辛基酚胁迫对雄性泥鳅抗氧化酶及卵黄蛋白原的影响

为研究辛基酚(OP)对雄性泥鳅抗氧化酶活性及血清卵黄蛋白原(VTG)含量的影响,将雄性泥鳅分别暴露于4种不同质量浓度OP(0.12、0.19、0.32、0.52 mg/L)中持续7、14、21 d和28 d,采用试剂盒检测肝脏超氧化物歧化酶(SOD)与过氧化氢酶(CAT)的含量,采用碱不稳定性蛋白结合磷法检测血清VTG的含量。结果表明,0.12 mg/L OP胁迫14 d,肝脏SOD和CAT含量均无显著变化,但是随着胁迫剂量增大和时间延长,SOD和CAT含量降低极其显著,在0.52 mg/L OP胁迫28 d时降到最低水平;泥鳅在0.12 mg/L OP中暴露7 d时,血清VTG含量就有极其显著升高,且随着胁迫剂量增大和时间的延长,VTG含量呈升高趋势。提示OP胁迫对SOD和CAT活性有显著的抑制作用,并随胁迫剂量增大和时间延长而抑制加剧,造成氧化损伤;OP胁迫可诱导VTG合成,并随暴露剂量增大和时间延长而诱导增强,具有明显的雌激素效应,这可能与其氧化损伤有密切关系。     

卵黄蛋白原的研究进展

卵黄蛋白原(vitellogenin,Vg)是一种普遍存在于卵生非哺乳动物中的糖磷脂蛋白,其作为一种重要的生殖蛋白参与卵生动物生殖、发育等重要生理过程。最新研究表明,卵黄蛋白原还参与动物的免疫防御过程,是一种典型的模式识别分子。本文系统全面的介绍了卵黄蛋白原的研究进展,包括卵黄蛋白原的激素诱导、合成方式、分解代谢、结构特性以及生物学功能等,此外还详细介绍了其作为生物标志物在环境激素检测中的应用。对卵黄蛋白原近期的研究成果进行综述可为今后开展卵黄蛋白原相关研究提供可靠的参考和指导。     

鲤和团头鲂幼鱼卵黄蛋白原的诱导、纯化及电泳比较

采用腹腔注射人工雌激素17α-乙炔基雌二醇(17α-ethinyloestradiol,EE2)的方法,在鲤幼鱼和团头鲂幼鱼体内诱导生成卵黄蛋白原;并结合利用阴离子交换介质DEAE—Sephrarose CL6B和液相层析技术分离纯化了诱导后的鲤幼鱼和团头鲂幼鱼血浆中的卵黄蛋白原;SDS-PAGE电泳结果表明所分离的两种鲤科鱼类卵黄蛋白原其亚基的分子量分别为170KD和150KD。     

EE2对稀有鮈鲫和斑马鱼幼鱼体内卵黄蛋白原诱导的比较

利用卵黄蛋白原(Vtg)作为类雌激素污染的生物标志物,比较研究了不同浓度的17α-乙炔基雌二醇(EE2)对斑 马鱼(Brachydanio rerio)和稀有鮈鲫(Gobiocypris rarus)幼鱼体内Vtg的诱导。研究结果表明:5ng/L,20ng/L和100ng/L EE2分别暴露5d后,稀有鮈鲫幼鱼体内的Vtg即可显著诱导,并且其含量随暴露时间的增加而增加,在暴露15d时 达到最大值;而斑马鱼幼鱼虽然100ng/LEE2暴露5d时可显著诱导体内Vtg的生成,但20ng/L EE2在暴露10d后, 5ng/L EE2在暴露15d后,才可显著诱导斑马鱼体内Vtg的生成。这一结果说明EE2暴露下对稀有鮈鲫体内Vtg的 诱导要比斑马鱼敏感。     

施氏鲟卵黄蛋白原及其相关蛋白的研究

采用凝胶柱层析、聚丙烯凝胶电泳、免疫印迹（Western blotting）和免疫扩散等方法对施氏鲟卵黄蛋白原（Vi-tellogenin,Vg）及其相关蛋白（Yolk protein,YP）进行了研究。结果表明,施氏鲟血清Vg是一种糖脂磷蛋白,其相对分子量为410kD,由分子量为205kD的两个同源亚基组成。Vg的3种相关蛋白YP1、YP2和YP3。其中YP1相对分子量为370kD,是一种糖脂磷蛋白,由相对分子量为97kD和33kD的两个亚基构成。YP2是一种相对分子量为144kD的磷脂蛋白,由相对分子量为94kD和45kD的2个亚基构成。YP3为相对分子量为66kD的磷蛋白,由相对分子量为30kD的同源亚基构成。     

双酚A诱导雄性中国林蛙肝细胞雌激素受体表达和卵黄蛋白原合成

为探讨双酚A(bisphenol A,BPA)对雄性两栖动物肝细胞中雌激素受体(estrogenreceptor,ER)表达和卵黄蛋白原(vitellogenin,VTG)合成的影响,将中国林蛙(Ranachensinensis)雄性成体分别持续暴露于10-7、10-6、10-5mol/LBPA水体中10、20、30d,设10-9、10-8mol/L雌二醇(E2)为阳性对照。用原位杂交技术检测ERmRNA在肝细胞中的表达定位,用免疫组织化学技术检测肝细胞中ER和VTG蛋白的表达。结果显示,各BPA和E2处理组肝细胞中均有ERmRNA阳性反应,ER和VTG蛋白的表达相对值均显著高于空白对照组。在同一暴露时间,ER和VTG表达值是随着BPA浓度的增加呈增高趋势。在同一BPA浓度,VTG表达值是随暴露时间的延长呈增高趋势,而ER表达值则无显著性变化。这些表明BPA可以通过诱导雄性中国林蛙肝细胞的ER高调导致VTG合成,但其效应远低于E2。     

日本沼虾卵黄蛋白原合成部位的初步研究

十足目卵巢中卵黄的来源,在过去的几十年研究中一直存在争议,内源性合成和外源性合成均有报道。以雌性日本沼虾为实验材料,根据外形观察和组织学研究确定其发育阶段,可以分为:卵原细胞增殖期;卵黄发生前期;初级卵黄发生期;次级卵黄发生期;成熟期和抱卵期(消退期)。从处于不同发育期的卵巢和肝胰腺中提取总RNA,用RT-PCR方法探讨不同发育期的日本沼虾卵巢和肝胰腺卵黄蛋白原mRNA表达,确定是否有卵黄蛋白原的合成功能。检测结果可以初步判定日本沼虾卵巢和肝胰腺都具有卵黄蛋白原mRNA表达功能,都是卵黄蛋白原的合成部位,其合成的量与沼虾卵巢的发育阶段相关。     

双须骨舌鱼卵黄蛋白原基因vtg的克隆、组织表达和原核表达分析

为探究双须骨舌鱼(Osteoglossum bicirrhosum)卵黄蛋白原vtg基因的结构和表达特征,本研究运用RACE技术克隆双须骨舌鱼vtg全长c DNA序列。序列分析发现,双须骨舌鱼vtg的c DNA全长为5 325 bp,开放阅读框(ORF)为5 121 bp,其5′和3′非编码区(UTR)分别为46 bp和159 bp,共编码1 706个氨基酸,预测蛋白质分子量约为186.79 ku。同源性分析发现,双须骨舌鱼与同科的美丽仆骨舌鱼(Scleropages formosus)相似度达到84.78%,与鲤形目、鲈形目、鲱形目、鲽形目鱼类的相似度均高于50%。系统进化树分析结果显示,本文获得的双须骨舌鱼vtg基因与骨舌鱼目聚为一类,与鳗鲡目硬骨鱼类亲缘关系较近,与双须骨舌鱼进化地位相符。荧光定量PCR检测结果表明,在雌雄鱼性腺和肝中vtg均有表达,且在雌鱼肝中的相对表达量显著高雄鱼(P0.05),卵巢和精巢表达量无显著差异(P0.05);在雌雄鱼鳃、脾、肾、肌肉、心、头肾、脑等7个组织中均极微量表达,且无显著差异(P0.05)。在Ⅱ、Ⅲ、Ⅳ期卵巢中vtg相对表达量依次增加,Ⅳ期显著高于Ⅱ和Ⅲ期(P0.05),也显著高于精巢Ⅳ期(P0.05);在雌鱼肝组织中呈先增后减趋势,且Ⅱ、Ⅲ、Ⅳ期之间无显著差异(P0.05),但每个时期都显著高于雄性(P0.05)。成功构建了原核表达载体p EASY-Blunt E2-vtg,并转入Transetta(DE3)表达感受态细胞中成功诱导出融合蛋白。Western-blotting检测显示,融合蛋白可被抗His标签特异性识别。综上表明,vtg表达水平高低与性别相关,并与性腺发育程度有关。此外,本研究结果也为后续深入研究卵黄蛋白原的生理功能打下基础。     

蜜蜂卵黄原蛋白的作用

卵黄蛋白不仅为胚胎发生提供营养物质,它在生物体内还具有其他的生物学功能。昆虫卵黄蛋白是昆虫卵内的营养储备,它的前体主要来源于脂肪体的雌性特异血蛋白——卵黄原蛋白(Vg),它是近年来昆虫生理学和生化学最活跃的领域之一,文章介绍蜜蜂卵黄原蛋白在蜜蜂生殖过程中的作用,以及与蜜蜂社会性生活及寿命的关系。     

一种特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达方案

为了实现特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达,将不能以可溶性蛋白形式表达的、只能以噬菌体展示形式特异性识别斑马鱼卵黄蛋白原的单链抗体F5的基因,克隆到pET 32a载体并转化入大肠杆菌ori DE3中。结果表明,通过诱导表达,可获得可溶性的并且仍特异性识别斑马鱼卵黄蛋白原的单链抗体32a-F5。噬菌体展示单链抗体不能可溶性表达是噬菌体展示技术应用中常见的问题,该方法提供了一种表达可溶性单链抗体的可行性方案。     

泥鳅性腺发生和分化的组织学研究

通过人工繁殖技术获得泥鳅幼体,采用石蜡显微切片技术对幼体性腺发生和分化的组织学特征进行了系统观察.结果表明泥鳅性腺发生于出膜后12 d,40日龄卵巢开始分化,至55日龄卵巢分化完全;精巢于出膜后55 d左右开始分化,100 d左右分化完全.卵巢分化早于精巢.从性腺分化开始,将要发育为卵巢的性腺还表现为体积快速增大,向体腔中间靠拢,横截面变宽;而将要发育为精巢的性腺则呈两端尖中间稍突的梭形,增生并不明显.这些特征可能与雌雄性腺的发育及生殖细胞的分化速度有关,可以作为泥鳅性腺早期分化的形态特征.     

The first record of Misgurnus anguillicaudatus in Germany

Oriental weatherfish Misgurnus anguillicaudatus , from southern China, have been imported to Germany in large numbers for the garden pond trade. The first record of M. anguillicaudatus from Central Europe, a population that has been established for at least 14 years in a small nature reserve in Germany, is detailed.     

Microsatellite-centromere mapping in the loach, Misgurnus anguillicaudatus

Primer sets for 15 polymorphic microsatellite loci were developed in the loach, Misgurnus anguillicaudatus (Cobitidae) by molecular cloning and sequencing techniques. Mendelian inheritance was confirmed for the 15 loci by examining the genotypic segregation produced with the primer sets in two full-sib families. The loci were mapped in relation to their centromere in four gynogenetic diploid lines, which were induced by inhibition of the second meiotic division after fertilization with genetically inert sperm. Microsatellite-centromere recombination rates ranged between 0.06 and 0.95 under the assumption of complete interference. Thus, these loci are distributed from the centromeres to the telomeres of their respective chromosomes. The success of mitotic gynogenesis, produced by suppression of the first cleavage, was verified by homozygosity at three diagnostic microsatellite loci that exhibited high gene-centromere meiotic recombination rates in the same family. The differences in heterozygosity levels observed with these markers were attributed to differences in the temporal application of heat shock following inert sperm activation.     

Protein polymorphism and geographic variation in the loach Misgurnus anguillicaudatus

Electrophoretic variations of enzymatic and non-enzymatic proteins were investigated in 8 populations of the loach Misgurnus anguillicaudatus. The cathodic hemoglobin and the muscle protein in the region II were shown to be polymorphic, in addition to the five polymorphic proteins previously reported (the anodic hemoglobin, muscle protein in the region III, 6-phosphogluconate dehydrogenase, phosphoglucomutase and aspartate aminotransferase; Kimura, 1976 & 1977). The muscle proteins in the regions I, IV and V were monomorphic. The values of genetic distances between loach populations, calculated over 10 loci, indicated that M anguillicaudatus should be classified into two local races.     

Protein polymorphism and geographic variation in the loach Misgurnus anguillicaudatus

Electrophoretic variations of enzymatic and non-enzymatic proteins were investigated in 8 populations of the loach Misgurnus anguillicaudatus . The cathodic hemoglobin and the muscle protein in the region II were shown to be polymorphic, in addition to the five polymorphic proteins previously reported (the anodic hemoglobin, muscle protein in the region III, 6-phosphogluconate dehydrogenase, phosphoglucomutase and aspartate aminotransferase; Kimura, 1976 & 1977). The muscle proteins in the regions I, IV and V were monomorphic. The values of genetic distances between loach populations, calculated over 10 loci, indicated that M. anguillicaudatus should be classified into two local races.     

Electrophoretic patterns of aspartate aminotransferase in the loach Misgurnus anguillicaudatus

The loach Misgurnus anguillicaudatus is a fresh-water teleost and is known to be widely distributed through Japan, Formosa, Korea and into eastern Asia. The author has reported electrophoretic variation in five different proteins in the loach (Kimura, 1976a, b, c). This note describes a sixth example, electrophoretic variation of aspartate aminotransferase (AAT; EC 2.6.1.1.).     

Geographical variation of lactate dehydrogenase and phosphoglucomutase in the loach Misgurnus anguillicaudatus

One of the two loci controlling muscle lactate dehydrogenase (LDH) was shown to be polymorphic in the loach Misgurnus anguillicaudatus. The result of the survey on four populations suggests that the differences in the LDH gene frequencies are correlated with the geographical distribution of the loach. A fourth phosphoglucomutase allele ( PGM ^D) was found in one of the loach populations examined.     

Geographic variation of muscle adenylate kinase in the loach Misgurnus anguillicaudatus

The electrophoretic pattern of the loach muscle adenylate kinase was composed of one or two major bands. Each major band was preceded by two minor bands. Three codominant alleles were postulated to segregate in loach. Each allele coded for one major band with different mobility.
Adenylate kinase (AK. E.C. 2.7.4.3.) catalyses the reaction 2ADP ATP + AMP. and is known as a heat stable protein constituent of skeletal muscle. Electrophoretic variation of AK has been reported in the pika Ochotona r. rufescens (Vergnes et al. 1974). the teleostean fish Zoarces viviparus (Frydenberg & Si-monsen, 1973), the mussel Mvtilus edulis (Ahmad et al. 1977). and the tunicate Ciona intestinalis (Schmidtke & Engel. 1980). In this note, individual variation of AK in muscle extracts of the fresh-water fish Misgurnus anguillicaudatus is described.
Loach were collected in ponds or purchased from fish shops (Table 1 & Fig. 1). Three populations (OS. AS and KN) were purchased from fish shops in Osaka. Akashi and Kanazawa cities, respectively. Their exact sampling locations were not known. For reference, the locations of these cities are indicated by an open circle in Fig. i. Fish were collected and stored frozen at – 20 ^oC at the sampling time given in Table 1. Muscle extracts were prepared and examined in the period February-April 1981.The method for preparing muscle extract and-the starch gel electrophoretic procedures were the same as those reported previously (Kimura, 1976). The amine-citrate buffer system as described by Clayton & Tretiak (1972) was used. AK was stained by the method of Allendorf et al. (1977). After electrophoresis, an inhibition test was also performed by immersing the gel in 10^-3 M 5.5'-dithiobis-(2-nitrobenzoate) solution for 30 minutes at room temperature.
Under the electrophoretic condition used in the present study, all of the AK     

饥饿和再喂食对泥鳅生化组成的影响

研究了泥鳅(Misgurnus anguillicaudatus)在18±0.5℃下,饥饿20 d和恢复喂食8 d的生化组成变化。结果表明,在饥饿初期,泥鳅鱼体的脂肪和蛋白质含量均明显地下降,饥饿15 d后,脂肪的降低量减少并趋于稳定,而蛋白质的含量则进一步显著降低。这表明在饥饿过程中,泥鳅首先动用机体的脂肪和蛋白质提供能源,饥饿至一定程度后则主要利用蛋白质。恢复喂食后这两种物质的含量均迅速回升,其中恢复喂食5 d后,脂肪和蛋白质的含量均达到初始水平。水分和灰分在饥饿过程中呈上升趋势,恢复后便开始下降;饥饿20 d后,鱼体的饱和脂肪酸含量基本保持不变,而多不饱和脂肪酸和单不饱和脂肪酸含量分别有一定的增加和降低,另外n-3系列脂肪酸含量升高,而n-6系列脂肪酸几乎不变。     

Geographical variation of lactate dehydrogenase and phosphoglucomutase in the loach Misgurnus anguillicaudatus.

One of the two loci controlling muscle lactate dehydrogenase (LDH) was shown to be polymorphic in the loach Misgurnus anguillicaudatus. The result of the survey on four populations suggests that the differences in the LDH gene frequencies are correlated with the geographical distribution of the loach. A fourth phosphoglucomutase allele (PGMD) was found in one of the loach populations examined.