单环刺螠呼吸肠JNK通路对硫化物的应激反应

JNK通路是MAPK信号通路的子通路之一,参与生物体的多种应激反应。本研究分析了单环刺螠JNK分子的特征,并对50和150μmol/L硫化物应激下单环刺螠呼吸肠中JNK的反应进行了研究。单环刺螠JNKcDNA全长2 102bp,编码403个氨基酸,推导的氨基酸序列具有JNK家族的典型特征。Western blot(蛋白免疫印迹)检测显示,单环刺螠呼吸肠中JNK总蛋白数量在硫化物暴露期间未见明显的变化;当单环刺螠暴露于硫化物环境中时,其呼吸肠细胞中磷酸化JNK的含量呈现显著性地持续升高趋势,其起始显著升高发生在单环刺螠暴露0.5h,随着暴露时间的延长,磷酸化JNK的含量持续升高,并在150μmol/L硫化物暴露48h时其含量增至最高,为对照组的10.2倍;此外,暴露于50μmol/L硫化物中的单环刺螠呼吸肠磷酸化JNK含量普遍低于150μmol/L硫化物相同暴露时间的磷酸化JNK的含量。研究结果表明,单环刺螠JNK基因拥有进化保守性,且JNK信号通路在单环刺螠呼吸肠应对硫化物中发挥作用。     

单环刺螠中肠和后肠交替氧化酶对硫化物的应激反应

外源性硫化物可通过抑制或阻断线粒体电子传递经典途径而对生物体产生损伤甚至致死,交替氧化酶(Alternative Oxidase,AOX)是线粒体硫化物氧化电子传递分支途径中的1个关键酶。为了研究硫化物环境中单环刺螠的生存对策,本文利用间接竞争性ELISA和酶活性分析等技术检测了单环刺螠在硫化物应激前后中肠和后肠中AOX的蛋白含量以及活性的变化。结果显示:在未处理的单环刺螠中,后肠的AOX蛋白含量和酶活性均高于中肠。当单环刺螠暴露在硫化物(50和150μmol·L-1)环境中时,2个器官的AOX含量和酶活性水平均随着硫化物浓度的提高和暴露时间的延长而提高,并且150μmol·L-1硫化物处理组的单环刺螠2个器官AOX蛋白含量和酶活性普遍高于相同处理时间下的50μmol·L-1组。结合已报道的单环刺螠硫化物应激下细胞色素c氧化酶活性的变化,提出单环刺螠体内存在线粒体电子传递分支途径;提高组织线粒体中AOX活性是其应对环境硫化物毒性的对策之一。     

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。     

铜离子对单环刺螠的毒性及对体壁抗氧化酶活性的影响

本文研究了重金属Cu2+对单环刺螠毒性效应。通过急性毒性试验,确定了Cu2+对单环刺螠24h、48h、72h及96h的半致死浓度,并计算得到其安全浓度为0.0675mg/L,高于渔业水质。通过酶活测定,发现Cu2+对单环刺螠抗氧化酶活性有影响。实验结果表明当暴露于0.25mg/L及0.50mg/L浓度的Cu2+中,24h时,单环刺螠体壁SOD、GPx活性显著提高(P<0.05);之后,随着暴露时间的增长,SOD、GPx、CAT三种酶活性均有所下降,除SOD外,其余两种酶活性均低于对照组,其中0.50mg/L组96h时CAT的活性显著低于对照组(P<0.05),说明Cu2+抑制了GPx及CAT的活性,并导致了虫体的死亡。     

单环刺螠Bcl-xL基因的鉴定及对硫化物的应激反应

Bcl-xL是Bcl-2家族中一类重要的抗凋亡因子,其功能主要是抑制细胞凋亡、促进肿瘤发生。为了探知Bcl-xL在生物硫化物应激中的作用,本实验以耐硫生物单环刺螠(Urechis unicinctus)为研究对象,RACE扩增获得一个长度为1 426 bp的单环刺螠Bcl-xL全长c DNA序列,推导的氨基酸序列包含Bcl-xL4个保守的BH结构域和1个跨膜区域。使用该c DNA的完整开放阅读框序列,构建了原核表达载体p ET28a-Bcl-xL,体外诱导表达该重组蛋白;镍柱纯化得到纯度为90%的纯化蛋白,并制备多克隆抗体。Western blotting结果显示:单环刺螠暴露于50μmol/L硫化物中2 h-24 h内其呼吸肠中Bcl-xL含量显著增加(P0.05),然而在150μmol/L硫化物中48 h-72 h内其呼吸肠Bcl-xL含量显著降低(P0.05),暗示Bcl-xL抑制细胞凋亡途径在单环刺螠应对低浓度硫化物暴露时发挥作用,而在高浓度硫化物暴露下Bcl-xL不能发挥抑制细胞凋亡的作用。     

久效磷对单环刺螠体内几种酶活性影响的初步研究

运用冰冻切片技术和酶组织化学的方法研究了长期暴露于不同质量浓度久效磷(0,50,100,150ng/L)下的单环刺螠(Urechisunicinctus)体内的乙酰胆碱酯酶(AChE)、细胞色素C氧化酶(CCO)、碱性磷酸酶(AKP)的活性变化。实验表明,AChE在单环刺螠的体壁上呈现出较强的酶活性,活性位点处为红棕色的颗粒沉淀、并随久效磷胁迫浓度的提高显色越来越深;消化道切片上基本上没有AChE的活性。CCO在单环刺螠的体壁及肠道上都呈现出较强的酶活性,活性位点显示为棕色;肠道上,不同质量浓度组由低到高其CCO活性位点依次增多、显色也依次加深。低质量浓度久效磷(<150ng/L)胁迫对单环刺螠体内的AKP活性没有明显的影响。     

单环刺螠硫醌氧化还原酶的表达、纯化和复性

硫化物是1种广泛分布的有毒物质。硫醌氧化还原酶(SQR)是硫化物代谢的关键酶。单环刺螠是1种生活在潮间带下区及潮下带中的底栖生物。本研究为了获得具有活性的单环刺螠SQR,将其基因的开放阅读框cDNA克隆到原核表达载体pET28a中,转化大肠杆菌BL21(DE3)进行重组蛋白的表达,后采用稀释复性的方法对重组蛋白进行复性。经IPTG诱导后该重组蛋白主要以包涵体形式存在。用8 mol/L的尿素溶解包涵体后,通过镍离子金属螯合柱纯化获得重组蛋白。首次通过蛋白复性的方法获得具有活性的单环刺螠SQR,确定最佳稀释复性条件为pH=8.0,蛋白浓度20μg/mL,L-精氨酸浓度0.2 mol/L,还原型谷胱甘肽∶氧化型谷胱甘肽=10∶1,复性时间为96 h。     

硫应激单环刺螠差异表达的初步研究

采用mRNA差异显示技术(DDRT-PCR)研究了100μmol/L硫化物环境与正常海水中耐硫生物单环刺螠mRNA的表达差异,初步筛选出7条体腔液细胞的差异表达基因,其中1个cDNA克隆片段与其它物种的MAP kinase kinase kinase(MAPKKK)有较高同源性。进一步采用RT-PCR技术对其在硫化物刺激前后的表达进行了检测,结果显示该基因在对照和应激后2 h6、h的个体中表达较弱;刺激12 h后表达量增高,并随应激时间增加,呈明显上调趋势。表明单环刺螠MAPK信号通路可被硫化物刺激激活,进而通过该信号通路调节下游生理反应,为进一步研究单环刺耐硫特性的分子机制奠定了基础。     

苯并(a)芘影响黑鲷肝脏EROD活性变化的动力学研究

研究了黑鲷(Sparusmacro cephalus)在实验生态条件下暴露于不同浓度苯并(a)芘(benzo(a)pyrene,BaP)后,肝脏乙氧基异吩噁唑酮脱乙基酶(EROD)活性在不同暴露时相的动力学变化情况.EROD活性采用动力学方法测定.实验结果表明,暴露于较低质量浓度BaP(0.5,1.0μg/L)黑鲷肝脏的EROD活性在2 d后极显著高于对照组(P<0.01),2.0μg/L组则在2 h后极显著高于对照组,并且随暴露时间的延长,诱导程度表现出不同的下降趋势,而暴露于高质量浓度组(5.0 μg/L)的肝脏EROD活性极显著性诱导出现在12h.总体上较高质量浓度组(2.0,5.0μg/L)EROD活性出现极显著性诱导的时间比低质量浓度组(0.5,1.0μg/L)早,诱导程度也比低质量浓度组的高.在净化实验中各暴露组肝脏EROD活性均下降,与空白对照组比较均没有显著性差异,说明试验浓度没有超出严重损伤肝脏自身恢复系统的范围,在此浓度范围内污染的鱼通过净化可清除BaP污染.     

干露胁迫对青蛤呼吸代谢及抗氧化相关酶活力的影响

研究了不同时间和温度下干露对青蛤(Cyclina sinensis)失水率、存活率、呼吸代谢相关酶及抗氧化相关酶的影响。结果表明,随着干露时间的增加,青蛤的失水率显著增加,且高温状态下青蛤失水加剧;在3℃和10℃条件下干露48 h未出现死亡现象。在20℃条件下,干露时间超过24 h后,青蛤出现死亡现象,死亡率为3.33%,至48 h死亡率增加至5.33%;随着干露时间的增加,青蛤的呼吸代谢相关酶的活性整体呈现显著下降趋势(P0.05),10℃条件下青蛤的呼吸代谢相关酶的活性整体高于3℃和20℃;随着干露时间的增加,青蛤的抗氧化相关酶的活性呈现与青蛤呼吸代谢相关酶相似的变化趋势,且不同温度条件下,干露时间达到48 h时,青蛤的抗氧化相关酶活性显著低于对照组(P0.05)。综上表明青蛤受干露胁迫时具有一定的自我调节能力,但干露时间超过其耐受限度时会对青蛤的生理状态造成影响。     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.