线纹海马Hippocampus erectus的tlr21基因结构及其对CpG-ODN的应答特征

作为先天性免疫反应中重要的模式识别受体之一, TLR(toll-like receptor)基因家族介导的先天性免疫是脊椎动物抵御病原微生物的重要防线。研究分析了线纹海马Hippocampus erectus tlr21基因的序列特征, 并基于抗原注射实验探讨了海马tlr21的免疫功能。线纹海马tlr21基因的开放阅读框长度为2946bp, 编码981个氨基酸, 预测其编码蛋白相对分子量为246.98kDa, 理论等电点为4.78。tlr21编码蛋白主要包含1个信号肽和3个功能结构域: 胞外区具有14个富含亮氨酸重复序列的结构域(LRR), 跨膜区具有富含半胱氨酸的结构域, 胞内区则具有TIR结构域。同源性比较和系统进化分析表明, 线纹海马tlr21基因与虎尾海马Hippocampus comes tlr21基因的同源性最高, 其次与斜带石斑鱼Epinephelus coioides、牙鲆Paralichthys olivaceus、大菱鲆Scophthalmus maximus和红鳍东方鲀Takifugu rubripes的tlr21基因聚为一枝。tlr21基因在线纹海马的脑、鳃、肝、肠、肾、性腺、肌肉和育儿袋各组织均有分布, 肾脏中表达量最高。抗原注射实验结果发现, 线纹海马Tlr21对不同类型CpG寡脱氧核苷酸(CpG-ODNs)的识别能力存在差异, 其中, CpG-2007和CpG-HC4040对海马肾脏tlr21的mRNA表达量具有显著的促进作用(P<0.05)。研究表明, 线纹海马Tlr21能够通过识别含CpG序列的DNA发挥免疫识别作用, 研究结果将有助于系统认识海马免疫系统中TLR家族基因的功能特征, 为建立海马病害防治策略提供理论依据。     

浮游植物metacaspase的分布、结构及其功能特性

精氨酸/赖氨酸特异性半胱氨酸蛋白酶(Metacaspase)在浮游植物程序性细胞死亡(programmed cell death.PCD)过程中起着关键作用。本文利用生物信息学方法分析metacaspase基因在浮游植物中的分布,并以海洋球石藻(Emilianiahuxleyi)metacaspases(EhMCs)为代表分析其结构和功能,依据Uniprot和String数据库挖掘与metacaspase功能相关并参与浮游植物PCD过程的互作蛋白。结果发现:共计29条、四种类型的metacaspases分布于11种浮游植物中,它们的metacaspase蛋白一级结构存在明显差异,但位于p20的组氨酸和半胱氨酸活性位点均具有高度的保守性;EhMCs三级结构与酵母的metacaspase十分相似,暗示它们具有类似的功能;浮游植物metacaspases与多种蛋白存在直接或间接的互作关系,在细胞PCD过程、响应环境胁迫和能量代谢等方面发挥重要作用。另外.metacaspase的磷酸化修饰可能在浮游植物PCD信号转导过程中起调节作用。     

日本囊对虾Cathepsin L基因的鉴定及其在幼体发育中的表达研究

本研究从日本囊对虾（Marsupenaeus japonica）中获得半胱氨酸蛋白酶家族中的cathepsin L基因,命名为Mj-Cathepsin L。Mj-Cathepsin L基因cDNA序列全长1191bp,其开放阅读框编码的Cathepsin L蛋白前体包含327个氨基酸,由信号肽,蛋白前体和成熟蛋白三部分组成。同源序列比对发现日本囊对虾Cathepsin L蛋白与其他已知物种的cahtepsin L蛋白具有较高的同源性。对Mj-Cathepsin L基因在不同组织以及早期幼体幼体发育阶段的研究发现,Mj-Cathepsin L主要在肝胰腺中大量表达,且在幼体发育不同阶段,其表达水平受发育阶段调控,且Mj-Cathepsin L基因显示出与其五个蜕皮时期相关的表达变化,说明Mj-Cathepsin L有可能在日本囊对虾个体发育过程中扮演不同的重要作用。并通过Western Blot方法在日本囊对虾肝胰腺,胃和肠中检测到Cathepsin L酶原和成熟Cathepsin L两种蛋白存在形式。     

厚壳贻贝(Mytilus coruscus)金属硫蛋白MT-10:cDNA克隆、结构分析及铜离子胁迫下的表达

近年来我国沿海海域重金属污染日趋严重,软体动物尤其是双壳贝类响应重金属污染胁迫的研究发展成为目前的研究热点。金属硫蛋白是一类广泛存在于生物体内富含半胱氨酸(Cys)的小分子蛋白,具有结合金属离子的能力,能有效调节生物体细胞内的金属离子平衡,具有清除自由基,重金属解毒的功能,关于厚壳贻贝MT蛋白的研究尚未见报道。本文首次利用RT-PCR和c DNA末端快速扩增技术克隆了厚壳贻贝MT-10蛋白c DNA全长(Mc-MT-10),其包含101bp的5′UTR区,108bp的3′UTR区以及222bp的ORF区,编码73个氨基酸。经比对发现Mc-MT-10富含Cys,且具有9个软体动物中常见的CXC结构以及CKCXXXCXCX结构,系统进化树中同科或属中MT-10聚合成一个分支,显示出高度保守性。采用Q-PCR技术分析了MT-10 mRNA在Cu~(2+)胁迫下厚壳贻贝外套膜及消化腺中表达变化,结果表明Mc-MT-10 mRNA在Cu~(2+)胁迫下两种组织中的表达量急剧升高,说明Cu~(2+)能够诱导Mc-MT-10 mRNA的表达,并且表现出诱导具有阈值性特点。研究结果可为深入研究厚壳贻贝MT蛋白的功能奠定基础,为厚壳贻贝抗重金属污染新品种选育提供数据支持。     

单环刺螠呼吸肠中Caspase-3对硫化物应激的表达特征

Caspase-3是细胞凋亡过程中的关键执行蛋白。本文采用RACE技术克隆了单环刺螠Caspase-3cDNA序列,其全长为1616bp,开放阅读框长759bp,编码252个氨基酸,其中含有Caspase家族保守的结构域(QACRG)和Caspase-3两个特异的切割位点(D96-L97、D135-S136)。体外原核诱导表达获得Caspase-3重组蛋白并制备其多克隆抗体。免疫印迹(Western Blot)检测结果显示,单环刺螠暴露在50μmol/L硫化物环境中,其呼吸肠Caspase-3蛋白含量未见显著变化;而当单环刺螠暴露在150μmol/L硫化物环境中时,其呼吸肠中Caspase-3蛋白的含量于暴露后24h显著提高,暴露72h其含量是同时间对照组的1.8倍。表明低浓度的硫化物对单环刺螠呼吸肠细胞的存活没有明显影响,而高浓度硫化物的长期暴露可能对单环刺螠呼吸肠细胞产生一定程度的致死影响。     

三硬骨鱼类多聚免疫球蛋白受体(pIgR)结构与功能研究进展

多聚免疫球蛋白受体(Polymeric immunoglobulin receptor,pIgR)是介导分泌型免疫球蛋白(Ig)多聚体跨过上皮细胞转运的I型跨膜糖蛋白,已在多种高等脊椎动物中得到克隆。2007年以来,陆续在红鳍东方鲀、斑马鱼及鲤鱼等多种硬骨鱼中克隆得到pIgR基因,并对其进行了表达分析。硬骨鱼类pIgR包含2个免疫球蛋白样功能域,含有4个保守的半胱氨酸(Cys)残基,能很好地结合Ig多聚体,进而介导其向皮肤或肠等黏液中的转运。但目前对pIgR基因功能及作用机制仍了解较少。本文拟对硬骨鱼中pIgR基因分子结构、转胞吞作用、功能及表达模式差异做简要概述,以期为今后深入研究pIgR在黏膜免疫中的功能及转运机制提供指导。     

紫露草微核技术在啤酒酵母菌解毒重金属离子中的观察

金属硫蛋白对某些重金属离子有解毒作用已有不少研究。这类蛋白质相继在哺乳动物、鸟类、鱼类、软体动物组织以及链孢霉、酵母等微生物中发现。它是铁蛋白以外金属含量最高的蛋白质，也是半胱氨酸含量最高的蛋白。用生物方法测定这类蛋白质解毒重金属离子的作用已有报道，而用啤酒酵母发酵液解毒重金属，并用紫露草微核技术测定解毒作用的生物效应还是首次。本文报道这项研究的初步结果。     

VAMP-2在鲈鱼组织中的表达分布

VAMP-2是真核生物中的一种膜受体蛋白,该蛋白在真核生物的胞吐、信号传导、胞内运输和分泌等活动中起到重要作用.采用免疫印迹方法和原位杂交技术,研究了VAMP-2在鲈鱼组织中的表达情况,分别对鲈鱼8种组织中的VAMP-2进行了检测.结果表明,除脑组织外,在肠、肝脏、肾脏、皮肤、脾脏、性腺等组织中也检测到VAMP-2的表达,各组织间的表达水平亦存在差异,肌肉组织未检测到表达.研究表明,鲈鱼的组织中表达的VAMP-2蛋白可能广泛参与鲈鱼的各种生理活动,并影响其生长、发育、繁殖和防卫等各项重要的生物学功能.     

镉对褐菖鲉金属结合蛋白的诱导

金属结合蛋白是一类富含半胱氨酸、不含芳香族氨基酸和组氨酸的低分子量蛋白质。在紫外254nm处具强的吸收。用不同剂量的氯化镉诱导褐菖，通过凝胶过滤从褐菖肝脏提取金属结合蛋白，并测定其结合的稿以及硫醇含量与紫外254nm处的吸收值。结果表明，镉能诱导褐菖金属结合蛋白的合成。     

菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白基因克隆及Cd~(2+)胁迫下的表达分析

金属硫蛋白(Metallothionein,MT)是一类富含半胱氨酸的小分子蛋白质,参与机体重金属解毒和金属元素代谢等生理过程。本研究采用RACE技术,克隆获得了菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白(RpMT)的全长cDNA序列。RpMT的cDNA全长为570bp,编码75个氨基酸,包含15个MT所特有的Cys-Xn-Cys结构。采用实时荧光定量PCR技术,分析了两种壳色菲律宾蛤仔(白蛤和斑马蛤)RpMT基因在Cd2+暴露后的表达变化。结果发现:Cd2+急性和亚慢性暴露均可导致两种壳色蛤仔消化腺和鳃组织RpMT基因表达量的显著上调;暴露后两种壳色蛤仔鳃组织RpMT基因表达量的增加幅度均高于消化腺组织,且以白蛤鳃组织基因表达水平的上调幅度较高。上述结果表明,RpMT可能在菲律宾蛤仔抵御Cd2+胁迫过程中发挥了重要作用。     

青蛤(Cyclina sinensis) TRAF6基因克隆及其在Poly I:C胁迫下的免疫应答

对青蛤(Cyclina sinensis)转录组文库筛选并克隆得到青蛤TRAF6(Cs TRAF6)基因的c DNA序列,其开放阅读框为2337bp,编码778个氨基酸,分子量为86.59k Da。Cs TRAF6蛋白包含RING型锌指结构,两个TRAF型锌指结构,coiled-coil结构域以及MATH结构域。经生物信息学及分子系统学分析表明Cs TRAF6属于TRAF6家族的一员。利用实时荧光定量PCR方法检测了Cs TRAF6基因在青蛤各个组织以及在Poly I:C侵染下在血淋巴中的时序性表达情况。结果表明,Cs TRAF6基因在青蛤血淋巴、闭壳肌、鳃、性腺、外套膜和肝脏中普遍表达,其中在血淋巴中的表达水平最高。在Poly I:C刺激后的3h,青蛤血淋巴中Cs TRAF6的表达量迅速升高,在6h达到最大值,说明青蛤的Cs TRAF6基因参与了病毒类似物侵染下的免疫应答反应。     

饲料中锌含量对斜带石斑鱼(Epinephelus coioides)生长、饲料效率、矿物质含量和体组成的影响

以ZnSO4·7H2O为锌源,用锌添加量为0、20、40、60、80和100mg/kg的实验饲料投喂斜带石斑鱼12周,研究其对斜带石斑鱼生长、饲料效率、矿物质含量和体组成的影响.结果表明,各实验组的增重率,饲料效率,存活率和全鱼、脊椎骨、鳞片的锌含量无显著差异.全鱼、脊椎骨和鳞片的锰含量均随着饲料中锌的添加而降低,锰和锌存在明显的拮抗关系.全鱼的铁含量随着饲料中锌的添加而降低.全鱼、脊椎骨和鳞片的钙、磷、镁含量均不受饲料中锌含量的影响.饲料中添加锌对全鱼水分、灰分,肌肉水分、脂肪,形态学指标,红细胞比容和血红蛋白含量没有显著影响.饲料中不添加锌时,全鱼的蛋白、脂肪和肌肉的蛋白、灰分含量较低.实验结果表明基础饲料中33mg/kg的锌已经满足斜带石斑鱼生长的需求,能使鱼体锌达到饱和.     

Zinc and cadmium metabolism in euglena gracilis: Metal distribution in normal and zinc-deficient cells

The effect of cadmium (5 μ/ml) on the growth of Euglena gracilis is moderated by the addition of supplemental zinc levels above the normal 1 μ/ml level found in the growth medium. Gel chromatography reveals that th majority (ca. 85%) of cytosolic cadmium is localized in a cadmium-binding protein (CdBP). The CdBP appears to elute in a position slightly lower in molecular weight than mammalian metallothionein, contains thiol, and upon ion-exchange chromatography elutes in multiple peaks at ionic strengths greater than mammalian metallothioneins. The remaining cadmium is associated with high molecular weight components. Cytosolic zinc is found primarily (> 80%) in a low molecular weight peak distinct from cadmium, with the remainder as high molecular weight species. As the cells become zinc-deficient and cease proliferation, zinc is lost from the low molecular weight peak while, based on cytosolic protein, the zinc content of the high molecular weight peak remains essentially constant.     

Zinc concentration effect at the organismal,cellular and subcellular levels in the eastern oyster

The purpose of this research was to demonstrate a concentration effect of zinc exposure at organismal, cellular and sub-cellular levels in the eastern oyster and to find associated protein expression signatures (PES) for each concentration of zinc. Oysters were exposed to six concentrations of zinc for 48 h in a controlled environment. At the organismal level, fecal material was observed as a measure of physiological health during metal exposures. At the cellular level, lysosomal destabilization was measured using hemolymph. This cellular response was significant only at the highest concentration, when the fecal index was lowest. Protein responses were monitored in the oyster following exposure to zinc. Gill tissue was excised and homogenized, and then analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and digital image analysis. Protein expression signatures were found to be specific to each concentration. The protein responses were linked to the other biological parameters measured, each of which followed a concentration gradient of zinc.     

Biochemical alterations in juvenile carp (Cyprinus carpio) exposed to zinc: glutathione reductase as a target

The aim of this study was to investigate biochemical changes in juvenile carp (Cyprinus carpio) exposed to zinc chloride (10, 30 and 100 microM) for a period of 48 h. Zinc exposure caused a concentration-dependent reduction in glutathione reductase (GR) activity in gills, liver and brain. Gill glutathione S-transferase (GST) was reduced when animals were exposed to the highest concentration of 100 microM zinc. The phosphorylation of p38(MAPK) increased in the brain of fish exposed to zinc 100 microM, while phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and c-Jun N-terminal protein kinase 1/2 (JNK1/2) remained unchanged. Expression of proteins HSP60 and HSP70 were not affected by zinc exposure. Considering the significant concentration-dependent inhibition of GR in all tissues analyzed, this enzyme could be a potential biomarker of exposure to zinc, which has to be confirmed.     

Influence of capture stress, salinity and reproductive status on zinc associated with metallothionein-like proteins in the livers of three marine teleost species

The influence of capture stress, salinity and reproductive status on zinc associated with the metallothionein-like (MT-like) proteins in the livers of three marine teleost species was investigated. In striped mullet, Mugil cephalus L., the zinc associated with the MT-like protein, as determined by gel-permeation HPLC, increased significantly from approximately 102 to 1162 nmol zinc bound/g wet wt 7 days following capture in the field, handling, transportation to the laboratory and transfer to holding tanks. Significant increases were also observed following exposure to reduced salinity for 7 days. Smaller increases were observed in cultured red drum, Sciaenops ocellatus, livers following exposure to reduced salinity and physical trauma for 7 days. Finally, there was approximately a 2·5-fold increase in the zinc associated with the MT-like protein in livers of female spotted seatrout, Cynoscion nebulosus, collected during the reproductive season compared to regressed animals. These findings emphasize the need for establishing the natural fluctuations in MT levels as well as the response to common physical environmental variables before measurement of MT can be used reliably as a biochemical indicator of environmental contamination.     

The impact of metals on the reproductive mechanisms of the ascidian Ciona intestinalis

Environmental pollution due to anthropogenic activities may exert an adverse impact on the reproductive mechanisms of animals. In particular, environmental chemicals introduced into seawater are able to disrupt the normal development and function of the reproductive system of marine animals. In this study, we have used the whole‐cell voltage clamp technique to examine the effects of four metals – lead, cadmium, mercury, and zinc – on reproductive mechanisms of the marine ascidian Ciona intestinalis (tunicates). In particular, we measured the effect of metals on plasma membrane electrical properties, the steady‐state conductance, the fertilization current in the mature oocyte, and larval development. Results show that oocyte voltage‐gated sodium currents are significantly reduced by all four metals, steady‐state conductance is affected only by zinc, and post‐fertilization contraction is inhibited only by lead. The fertilization current is suppressed in the presence of zinc and mercury. Embryo development up to larval stage is inhibited by zinc and mercury exposure with a reversible effect; however, a long‐term effect on larval morphology was observed. After exposure to cadmium, fertilization occurs but gives rise to abnormal larval development. These findings highlight the point that exposure to metals represents a significant risk factor for the physiology of reproduction of marine species and suggest a possible role of the C. intestinalis as a bioindicator for marine pollution monitoring.     

Influence of two different dietary zinc sources in freshwater prawn Macrobrachium rosenbergii post larvae

A feeding experiment was conducted to evaluate the ef fects of bulk zinc(B-Zn) and zinc nanoparticles(Zn-NPs) on survival, growth, compositions of amino acid and fatty acid, and nonspeci?c immune responses of the freshwater prawn, Macrobrachium rosenbergii post larvae. The B-Zn(size 10 μm)and Zn-NPs(size 50 nm) were supplemented separately with the basal feeds of M. rosenbergii at 60 mg/kg and feed without supplementation of any Zn source was served as a control. M. rosenbergii were fed these feeds for 90 days and the results showed that signi?cant( P <0.05) improvements in survival, growth, feed intake,speci?c growth rate, essential amino acids, unsaturated fatty acids, nonspeci?c immune responses(total haemocytes and dif ferential haemocytes count) of M. rosenbergii fed with B-Zn and Zn-NPs supplemented feeds when compared to control. Among these Zn sources, Zn-NPs supplemented feeds fed prawns showed signi?cantly( P <0.05) better performance than that of B-Zn and control. Hence, present study suggests that the 60 mg/kg Zn-NPs can be supplemented with basal feeds of M. rosenbergii for regulating better survival and growth.     

Effects of heavy metals on the development and survival of abalone Haliotis midae larvae

Despite a growing abalone Haliotis midae industry in South Africa, few studies have measured the effects of heavy metals on larval survival and growth in the face of recent increases in marine pollution. The aim of this study was to quantify the effect of copper on survival and zinc on development of H. midae larvae. Larvae 24 hours old were exposed to either copper (0–30 μg l^–1) or zinc (0–200 μg l^–1) for 48 h before mortality or larval development respectively were quantified. Copper significantly reduced survival, with an LC₅₀ of 5.58 μg l^–1 (5.07–6.15, 95% CI). Although the concentrations of zinc used in the study did not have a significant effect on mortality, there was a significant effect on the incidence of abnormal larvae with an EC₅₀ of 102.25 μg l^–1 (96.68–105.94, 95% CI). At concentrations above the EC₅₀, more than 50% of the larvae showed severe developmental abnormalities. Results suggest that copper poses the greatest risk to abalone larvae as the LC₅₀ was similar to the target concentration for this metal for South African coastal waters. By contrast, zinc is potentially less problematic, with the EC₅₀ for larval development being four times the target concentration for this metal.     

多管藻化学组成特点研究

用普通化学分析法方法对红藻类的一种特殊海藻－－多管藻进行了全面系统的化学成分测定，结果表明，多管藻中铁，锌含量超常丰富，尤其铁元素含量是其他藻类的近千倍，而Ｐｂ、Ｃｄ等重金属含量极微；蛋白质含量在海藻中达３５％以上，这在中国大型海藻中是极为罕见的现象。