensSchroct．）经过Tris－HCl缓冲液浸提,硫酸铵分级沉淀,离子交换,卵粘蛋白－Sepharose4B亲和层析以及Sephacryl-S100分子筛等步骤,纯化得到纯度为95％以上的雷扎凝集素（简称OLL）。比活提高45.8倍,活力回收2.5％。雷丸凝集素是单一肽链的蛋白质,分子量为12kDa,等电点为7.5,可被半乳糖抑制。具有热稳定性及酸碱（pH1～12）稳定性。2mmol／L氯化锌可使其比活提高4倍。同兔血相比,与人血进行凝集反应,其比活可提高4倍。进行圆二光谱测定,α－螺旋和β-折叠含量较高。与半乳糖间的抑制反应瞬间内完成,且空间结构变得紧密。     

红藻条斑紫菜凝集素的纯化及其色氨酸化学修饰对其活性的抑制作用

为了探索条斑紫菜凝集素(Porphyra yezoensis Ueda lectin, PYL)的作用机理,对其进行了分离和纯化．条斑紫菜经磷酸盐缓冲液浸泡、20%～75%硫酸铵分级、DEAE 纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到PYL纯品. Sephadex G-200分子筛层析测得其分子量为63.2 kD,在非还原SDS-PAGE上显示1条蛋白染色带,分子量为63.1 kD,还原SDS-PAGE显示1条蛋白染色带,亚基分子量为15.8 kD．PYL在对兔、大鼠、鸡、羊、狗血细胞的凝集作用中,对大鼠红细胞的凝集活性最高.PYL在pH 6.50～10.53范围内均有活性,在pH 8.40～8.91活性最高．经42 ℃热处理10 min后,仍然对大鼠红细胞血凝活性保留12.5%,其活性最大温度范围为4 ℃～20 ℃, 48 ℃加热10 min后,其活性完全丧失．EDTA对PYL的凝集活性有抑制作用,最小抑制浓度为156 mmol/L,而 Ca2+和Mg2+未发生凝集抑制现象．PYL凝集大鼠红细胞的作用不被D 果糖、葡萄糖、D-半乳糖、D-甘露糖、菊粉、γ球蛋白、牛甲状腺球蛋白等所抑制,但可被蔗糖和麦芽糖抑制,最小抑制浓度蔗糖为20 mmol/L,麦芽糖为40 mmol/L．用N 溴代丁二酰亚胺(NBS) 对PYL分子中的Trp残基进行化学修饰,有2.1个Trp残基被修饰,修饰后PYL活性丧失, 表明Trp残基是PYL凝集活性所必需的基团．     

植物性雌激素genistein对哇巴因所致豚鼠乳头状肌迟后去极化及触发活动的效应

应用标准玻璃微电极技术,研究了植物性雌激素genistein(GST)对哇巴因所引起的豚鼠乳头状肌迟后去极化（DAD）及触发活动（TA）的效应,结果如下：（1）预先给予是GST（10,50,100umol/L)剂量依赖性地抑制哇巴因（1umol/L)所引起的鼠乳头状肌DAD及TA;（2）预先应用一氧化氮合酶抑制剂L－NAME（1mmol/L),不影响GST（50μmol/L）对DAD及TA的效应;（3）单独应用17β－雌二醇（E2,5μmol/L）或GST（10μmol/L）对DAD及TA无明显影响,而联合应用相同剂量的GST和E2则产生明显儿应,以上结果提示,GST可能通过抑制钙离子内流从而具有抗心律失常作用,这对于心血管系统的保护有一定意义。     

外源半胱氨酸对铜胁迫下小麦幼苗生长、铜积累量及抗氧化系统的影响

采用滤纸培养法,研究了不同浓度的L-半胱氨酸（L-Cys）对200μmol/L铜离子胁迫下小麦幼苗生长、铜积累量、和抗氧化系统的影响。结果表明,（1）200μmol/L的铜离子可抑制小麦幼苗生长,使根长、生物量、总叶绿素含量极显著下降,可溶性蛋白和还原性谷胱甘肽（GSH）含量,超氧化物歧化酶（SOD）和抗坏血酸氧化酶（APX）活性略微上升,丙二醛（MDA）含量和细胞膜透性极显著上升。（2）外源Cys在1.0—5.0mmol/L时,受铜胁迫的小麦幼苗生长势与对照无差异,在1.0和2.5mmol/L下,根长、生物量、叶绿素a和总叶绿素含量与对照无显著差异,与Cu处理组差异显著（P<0.01）。（3）高于1.0mmol/L的外源Cys可极显著增加铜胁迫下小麦叶片和根系中的铜积累量。（4）外源Cys极显著提高了铜胁迫下小麦幼苗可溶性蛋白和GSH含量,并使SOD和APX活性持续维持在较低水平;外源Cys浓度低于2.5mmol/L时,MDA含量极显著下降,低于5.0mmol/L时,细胞膜透性极显著升高;多酚氧化酶（PPO）活性先上升后下降,除Cys为0.5mmol/L处理外,其它各处理间PPO活性均无显著差异。综合来看,喷施1.0—2.5mmol/L的外源Cys可提高小麦幼苗对铜胁迫的耐受性。     

镉对平菇菌丝生长及同工酶表达的影响

采用液体培养研究了不同浓度镉（Cd）处理7d对平菇（Pleurotus ostreotus）菌丝体生长及其同工酶表达的影响.结果表明,50 μmol/L Cd处理对平菇菌丝生长抑制率为55.6%,2000μmol/L Cd为菌丝生长致死浓度.同工酶活性电泳图谱显示,Cd处理不仅改变酯酶（EST）、乳酸脱氢酶（LDH）、过氧化物酶（POD）和超氧化物岐化酶（SOD）同工酶带数,而且也影响各酶带的表达强度.50 μmol/L和100μmol/L Cd处理分别诱导出2条和3条新的POD同工酶带,而抑制一条分子量较大的POD酶带的表达,但明显增强总的POD活性.正常生长的平菇菌丝体LDH同工酶谱只出现2条酶带,50 μmol/L以下Cd处理不影响其同工酶的表达,100 μmol/L Cd处理组2条同工酶带均消失.50和100μmol/L Cd处理能够显著增强SOD活性,且诱导2条SOD同工酶表达.100μmol/L及其以下浓度Cd处理均能提高EST的活性,这可能具有加速细胞内酯类化合物水解而增加羧基的数量以螯合更多的游离Cd离子而解Cd毒的作用.Cd浓度在50μmol/L以下时,随着处理浓度的增加,对金属硫蛋白的诱导作用呈逐渐增强的趋势,而100 μmol/L Cd的诱导作用减弱.     

蓝藻Anabaena sp.PCC7120 羧体碳酸酐酶的鉴定

在丝状蓝藻Anabaena sp.PCC7120细胞粗提液的碳酸酐酶(CA)分析中,发现了两种形式的CA活性.高CO_2下生长的细胞,在35μmol/L EZ(Ethoxyzolamide,碳酸酐酶的抑制剂)存在的情况下,CA总活性的85％左右被抑制,其半抑制浓度I_(50)为7.4μmol/L;随着EZ浓度的继续增加,CA活性在EZ浓度达到约150μmol/L处出现了第二个抑制峰,在250μmol/L处抑制程度达到最大,使CA总活性的15％被抑制,其半抑制浓度I_(50)为190μmol/L。在空气条件下生长的细胞中也出现了CA的两个抑制峰:低I_(50)为6μmol/L,高I_(50)为120μmol/L,对羧体的分离及体外测试表明,在羧体制备物中的CA活性只有一个EZ的抑制峰,而且在EZ浓度达到35μmol/L,正如所期望的那样,该CA活性全部被抑制。其半抑制浓度I_(50)为5.2μmol/L左右。这个值跟空气或高CO_2条件下生长的细胞粗提物中的低I_(50)(6μmol/L或7.4μmol/L)十分相似。说明低浓度的EZ可以特异性地抑制定位于羧体的CA活性。另外一种形式的CA,具有高I_50(120—190μmol/L),约占CA总活性的15—20％,则有可能定位于细胞质膜。     

腺苷减少豚鼠心室肌细胞内游离钙浓度

目的：研究腺苷对豚鼠心室肌细胞内游离钙浓度（[Ca^2＋]i）的影响并探讨其可能机制。方法：用激光共聚焦显微镜探测细胞内游离钙浓度,结果用相对荧光强度（（FI-FI0）／FI0,％;FI0：对照;FI：给药）表示。结果：①在正常台氏液和无钙台氏液中,腺苷（10,50,100μmol／L）浓度依赖性地降低[Ca^2＋];。②含30mmol／L KCl的台氏液（高钾台氏液）能够增加[Ca^2＋]i。腺苷（10,50,100μmol／L）能够显著抑制KCl引起的[Ca^2＋]i的增加。③预先应用选择性腺苷AI受体拮抗剂DPCPX（1μmol／L）,可大部分取消腺苷（100μmol／L）在高钾台氏液中的作用。腺苷（100μmol／L）在高钾台氏液的作用也可被预先应用一氧化氮（No）合酶抑制剂L-NAME（1mmol／L）所部分减弱。④腺苷（100μmol／L）能明显抑制无钙台氏液中由低浓度ryanodine引起的[Ca^2＋];增加。⑤当细胞外液钙浓度由1mmol／L增加到10mmol／L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,腺苷（100μmol／L）可降低钙波发生的频率和持续时间,最终阻断钙波并降低[Ca^2＋];。结论：腺苷可通过抑制外钙内流和减少肌浆网内钙释放从而降低[Ca^2＋],其减少外钙内流可能是由于腺苷A1受体介导的电压依赖性Ca^2＋通道的抑制,NO可能参与这一过程。     

Hydrogen sulfide delays nicotinamide-induced premature senescence via upregulation of SIRT1 in human umbilical vein endothelial cells

The present study was designed to investigate the effect of hydrogen sulfide on cellular senescence of human umbilical vascular endothelial cells (HUVECs CC-2517) and its underlying mechanism. The premature senescence-like phenotype HUVECs (the fourth passage) was induced by treatment with nicotinamide (NAM, an inhibitor of SIRT1, 5 mmol/L, 12 h). Cells were cultured with sodium hydrosulfide (NaHS, 12.5, 25, 50 and 100 μmol/L) for 48 h in premature senescence-like phenotype HUVECs. The fourth passage of HUVECs was considered as young group. Senescence-associated (SA)-β-galactosidase activities were detected to evaluate cell senescence, and the expression of SA heterochromatin foci (SAHF) was visualized by DAPI DNA staining. The mRNA and protein levels of SIRT1 were detected using RT-PCR and western blotting analysis, respectively. The results showed that β-galactosidase positive cells and the formation of SAHF were markedly increased after treatment with NAM (5 mmol/L) for 12 h. We also found that NaHS (12.5 μmol/L) had no effect on the percentage of SA β-gal positive cells and the expression of SAHF, and the hallmarks decreased at the concentration of 25 and 50 μmol/L, reaching the minimum at 50 μmol/L, while the percentage of SA β-gal positive cells and the expression of SAHF increased at the concentration of 100 μmol/L. Furthermore, we found that both on protein and mRNA levels of SIRT1 in the Y+N+S50 group was significantly increased compared with that in Y+N group. In conclusion, NaHS delays senescence of HUVECs induced by NAM via upregulation of SIRT1 expression.     

FD耐热逆转录酶的部分酶学性质研究

ＦＤ耐热逆转录酶（ＦＤ－ＴＲＴ）来自一株栖热杆菌菌株．通过ＲＴ－ＰＣＲ方法,论文对ＦＤ－ＴＲＴ的部分酶学性质进行了研究．ＦＤ耐热逆转录酶能耐受９５℃的高温,最适反应温度在６５～７５℃左右,这时ＲＮＡ的高级结构能解开,可以提高逆转录反应的效率;同时引物和ＲＮＡ模板识别的专一性强,可大大提高逆转录的特异性．实验表明ＦＤ－ＴＲＴ逆转录反应的最适条件为：２５ｍｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．８,１５ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４,１００μｇ／ｍｌ明胶,５００μｍｏｌ／ＬｄＮＴＰｓ,２５ｐｍｏｌ逆转录引物,１ｍｍｏｌ／ＬＭｎＣｌ２,２ＵＦＤ耐热逆转录酶,反应在６５～７０℃下进行．在上述条件下,用ＲＴ－ＰＣＲ可检出少于５ｐｇ外周血总ＲＮＡ中特异的α珠蛋白ｍＲＮＡ．     

First report of a xylose-specific lectin with potent hemagglutinating, antiproliferative and anti-mitogenic activities from a wild ascomycete mushroom

From fresh fruiting bodies of the wild ascomycete mushroom (Xylaria hypoxylon) a lectin with N-terminal sequence resemblance to a part of Aspergillus oryzae genome and only slight similarity to fungal immunomodulatory protein from the mushroom Flammulina velutipes was isolated. The protocol comprised extraction with water, precipitation from the aqueous extract using 80% saturated (NH(4))(2)SO(4), ion exchange chromatography on DEAE-cellulose and CM-cellulose, and then gel filtration by fast protein liquid chromatography on Superdex 75. Lectin activity was adsorbed on DEAE-cellulose and unadsorbed on CM-cellulose. The lectin appeared as a single band with a molecular mass of 14.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single 28.8-kDa peak in gel filtration on Superdex 75. The lectin exhibited highly potent antiproliferative activity toward tumor cell lines, and exerted a potent anti-mitogenic action on mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by inulin and xylose. It was stable up to 35 degrees C. At 40 degrees C its hemagglutinating activity was reduced by 50%, and it dwindled to 12.5% of the original activity at 50 degrees C. The hemagglutinating activity was also sensitive to NaOH and HCl solutions. The hemagglutinating activity was unaffected by CaCl(2) and ZnCl(2), and was potentiated substantially in the presence of AlCl(3) and FeCl(3). The distinctive features of this lectin comprise a unique sugar specificity, and highly potent hemagglutinating, antiproliferative and anti-mitogenic activities. X. hypoxylon lectin differs in molecular mass, N-terminal sequence and sugar specificity from previously reported ascomycete mushroom lectins.     

Selenium and Zinc against Aβ<Subscript>25–35</Subscript>-Induced Cytotoxicity and Tau Phosphorylation in PC12 Cells and Inhibits γ-cleavage of APP

Amyloid beta (Aβ) is the main component of the amyloid plaques that accumulate in the brains of Alzheimer patients. The present study was conducted to investigate whether the combined treatment with selenium (Se) and zinc (Zn) offers more beneficial effects than that provided by either of them alone in reversing Aβ_25–35-induced neurotoxicity in PC12 cells. Cells were pretreated with 0.1 μmol/L of Se and Zn for 4 h, after treated with 10 mmol/L Aβ_25–35 for 24 h. Cells were divided into control and five treated groups, and received either 10 mmol/L Aβ_25–35,10 mmol/L Aβ_25–35 + 0.1 μmol/L Se, 10 mmol/L Aβ_25–35 + 0.1 μmol/L Zn, 10 mmol/LAβ_25–35 + 0.1 μmol/L Se + 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. The result showed that cell viability was decreased in MTT metabolic rate; LDH release and MDA, H₂O₂, and NO levels were increased and the GSK-3β and phosphorylated tau protein level were increased in Aβ_25–35-treated group (P < 0.05 or P < 0.01), which whole changes were attenuated by Se and Zn and Se combined Zn. In order to evaluate whether the Se and Zn have an effect on processing pathway of amyloid precursor protein (APP), we examined the activity of γ-secretase in primary cultured cortical neuron cells. ELISA analysis showed that Se and Zn could inhibit the activity of γ-secretase. Then we also investigated the effect of Se and Zn on the Aβ_1–40 concentration and APP-N-terminal fragment expression from APP695 stably transfected Chinese hamster ovary (CHO) cells. APP695 stably transfected CHO cells were treated with 0.1 μmol/L Se and Zn; cells were divided into control and four treated groups, which received either 0.5 M DAPT, 0.1 μmol/L Se, 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. Se and Zn could decrease Aβ_1–40 production and increase the APP-N-terminal fragment protein expression. These experiments indicate that Se and Zn have a protective effect on AD pathology that a possible mechanism is inhibiting the activity of γ-secretase to decreasing Aβ_1–40 production further influencing the APP processing. Altogether, our findings may provide a novel therapeutic target to treat AD sufferers.