厦门海域岩相海岸带疣荔枝螺（Thais clavigera）与甲虫螺（Cantharus cecillei）性畸变

海产腹足类性畸变以其对有机锡的敏感性、特异性和不可逆性而成为海洋有机锡污染的理想指示种.调查研究了厦门海域岩相海岸带疣荔枝螺(Thais clavigera)和甲虫螺(Cantharus cecillei)的性畸变情况,通过生物学指标IOI(性畸变率)、RPSI(相对阴茎发展指数)、VDSI(输精管发展指数)及SRI(性比指数)综合评价其性畸变发展程度,间接指示厦门周边海域的有机锡污染程度.研究结果发现,在采集疣荔枝螺的17个站点中有5个站点种群性畸变率为100%,其中宝珠屿种群性畸变程度最为严重,VDSI达4,采集甲虫螺的4个站点的种群性畸变率均为100%.性畸变程度较严重的种群普遍集中于厦门西港,并且表现出由港内到港外逐渐减轻的趋势,而白城、会展中心和大嶝岛等位于开阔的厦门东海域的种群性畸变程度最轻,与往年厦门西海域有机锡污染监测数据相比一致,体现出性畸变程度与有机锡污染程度及海港码头远近之间的相互关系.     

桶形芋螺毒液蛋白质组分析及二硫键异构酶的鉴定

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶。在热带药用海洋生物芋螺的毒液中,富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内氧化折叠至关重要。研究上样量、水化方式与时间、除盐步骤、聚焦电压和时间、平衡时间、凝胶电泳方法和染色方法等方面,优化并建立了较为理想的芋螺毒液蛋白样品双向电泳的实验条件与方法;通过双向电泳和MALDI-TOF-MS质谱分析技术,从海南产桶形芋螺(Conus betulinus Linnaeus)毒液蛋白中成功分离鉴定出PDI等9种蛋白;通过双向电泳和PDQuest软件分析了并比较了三种不同大小桶形芋螺毒液总蛋白的差异性,并质谱鉴定出5个明显的差异蛋白点。研究建立了桶形芋螺毒液蛋白双向电泳分析及质谱鉴定的技术方法,为后续大量分离纯化出天然的芋螺PDI酶以及利用蛋白质组学技术方法深入研究芋螺毒液特征提供了重要的基础。     

几种南海芋螺二硫键异构酶的克隆及进化分析

目的:从来自中国南海的4种芋螺中克隆出包含完整3’和5’非翻译区的蛋白质二硫键异构酶(PDI)全基因序列,并对其进行序列及进化分析。方法:根据各种生物PDI基因的保守区域设计引物,利用3’和5’cDNA末端快速扩增(RACE)方法克隆出PDI全基因序列,并通过生物信息学方法对各芋螺PDI序列进行分析。结果与结论:从中国南海玉女芋螺、黑星芋螺、堂皇芋螺、桶形芋螺cDNA中克隆出包含有完整3’和5’非翻译区的PDI全基因序列;分析结果表明各芋螺之间的同源性大于90%,而与对虾、人类、酿酒酵母的同源性均小于60%;各芋螺PDI与其他生物的2个活性位点序列高度保守,而底物结合位点具有物种特异性,进化树显示各芋螺PDI的特征可能受其捕食食性影响。     

海南产桶形芋螺蛋白质二硫键异构酶的鉴定

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶.在热 带药用海洋生物芋螺的毒液中富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内 氧化折叠至关重要.本研究主要采用凝胶过滤层析和制备型Rotofor液相等电聚焦 电泳等多种方法,从海南产桶形芋螺（Conus betulinus Linnaeus）毒管中分离 纯化天然的PDI酶蛋白,经电泳和MALDI-TOF MS质谱鉴定分析确证获得了高纯度 的桶形芋螺PDI酶,建立了天然芋螺PDI酶分离纯化的技术方法. 以芋螺毒素线性 肽K412为底物进行了PDI酶活性鉴定.结果表明,该分离纯化的PDI酶能够促进K412 的氧化折叠.由于芋螺毒素的氧化折叠非常复杂,且氧化折叠后具有正确二硫键连 接方式的芋螺毒素才具有各种药理活性,因此,本研究结果为后续PDI酶在种类繁 多的芋螺毒素氧化折叠中的应用及其作用机制研究提供了重要的物质基础.     

织锦芋螺毒素的研究进展

芋螺毒素是来源于芋螺的毒液的活性多肽,由于其分子质量小、结构多样、作用靶点广泛、功能专一、组织特异性强等优点,广泛地被用作细胞中各种具有重要生理功能靶点的探针,以及作为新药的先导化合物甚至直接作为新药开发.芋螺可以分为食鱼、食软体动物和食虫3种类型,织锦芋螺是一种分布广泛的食软体动物芋螺.作为毒性最强的芋螺品种之一,织锦芋螺毒素成为食软体动物类芋螺毒素研究的代表.现对20世纪90年代末至今的织锦芋螺毒素方面的研究进行了综述.     

芋螺毒素MⅦA研究进展

来自芋螺(Conus)的芋螺毒素(conotoxin)是一类具有独特神经药理活性作用的多肽,它们能够高特异选择性识别、作用其受体,这是在五千万年进化史中与其受体相互作用形成的。其中,w-芋螺毒素MⅦA来自致幻芋螺(Conusmagus),是一种高选择性的N-型电压敏感型Ca^2 通道阻断剂。研究表明它具有强大的镇痛活性,并对脑缺血所造成的神经损伤具有保护作用。本综述芋螺毒素MⅦA的相关基础、临床前及临床研究。     

海南产桶形芋螺线粒体基因组DNA提取条件的优化

提取海南产桶形芋螺线粒体基因组完整DNA (mtDNA),并对提取条件进行优化。以桶形芋螺腹足肌肉、毒腺和肝胰脏三个不同组织为材料,分别采用改进高盐沉淀法、细胞器/磁珠法和试剂盒提取三种方法,提取桶形芋螺mtDNA,并利用琼脂糖凝胶电泳和紫外分光光度计对提取mtDNA的纯度和浓度进行测定。以coxⅠ-rRNA小亚基基因和α-芋螺毒素基因设计引物,通过PCR反应来确证所提取的DNA确实是mtDNA。试剂盒法提取肝胰脏、高盐沉淀法提取肝胰脏和腹足肌肉组织这三种方法的产率很高,分别为44.4μg/mg、43.3μg/mg和32.6μg/mg。A260/280比值表明,改进高盐沉淀法提取毒腺和腹足肌肉组织,细胞器磁珠法提取腹足肌肉组织的mtDNA纯度很高。综合比较,采用改进高盐沉淀法,利用桶形芋螺腹足肌肉组织所提取的mtDNA产率高、质量好、纯度高。高质量芋螺mtDNA的获取为利用分子生物学方法对芋螺进行遗传进化分析和系统分类提供了基础。     

重组芋螺毒素GeXIVAWT的表达、纯化和鉴定

芋螺毒素GeXIVAWT是来自食虫将军芋螺(Conus generalis Lonnaeus)毒管中,具有两对二硫键的28肽.通过化学合成这种芋螺毒素产量低、成本高、难以纯化.利用简单快速的重组表达来生产富含二硫键的芋螺毒素有可能成为有效的新途径.通过对芋螺毒素GeXIVAWT的基因进行密码子优化,人工合成引物来构建表达载体pET22b(+)/pelB-GeXIVAWT.融合了信号肽的重组芋螺毒素以包涵体的形式在大肠杆菌中获得了高效表达.利用低浓度尿素洗涤和超滤管进行纯化无组氨酸标签的重组芋螺毒素pelB-GeXIVAWT,再利用稀释复性的方法将无活性的包涵体转变成具有活性的重组芋螺毒素.复性后的重组蛋白采用反相高效液相色谱进一步纯化后进行了质谱鉴定.活性实验表明重组芋螺毒素pelB-GeXIVAWT具有抑制昆虫细胞Sf-9的生长,为研究芋螺毒素作为生物杀虫剂奠定基础.     

桶形芋螺毒管蛋白质组双向电泳条件优化

目的：优化双向电泳的条件,建立适用于桶形芋螺毒管蛋白质组分析的双向电泳方法。方法：对毒管蛋白的提取、上样量及SDS-PAGE凝胶浓度等影响因素进行优化。结果：乙酸提取法适宜于毒管蛋白的提取,对于pH3～10、17cm的IPG胶条,当上样量为0.75mg,聚焦70000Vhr,SDS-PAGE凝胶浓度为15%时,可提高双向电泳的分离效果,所得蛋白点清晰、数目达到1003个。结论：采用优化的条件进行双向电泳,能得到分辨率高、重现性好、完整的双向电泳图谱,为后续桶形芋螺毒管蛋白质组学研究打下基础。     

μ芋螺毒素基因的串联表达

芋螺毒素是一类小分子多肽,专性作用于生物系统的离子通道及其它神经递质。针对自然产芋螺毒素产量小、采集、提取困难等因素,采用基因表达的方法,将μ-芋螺毒素基因进行串联,在大肠杆菌中得到了有效地表达。     

The neuropeptide APGWamide induces imposex in the mud snail, Ilyanassa obsoleta

We investigated whether neuropeptides which control sexual differentiation in mollusks can induce imposex-a condition where female snails grow male accessory sex organs after exposure to tributyltin (TBT). Mud snails, Ilyanassa obsoleta, were dosed with one of four neuropeptides: APGWamide, conopressin, LSSFVRIamide, or FMRFamide for seven or fourteen days. TBT and testosterone (T) were used as positive controls and induced imposex as expected. APGWamide significantly induced imposex, with a threshold dose near 10(-)(16) moles. The other neuropeptides had no effect on imposex induction. We propose that TBT could act as a neurotoxin to induce imposex via abnormal release of APGWamide.     

New implications in the use of imposex as a suitable tool for tributyltin contamination: experimental induction in <Emphasis Type="Italic">Hexaplex trunculus</Emphasis> (Gastropoda,Muricidae) with different stressors

Imposex, i.e. the development of additional male sex organs (penis and/or vas deferens), in females of gonochorist marine and freshwater gastropods, is known to be caused by tributyltin (TBT), and it has been widely used as a biomonitoring tool in environmental surveys for TBT pollution assessment. In this study, we experimentally tested the potential to induce imposex by another endocrine disruptor (polychlorinated biphenyls [PCBs] mixture—Aroclor 1260). Adults of Hexaplex trunculus with low imposex level, coming from an Italian Marine Protected Area, were injected separately with different doses of tributyltin chloride (TBTCl) and Aroclor 1260. The compounds were dissolved in ethanol and the organisms were narcotised by immersion in MgCl₂ solution before injection. Before and after the experiment, butyltin compounds (BuTs) and PCB tissue concentrations were determined. A significant increase in imposex with respect to non-treated organisms was observed in all treatments, including artefact controls. No clear correlation was observed between BuTs and PCB tissue concentrations and indices of imposex incidence. Based on these results, no assumption can be formulated about PCB effect on imposex development. Nevertheless, they suggest that the imposex level increase, at least in H. trunculus, in laboratory conditions might not be caused by TBT only, but it would rather be a non-specific response to different stress stimuli.     

Imposex in Hexaplex trunculus at Some Sites on the North Mediterranean Coast as a Base-Line for Future Evaluation of the Effectiveness of the Total Ban on Organotin based Antifouling Paints

Imposex – the superimposition of male sexual organs (penis and vas deferens) onto female Neogastropods such as Hexaplex trunculus (Linné, 1758) – is used world-wide as a biomarker of ecological impact of organotin based antifouling biocides (TBT and TPhT). To limit the impact of organotin pollution, since January 1, 2003, the International Maritime Organization (IMO) has enacted a global ban on the use of organotin compounds in antifouling systems. It is important to record imposex levels and organotin contamination before the implementation of the ban, in order to assess the current situation and be able, in the future, to verify the effects of the International Protocol. In this paper, recent imposex data measured in populations of Hexaplex trunculus from three different Mediterranean regions are compared: the Ligurian Sea (Italy), the Lagoon of Venice (Italy) and the western coast of Istria (Croatia). In the two former locations, a partial ban on TBT has been in force for vessels less than 25 m since 1982, while in the latter region no restrictions on organotin antifouling paints have been applied yet. Gastropod samples collected from the Venice lagoon were analysed with an acid extraction followed by Grignard derivatisation, clean up and GC-MS determination, in order to relate the levels of TBT, TPhT and their metabolites with the imposex degree detected. Biological data show that the levels of imposex were very high (VDS from 4.3 to 5) in all the sampling sites considered, particularly in the Croatian coast stations. The concentrations of organotin compounds – butyltins and phenyltins – measured in the samples from the lagoon of Venice were found to partition differently in the visceral coil and in the rest of the soft body of the analysed organisms.     

Anti-androgenic effects of sewage treatment plant effluents in the prosobranch gastropod Nucella lapillus

Recent studies performed under laboratory conditions have shown that single exposure to high levels of several xenoestrogens is able to induce imposex in at least two neogastropod species. In an attempt to evaluate if xenoestrogens, at environmentally relevant conditions, do contribute to imposex induction, we have tested the effects of a mixture containing xenoestrogens (municipal sewage effluents) on imposex development in the dogwhelk Nucella lapillus. Exposure for 3 months to the raw (0.25% and 1%) and the final sewage effluent (12.5% and 50%) rendered no increase in the severity of imposex. Conversely, as exposure to high concentrations of natural steroids, estradiol and estrone, had previously been shown to partially rescue imposex development under laboratory conditions, we have also tested if exposure to the final sewage effluent could ameliorated the severity of imposex induction by tributyltin (TBT). The results demonstrated that co-exposure to the final sewage effluent leads to a decrease trend in the severity of imposex in the presence of TBT. Within the studied imposex parameters, the Relative Penis Size index (RPSI) was the most affected with a 50% decrease in the effluent 12.5% plus TBT exposed group and 25% decrease in the effluent 50% plus TBT, if compared with the TBT alone. Overall, our results give further support to the use of imposex in N. lapillus as a specific biomarker of TBT contamination. However, in areas of high inputs of sewage effluents, the assessment of TBT contamination by the use of the imposex phenomenon should ideally also include data on the tissue levels of butyltins.     

TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level

Tributyltin (TBT) exposure at different concentrations (5, 60, and 100 ng TBT as Sn/l) induces a concentration- and time-dependent imposex (=pseudohermaphroditism) development in femaleNucella lapillus andHinia reticulata. In both species the average imposex stage, termed as vas deferens sequence (VDS) index, and the average female penis length increases with increasing TBT concentration and duration of TBT exposure. Testosterone added at a concentration of 500 ng/l induces a faster and more intensive imposex development compared to that induced by the TBT concentrations used in the present experiments. Radioimmunological determination of endogenous steroid content reveals increasing testosterone titres in female gastropods exposed to TBT which correlate with the TBT concentration used and the duration of the experiment. The most marked and highest increase of the endogenous testosterone level is exhibited by females, of both species exposed to testosterone. Simulataneous exposure to TBT and to the antiandrogen cyproterone acetate which suppresses imposex development completely inN. lapillus and reduces imposex development strongly inH. reticulata proves that the imposex-inducing effects of TBT are mediated by an increasing androgen level and are not caused directly by the organotin compound itself. Further-more, TBT-induced imposex development can be suppressed in both snails by adding estrogens to the aqueous medium. These observations suggest that TBT causes an inhibition of the cytochrome P-450 dependent aromatase system which catalyses the aromatization of androgens to estrogens. The increase of the androgen content or the shift of the androgen-estrogen balance in favour of androgens induces the development of pseudohermaphroditism in marine prosobranchs. Artificial inhibition of the cytochrome P-450 dependent aromatase system using SH 489 (1-methyl-1,4-androstadiene-3,17-dione) as a steroidal aromatase inhibitor and flavone as a nonsteroidal aromatase inhibitor induces imposex development inN. lapillus as well as inH. reticulata.     

New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus

In an attempt to clarify the mechanism(s) of tributyltin-mediated imposex induction in females of the neogastropod Nucella lapillus, dogwhelks collected in an almost imposex free population were exposed to several treatments for a 3 month-period, and the effects on imposex induction and testosterone/estradiol levels were evaluated. As a positive control, tributyltin (50 ng TBT Sn/L) clearly induced imposex and led to a significant increase in the severity of the phenomenon. In contrast, although a selective P450 aromatase inhibitor (formestane at 0.3 mg/L) was capable of imposex induction, it failed to increase its severity. A vertebrate androgen receptor (AR) antagonist (cyproterone acetate at 1.25 mg/L) in combination with TBT completely blocked the imposex induction capacity of TBT. On the other hand, an estrogen receptor antagonist (tamoxifen at 0.3 mg/L) rendered no effect. The determination of steroid levels in female specimens revealed that TBT induces an elevation of free testosterone (but not the total amount, free+esterified), while the co-administration of the anti-androgen and TBT was able to rescue the increase of free testosterone levels. Despite a minor decrease in the amount of testosterone-fatty acid esters in the TBT group, significant differences in esterified testosterone were not found among treatments. On the contrary, free estradiol levels were elevated in the TBT, anti-androgens and TBT plus anti-androgens groups. These results indicate that free estradiol biosynthesis in TBT-exposed females does not seem to be affected. Overall, our results demonstrate that a selective aromatase inhibitor can induce imposex in N. lapillus but not to a similar extent of TBT, which may suggest the involvement of other mechanism in imposex induction, besides aromatase inhibition. Additionally, the study points to the involvement of AR receptors in imposex induction.     

Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds

Molluscs can conjugate a variety of steroids to form fatty acid esters. In this work, the freshwater ramshorn snail Marisa cornuarietis was used to investigate sex differences in endogenous levels of esterified steroids. Testosterone and estradiol were mainly found in the esterified form in the digestive gland/gonad complex of M. cornuarietis, and males had higher levels of esterified steroids than females (4-10-fold). Additionally, the ability of several xenobiotics, namely tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN) to interfere with the esterification of testosterone and estradiol was investigated. All three compounds induced imposex - appearance of male sexual characteristics in females. Exposure to TBT led to a decrease in both esterified testosterone (60-85%) and estradiol (16-53%) in females after 100 days exposure, but had no effect on males. Exposure to FEN and MT did not alter levels of esterified steroids in males or in females, although exposed females developed imposex after 150 days exposure. The decrease in esterified steroids by TBT could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, which catalyzes the esterification of steroids. In fact, ATAT activity was marginally induced in organisms exposed to TBT for 50 days (1.3-fold), and significantly induced in males and females exposed to MT for 50 days (1.8- and 1.5-fold, respectively), whereas no effect on ATAT activity was observed after 150 days exposure.     

三种前鳃亚纲海产腹足类性畸变现象的组织学研究

有机锡污染可以导致海产腹足类雌性个体产生性畸变现象。本文报道了阿文绶贝(Mauritia arabica)、褐棘螺(Chicoreus brunneus)和桶形芋螺(Conus betulinus)三种前鳃亚纲腹足类正常雄性个体和性畸变个体雄性生殖器官的组织学结构。结果表明,不同种间雄性个体的输精管和阴茎的结构存在开放和封闭两种类型,封闭型是由开放型进化而来。虽然性畸变个体的雄性生殖器官与正常雄性个体的在组织结构上无明显差异,但性畸变个体的雄性生殖器官并不完整,无法行使生殖功能。由于内分泌扰乱物质对人和动物影响的相似性,使得海产腹足类性畸变现象应受到人们的重视。     

Transcriptomics and in vivo tests reveal novel mechanisms underlying endocrine disruption in an ecological sentinel,Nucella lapillus

Anthropogenic endocrine disruptors now contaminate all environments globally, with concomitant deleterious effects across diverse taxa. While most studies on endocrine disruption (ED) have focused on vertebrates, the superimposition of male sexual characteristics in the female dogwhelk, Nucella lapillus (imposex), caused by organotins, provides one of the most clearcut ecological examples of anthropogenically induced ED in aquatic ecosystems. To identify the underpinning mechanisms of imposex for this ‘nonmodel’ species, we combined Roche 454 pyrosequencing with custom oligoarray fabrication inexpensively to both generate gene models and identify those responding to chronic tributyltin (TBT) treatment. The results supported the involvement of steroid, neuroendocrine peptide hormone dysfunction and retinoid mechanisms, but suggested additionally the involvement of putative peroxisome proliferator–activated receptor (PPAR) pathways. Application of rosiglitazone, a well‐known vertebrate PPARγ ligand, to dogwhelks induced imposex in the absence of TBT. Thus, while TBT‐induced imposex is linked to the induction of many genes and has a complex phenotype, it is likely also to be driven by PPAR‐responsive pathways, hitherto not described in invertebrates. Our findings provide further evidence for a common signalling pathway between invertebrate and vertebrate species that has previously been overlooked in the study of endocrine disruption.     

Inhibition of human cytochrome P450 aromatase activity by butyltins

Organotin compounds are widely used as antifouling agents and bioaccumulate in the food chain. Tributyltin chloride (TBT) has been shown to induce imposex in female gastropods. On the basis of this observation it has been suggested that TBT acts as an endocrine disrupter inhibiting the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. However, to date, the molecular basis of TBT-induced imposex and in particular its putative inhibitory effects on human aromatase cytochrome P450 activity have not been investigated. Therefore, we examined the effects of the organotin compounds tetrabutyltin (TTBT), TBT, dibutyltin dichloride (DBT) and monobutyltin trichloride (MBT) on human placental aromatase activity. TBT was found to be a partial competitive inhibitor of aromatase activity with an IC(50) value of 6.2 microM with 0.1 microM androstenedione as substrate. TBT impaired the affinity of the aromatase to androstenedione but did not affect electron transfer from NADPH to aromatase via inhibiting the NADPH reductase. DBT acted as a partial but less potent inhibitor of human aromatase activity (65% residual activity), whereas TTBT and MBT had no effect. The residual activity of TBT-saturated aromatase was 37%. In contrast, human 3beta-HSD type I activity was only moderately inhibited by TBT (80% residual activity). Moreover, neither TTBT or DBT nor MBT inhibited the 3beta-HSD type I activity. Together, these results suggest that the environmental pollutants TBT and DBT, both present in marine organisms, textile and plastic products, may have specific impacts on the metabolism of sex hormones in humans.