蟾蜍皮肤活性蛋白与多肽的研究

近年来，我们致力于蟾蛛（Bufo andrewski）皮肤活性组份的研究，构建了缥蛛皮肤cDNA文库，检测了蟾蛛皮肤分泌液中多种生物活性，进一步纯化得到了四个新的生物活性蛋白：溶菌酶、抗爱滋病毒蛋白以及两个丝氨酸蛋粼酶抑制剂，并，简述如下；I、第三章报导了我们从蟾蛛皮肤分泌液中分离得到的一个谊薇酚犷杰女呱BA一娜"zym"）·BA一lvs"Zym"经5DS一PAGE检测为一条带，其分子量约为巧k珍。·它是一个高效的溶菌酶，每毫克蛋白的溶菌活性为2．7xl护俪ts，还能抑制革兰氏阳性菌（金黄色葡萄球菌Slaj，匆褚ococcusaureus）和革兰氏阴性菌（大肠杆菌Esch邵ichiacoli）生长，其最小抑菌浓度（MIC）分别为1．36拼M和84并M。使用PCR筛选法，我们从蟾蛛皮肤c溯A文库中克隆得到编码BA一lyso即me的cDNA序列。BA一lysozymeN末端测序和肤质量图谱确认了蛋白和基因的网一性。它的蛋白全序列与鸡溶菌酶的相似性为5氏5％。系统发育分析显示，与其最相似的是来源于海龟的溶菌酶。11、第四章介绍了我们从蟋蛛皮肤分泌液中分离得到的一个新的抗lllV蛋白，命名为BAS一AH。BAS一AH是一个分子量为63kD。的单链蛋白，每摩尔蛋白质含有0．89摩尔血红素辅基。BAS一AH对人T淋巴细胞系CS166细胞的毒性（CCS。）为9．5尽M。BAs一AH具有较强的抗HIV活性，它对HIV感染和复制具有剂量依赖抑制效应，其选择指数（CCso／Ecs。）分别为14．4和11．4·BAS一麟J也能抑制HIV的逆转录酶，其1C5（）为L32冬以。BAs一AH的N末端氨基酸为NA以KADvIGKIsILLGQDI』slvAAM，与己知的抗Hlv蛋白没有同源性·表明它可能是一个新的抗川v蛋自。BAs一AH没有检测到抗菌活性、蛋自酶水解活性、胰蛋自酶抑制剂活性、L一氨基酸氧化酶活性和过氧化氢酶活性。班、第五伞报导了我们通过离子交换、分子筛和反向层析，从蟾赊皮肤中分离得到的一个新的胰蛋白酶抑制剂，命名为BATI。BATI是一个单链糖蛋自·其分子量为22kD。。它是吵～个热稳定的竞争性的抑制剂，能有效抑制胰蛋自酶·其抑制常数凡为14nM。B灯I对凝血酶、弹性蛋白酶以及糜蛋白酶都没有抑制作用。BATI的N末端序列为El犯ITD，不同于其它物种来源的蛋白酶抑制剂。W、第六章介绍了蟾蛛皮肤分泌液中纯化得到的另外一个蛋白酶抑制剂（命名为baserpin）。与上述BATI不同的是，basel咖n不可逆地抑制多种蛋白酶。它是一个分子量约为60kDa的单链糖蛋白，除了能抑制胰蛋白酶，还能有效抑制糜蛋白酶和弹性蛋白酶。它抑制上述三种酶的二级反应常数（编）分别为4．6x1护M一，s一l、8．9》1护M一15一I以及6．8xl护M一ls一l。BaserPin是第一个来源于两栖类皮肤的不可逆抑制剂，其N末端氨基酸序列为HTQYPDILIAKPxDK，与其它物种来源的蛋白酶抑制剂不同。本论文综述了蟾蛛皮肤中的活性组份，报导了我们近年来研究蟾蛛皮肤活性蛋白与多肤的进展，分四章详细介绍了蟾蛛皮肤中纯化得到的四个活性蛋白。BA一lysozyme是两栖类动物中第一个得到蛋白质全序列的溶菌酶，能有效抑制革兰氏阳性菌和革兰氏阴性菌生长；BA象AH是一个含血红素辅基的抗HIV蛋白，其独特的理化性质和功能证明它是一个新的抗病毒蛋白。根据所鉴定的性质判断，BATI和bos仰in分别属于竞争性抑制剂和不可逆抑制剂。其中，base印in是第一个从两栖类皮肤中分离得到的不可逆抑制剂。

Proteinogramas séricos de ratos Wistar experimentalmente infectados com Trypanosoma evansi

Estabeleceu-se o perfil eletroforético de proteínas séricas de ratos Wistar experimentalmente infectados com Tripanosoma evansi, utilizando-se 40 ratos, distribuídos em oito grupos de cinco animais cada. Um grupo foi mantido como testemunho (G1), e os demais (G2 a G8) foram inoculados, via intraperitoneal, com cerca de 10³tripomastigota de T. evansi. Amostras de sangue para obtenção de soro foram coletadas no quinto (G2), 10º (G3), 15º (G4), 30º (G5), 45º (G6), 60º (G7) e 75º (G1 e G8) dia após as inoculações. O fracionamento das proteínas foi realizado pela técnica SDS-PAGE. Foram identificadas 31 proteínas, sendo sete de fase aguda: ceruloplasmina (101KD), hemopexina (83KD), transferrina (75KD), albumina (66KD), antitripsina (60KD), haptoglobina (44KD) e glicoproteína ácida (38KD). As proteínas com pesos moleculares 12KD; 22KD; 25KD; 28KD; 32,5KD; 35KD; 53,5KD; 63KD e 72KD apareceram apenas nos ratos inoculados com T. evansi.

Nd, Pb, and Be isotopes of ferromanaganese crusts of the South Pacific

The application of radiogenic isotopes to the study of Cenozoic circulation patterns in the South Pacific Ocean has been hampered by the fact that records from only equatorial Pacific deep water have been available. We present new Pb and Nd isotope time series for two ferromanganese crusts that grew from equatorial Pacific bottom water (D137-01, 'Nova', 7219 m water depth) and southwest Pacific deep water (63KD, 'Tasman', 1700 m water depth). The crusts were dated using 10Be/9Be ratios combined with constant Co-flux dating and yield time series for the past 38 and 23 Myr, respectively. The surface Nd and Pb isotope distributions are consistent with the present-day circulation pattern, and therefore the new records are considered suitable to reconstruct Eocene through Miocene paleoceanography for the South Pacific. The isotope time series of crusts Nova and Tasman suggest that equatorial Pacific deep water and waters from the Southern Ocean supplied the dissolved trace metals to both sites over the past 38 Myr. Changes in the isotopic composition of crust Nova are interpreted to reflect development of the Antarctic Circumpolar Current and changes in Pacific deep water circulation caused by the build up of the East Antarctic Ice Sheet. The Nd isotopic composition of the shallower water site in the southwest Pacific appears to have been more sensitive to circulation changes resulting from closure of the Indonesian seaway.