广西眼镜王蛇毒两种酸性磷脂酶A2的cDNA克隆及序列分析

从广西眼镜王蛇毒腺中提取总RNA,利用RT－PCR进行体外扩增,获得磷脂酶A2基因（PLA2）,克隆至PUCm－T载体中,筛选出编码两种酸性PLA2（命名为APLA2－1和APLA2－2）的基因,经双向测序测定了它们基因的全序列,由此推导出编码的氨基酸序列,其中APLA2－1的N端15个氨基酸残基序列同其蛋白质直接测序的结果完全一致。利用计算机推算出它们的等电点与实际测定的等电点较为吻合。同源性比较表明,APLA2－1与福建眼镜王蛇和台湾眼镜王蛇毒PLA2成熟肽同源性极高,而APLA2－2与它们的同源性相对较低,并且APLA2－2与APLA2-1芨其他两种眼镜王蛇毒PLA2分子名有一个显著的差别即少一个62－66位的“胰腺环”,可能与物种进化和生物活性有关。     

广西眼镜王蛇毒酸性磷脂酶A2-1在不同载体的表达

目的构建广西眼镜王蛇毒酸性磷脂酶A2-1(APLA2-1)在不同载体的重组表达质粒,在E.coli中表达APLA2-1并比较不同表达系统对APLA2-1的表达效果。方法将广西眼镜王蛇毒酸性磷脂酶A2-1(AP-LA2-1)基因克隆至表达载体pBLMVL2和pET28a( ),分别转化入大肠杆菌RR1和BL21,经过诱导表达,应用SDS-聚丙烯酰胺凝胶(SDS-PAGE)及Western blot观察重组蛋白表达情况。结果成功构建了重组质粒pBLMVL2-APLA2-1和pET28a-APLA2-1。pBLMVL2-APLA2-1在SDS-PAGE上没见明显表达带,在Western blot上可见一14 kD的表达带。pET28a-APLA2-1在SDS-PAGE上有一明显的18 kD表达条带,表达产物AP-LA2-1约占细菌总量30%,并以包涵体的形式存在。结论APLA2-1可在大肠杆菌中表达,pET28a( )对APLA2-1的表达效果优于pBLMVL2。     

蛇志2001年第13卷总目次

第 1期 (2 0 0 1年 3月 )有用微生物提取物 (EM- X)对果蝇和小白鼠的抗老化作用柯　彬 ,比嘉照夫 ,钟正贤 ,等 (1 )……………广西驯养蛇类腐皮病病因调查的研究曾维铭 ,韦　平 ,周维官 ,等 (9)………………………………………广西眼镜王蛇毒酸性磷脂酶 A2 的肌毒性研究王秋雁 ,庄茂辛 ,林文珍 ,等 (1 2 )……………………………蕲蛇酶的中性己糖及唾液酸的含量测定王志红 ,刘广芬 ,王晴川 (1 5 )………………………………………中华眼镜蛇伤致局部组织损伤治疗的实验研究李景新 ,唐荣德 ,蒋三员 ,等 (1 7)……………………………蛇骨粉中…     

眼镜王蛇毒中毒发病机理的研究报告

采用CM-Sepharose CL-6B柱层■可从广西产眼镜王蛇毒中得到28个蛋白峰,研究表明眼镜王蛇毒是一种含有多种毒性组份的毒性蛋白,可引起动物的局部损伤及心、肝、肺、肾、脑、肋肌和神经、血液等多个器官系统的病理学改变。眼镜王蛇毒中毒死亡的主要原因为神经毒素致呼吸麻痹引起全身重要生命器官缺氧加上急性微循环衰竭的综合作用所致,并提出有关抢救新方法。     

广东眼镜王蛇毒酸性磷脂酶A2的分离纯化及抗血小板聚集作用

目的 研究眼镜王蛇毒中酸性磷酸脂酶Ａ２对血小板的作用。方法 采用ＣＭ－Ｓｅｐｈａｄｅｓｘ Ｃ－２５Ｓｅｐｈａｄｅｘ Ｇ－７５,ＤＥＡＥ－Ｓｅｐ「ｈａｄｅｘＡ－２５,Ｓｅｐｈａｄｅｘ Ｇ－７５多步柱层析法,聚丙烯酰胺产胶电泳,酸性磷脂酶Ａ２酶活性测定;血小板聚集实验。累进要从粗中分离纯化出一酸性磷脂酶Ａ２单体,分子量为１４ＫＤ;等电点ＰＩ３．８;ＰＬＡ２比值２０μｍｏｌ．ｍｇ＾－１．ｍｉｎ＾－１;小     

抗中华眼镜蛇毒鸡卵黄抗体IgY的动物保护作用研究

目的通过连续观察蛇伤大鼠多项生理指标的动态变化,探讨不同时段施予血清（125u血清／mg蛇毒）同等效价但量加倍的抗蛇毒IgY对其心、肺功能保护的时效性。方法SD大鼠分为80、100、120min IgY保护组和蛇毒组共4组,戊巴比妥钠腹麻,按2倍LD50（1．272mg／kg）的剂量注入舟山眼镜蛇毒。连续3h记录呼吸频率、心电图和肌电等生理指标的动态变化,于注毒后80、100、120min分3个时段注入抗眼镜蛇毒IgY,比较各组大鼠的平均存活时间、保护率及心、肺、腓肠肌功能的差异。结果蛇毒组大鼠注入2倍LD5。的眼镜蛇毒后,平均存活时间为124．4min,存活率为0;80、100min IgY的存活率均达到或超过50％。但120minIgY组的保护作用不明显。与生理盐水组相比,各组心电图异常（心率减慢、ST段抬高）、呼吸频率减慢及肌电减弱多出现在100min以后。大鼠死亡前均出现潮式呼吸直至呼吸衰竭。结论应用与血清（125u血清／mg蛇毒）同等效价但量加倍的抗蛇毒IgY,可有效地减低蛇毒对机体心、肺、骨骼肌功能的损伤,提高生存率;与抗血清（125u血清／mg蛇毒）对蛇伤大鼠保护作用的结果相比,加量后保护作用更强．保护的有效时段有一定的延长。     

广西眼镜蛇蛇毒磷脂酶A_2的分离纯化及其对HSC-T6细胞的作用

目的对广西眼镜蛇毒中磷脂酶A2(PLA2)进行分离纯化,测定其对肝星状细胞HSC-T6的增殖抑制作用。方法采用Sephadex G-50凝胶层析柱、CM-Sepharose CL-6B离子交换柱、Macro-prep High S预装柱结合的方法分离广西眼镜蛇粗毒,经平板法测定各峰的PLA2活性;经SDS-PAGE电泳鉴定终产物纯度并测定分子量,NanoLC-ESI-MS/MS鉴定其组分;CCK-8法测定PLA2对肝星状细胞(HSC-T6)的增殖抑制作用,确定其凋亡的最小毒性浓度。结果 Sephadex G-50凝胶层析柱、CM-Sepharose CL-6B离子交换柱、Macro-prep High S预装柱层析法,得到第Ⅲ峰具PLA2活性,且达到电泳纯,经NanoLCESI-MS/MS鉴定其为PLA2,分子量约为14.06kD;PLA2在0~1μg/ml的浓度下对HSC-T6细胞具有一定的促增殖作用,2μg/ml时细胞数达到最大值,4~16μg/ml时对细胞生长有抑制作用,且随浓度增大细胞数降低。结论采用Sephadex G-50、CM-Sepharose CL-6B、Macro-prep High S预装柱结合的方法对广西眼镜蛇毒进行分离纯化,得到电泳纯且具PLA2活性的磷脂酶A2;广西眼镜蛇毒PLA2对肝星状细胞HSC-T6增殖有抑制作用,PLA2对HSC-T6细胞的最小毒性浓度为2μg/ml。     

低剂量创伤弧菌感染小鼠心肌与骨骼肌电镜超微结构观察比较

目的:观察低剂量创伤弧菌感染小鼠心肌、骨骼肌的超微结构变化,比较创伤弧菌引起的特征性病变下肢水肿骨骼肌病变与心脏病变出现的次序,探讨下肢水肿是否存在与心肌病变有关.方法:16只6～8周ICR(清洁级)小鼠.实验组12只腹腔注射＜LD50(1.34×107个/ml)的菌量(4.45×105个/ml)0.2ml,4只注射生理盐水0.2 ml作为菌液对照.分别取1 h、3 h、6 h、12 h小鼠心肌、后肢骨骼肌肌肉组织0.1cm×0.1 cm×0.1 cm置电镜固定液,超薄切片观察超微结构.结果:引起的动物模型实验组小鼠主要的实质性病变在心肌.实验组3 h就发现肌原纤维间隙扩大,肌丝断裂,肌膜下水肿较明显.6 h肌丝排列紊乱,疏松,局灶性肌丝断裂溶解.12 h核固缩水肿,间隙水肿,肌丝断裂,线粒体肿胀.而骨骼肌肌肉超微结构变化不明显,3 h、6 h和12 h实验组未死亡小鼠只表现肌组织间质变化,肌浆网扩张,组织间质水肿,胶原纤维排列稀疏、溶解.结论:本实验心肌、骨骼肌水肿与临床下肢水肿的症状相吻合,并提示心肌的实质病变明显早于骨骼肌,比较而言创伤弧菌所致病变是以重要脏器心脏的损伤为主,早期创伤弧菌对骨骼肌肌肉的实质性损伤并不明显.由创伤弧菌所致的原发性败血症表现双下肢出血性水肿可能不是创伤弧菌初始病变.     

肌酸对游泳大鼠乳酸、糖原含量和乳酸脱氢酶活性的影响

为探讨肌酸对提高大鼠运动能力的作用 ,观察了肌酸对游泳大鼠血清、心肌和骨骼肌乳酸、糖原含量和乳酸脱氢酶 (LDH)活性的影响。实验用雄性wistar大鼠 2 4只 ,随机分为正常组、游泳对照组和游泳补充肌酸组。两个游泳组每天游泳训练 1h,9天后 ,游泳 4h ,测定血清、心肌和骨骼肌乳酸水平 ,测定血清和骨骼肌乳酸脱氢酶活性以及心肌与骨骼肌糖原含量。结果显示 :肌酸可抑制游泳运动后大鼠血清、心肌和骨骼肌乳酸浓度以及血清LDH活性的升高幅度 ,抑制心肌和骨骼肌糖原含量及骨骼肌LDH活力的下降。以上结果表明 ,肌酸可改善运动后机体乳酸和糖原的代谢 ,降低运动性疲劳 ,提高大鼠的运动能力     

蛇毒神经生长因子诱导PC12细胞分化超微结构的观察

目的观察蛇毒神经生长因子（Nerve growth factor,NGF）诱导大鼠肾上腺嗜铬细胞瘤细胞系（Pheo—chromocytoma cells。PC12）细胞分化后,细胞超微结构的改变。方法取对数生长期PC12细胞接种24孔板,设200ng／ml NGF实验组和对照组。培养72h,离心,分别收集细胞制成电镜标本,镜下观察各组细胞超微结构的改变。结果与对照组细胞相比,实验组细胞长出大量突起．并且胞质的细胞器逐渐消失．出现较多的脂滴。结论广西眼镜蛇毒神经生长因子可以促进PC12细胞增殖．并诱导其向神经样细胞分化,长出突触。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.