一种新型贻贝足丝抗氧化蛋白的原核重组表达及功能

贻贝足丝及其足丝蛋白相关研究对于开发新型水下生物粘附剂具有重要的仿生学意义。足丝蛋白在其粘附过程中需要维持一定的还原态,而目前已报道的足丝抗氧化蛋白仅有MFP-6。此前在厚壳贻贝足丝中鉴定到一种新型的富含半胱氨酸和甘氨酸的足丝蛋白质,该蛋白质被命名为Cys/Gly-Rich-Protein（CGRP）,但是CGRP蛋白在足丝中的作用及机制尚不明确。为此,针对CGRP蛋白,在序列分析基础上,利用原核重组表达手段获得其重组蛋白质,采用2,2-联苯基-1-苦基肼基（2,2-diphenyl-1-picryl hydrazyl radical,DPPH）法检测CGRP重组蛋白经不同条件处理后的抗氧化活性。序列分析结果表明,CGRP蛋白含16.5%的半胱氨酸和10%的甘氨酸,其序列中含有两段半胱氨酸位置保守的重复序列,结构预测表明,其优势构象以无规卷曲为主。同源蛋白质搜索结果表明,CGRP蛋白在数据库中尚无高同源性蛋白质存在。通过密码子优化结合原核重组表达策略成功表达出CGRP重组蛋白,所获得的CGRP重组蛋白具有明显的抗氧化活性,且该活性在其半胱氨酸还原后显著增强(0.91±0.05 vs 0.71±0.11, P<0.01)而在半胱氨酸烷基化之后显著下降(0.08±0.03 vs 0.71±0.11, P<0.01),表明CGRP蛋白的抗氧化活性与其序列中半胱氨酸的自由巯基有关。本研究提示,CGRP蛋白是足丝中一种新的具有抗氧化功能的蛋白质,在足丝粘附过程中推测与MFP-6一起参与了富含多巴的足丝粘附蛋白的还原态维持,对贻贝足丝在固化和粘附过程中防止提前粘附具有重要意义。     

Response of Heterotrophic Planktonic Bacteria to the Zebra Mussel Invasion of the Tidal Freshwater Hudson River

Abstract Invasions of aquatic ecosystems by exotic bivalves are known to cause dramatic changes in phytoplankton and some other groups, but their effect on the microbial component is unknown. The invasion of the tidal freshwater Hudson River by the exotic zebra mussel (Dreissena polymorpha) has caused large changes in several components of the Hudson's food web. Planktonic bacteria in the tidal freshwater Hudson are a major part of the food web, and mediate important processes in the carbon budget. We used a long-term data set, spanning four years prior to the zebra mussel (ZM) invasion and four years post-invasion, to describe ZM effects on planktonic bacteria. Small and meso-scale experiments were conducted to specifically examine direct consumption of bacteria by ZM, as well as effects on protozoans. Bacterial abundances in the Hudson have increased roughly 2× since the ZM arrived, making it clear that direct consumption by Dreissena is a minor process. Experiments show that ZM do not remove bacteria from Hudson River water, but are very effective at clearing flagellated protozoans, the major predator of bacteria. The observed changes in bacterial abundance have not been accompanied by equally large changes in bacterial productivity, suggesting growth is primarily limited by carbon supply. Bacterial production has not declined despite a dramatic decline of phytoplankton, confirming previous suggestions that bacteria and phytoplankton are not strongly linked in the Hudson. As a result of the increase in bacterial abundance and removal of phytoplankton, the absolute and relative contributions of bacterial carbon to living particulate organic carbon (POC) standing stocks have increased dramatically. The maintenance of the bacterial component of the Hudson River's food web may be one mechanism whereby consumers are ``insulated' from effects of zebra mussel consumption of phytoplankton carbon. Received: 24 October 1997; Accepted: 9 February 1998     

利用质谱技术结合EST库搜索鉴定厚壳贻贝新型足丝蛋白

贻贝利用足丝粘附于水下各种基质表面.作为一种具有优异粘附性能的生物材料,贻贝足丝蛋白在新型水下粘附剂及表面保护涂层的研制与开发中具有重要的仿生学意义.目前,已报道的贻贝足丝蛋白分子达11种,但是仍然有更多的足丝蛋白分子不为人知.为进一步探讨贻贝足丝蛋白的分子多样性,并从中筛选具有特殊生物学功能的足丝蛋白分子,本文采用鸟枪法-液相色谱-质谱/质谱技术（shotgun-LC-MS/MS）对厚壳贻贝足丝蛋白进行了蛋白质组学分析.将厚壳贻贝足丝分为足丝纤维和足丝盘两部分,每一部分均采用醋酸-尿素溶液,以及醋酸-盐酸胍溶液进行蛋白质抽提;抽提后的足丝蛋白经胰蛋白酶酶解,利用线性离子阱四级杆质谱（LTQ）进行鸟枪法质谱分析.二级质谱图（MS/MS）用以搜索公共数据库中的贻贝表达序列标签（expressed sequence tag,EST）数据库.采用上述方法,获得14种贻贝新型足丝蛋白的高可信度结果及其所匹配的部分或全长cDNA序列;经结构域分析,发现上述新型贻贝足丝蛋白分子的序列中多数包含各种类型的结构域,包括胶原蛋白结构域、C1Q结构域、C1Q结合结构域、微管蛋白辅助折叠结构域、蛋白酶拮抗结构域、VWA结构域、几丁质酶结构域等.在此基础上,对上述新型足丝蛋白在贻贝足丝形成以及粘附方面的功能进行了推测.上述结果对进一步了解贻贝足丝的分子组成以及粘附机理奠定了基础.     

Involvement of Apoptosis in Host-Parasite Interactions in the Zebra Mussel

The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp) can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism.     

A Glycosylated Byssal Precursor Protein from the Green Mussel Perna viridis with Modified Dopa Side-chains

Foot tissue of the green mussel Perna viridis contains a variety of byssal precursor proteins with the unusual redox-active amino acid, Dopa (L-β-3,4-dihydroxyphenyl-α-alanine). Eight proteins were detectable in acidic extracts of the Perna foot by a redox cycling assay with nitroblue tetrazolium. In one of these, however, P. viridis foot protein-1 (Pvfp-1), activity was not due to Dopa, but to another redox-active derivative. Based on specific colorimetric derivatization with Arnow's reagent, ninhydrin and phenylisothiocyanate (Edman), mass spectrometry, the redox-active derivative in Pvfp-1 is not consistent with any known modification. Another uncommon modification of Pvfp-1 involves O-glycosylation of threonine by mannose, glucose or fucose. As in previously characterized fp-1s, the primary sequence of the Pvfp-1 (apparent mass 89?kDa) has two consensus decapeptide motifs; one is APPKPX₁TAX₂K and the other is APPPAX₁TAX₂K, where P is Pro/Hyp, and X₁ and X₂ are difucosylated threonine and a redox sensitive derivative of tyrosine or Dopa, respectively. Of these two unusual residues, X₂ is unique to Pvfp-1, whereas O-glycosylated Thr has been previously detected in freshwater mussel fp-1. The sequence homology of Pvfp-1 with the common structural motifs of the fp-1 protein family strongly suggests that the Pvfp-1 functions as the byssal coating (lacquer) protein.     

Zebra Mussel Antifouling Activity of the Marine Natural Product Aaptamine and Analogs

Several aaptamine derivatives were selected as potential zebra mussel (Dreissena polymorpha) antifoulants because of the noteworthy absence of fouling observed on Aaptos sponges. Sponges of the genus Aaptos collected in Manado, Indonesia consistently produce aaptamine-type alkaloids. To date, aaptamine and its derivatives have not been carefully evaluated for their antifoulant properties. Structure–activity relationship studies were conducted using several aaptamine derivatives in a zebra mussel antifouling assay. From these data, three analogs have shown significant antifouling activity against zebra mussel attachment. Aaptamine, isoaaptamine, and the demethylated aaptamine compounds used in the zebra mussel assay produced EC₅₀ values of 24.2, 11.6, and 18.6 μM, respectively. In addition, neither aaptamine nor isoaaptamine produced a phytotoxic response (as high as 300 μM) toward a nontarget organism, Lemna pausicostata, in a 7-day exposure. The use of these aaptamine derivatives from Aaptos sp. as potential environmentally benign antifouling alternatives to metal-based paints and preservatives is significant, not only as a possible control of fouling organisms, but also to highlight the ecological importance of these and similar biochemical defenses.     

Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.     

Zebra Mussel Infestation of Unionid Bivalves (Unionidae) in North America

SYNOPSIS. In 1989, zebra mussels received national attentionin North America when they reached densities exceeding 750,000/m2in a water withdrawal facility along the shore of western LakeErie of the Laurentian Great Lakes. Although water withdrawalproblems caused by zebra mussels have been of immediate concern,ecological impacts attributed to mussels are likely to be themore important long-term issue for surface waters in North America.To date, the epizoic colonization (i.e., infestation) of unionidbivalve mollusks by zebra mussels has caused the most directand severe ecological impact. Infestation of and resulting impactscaused by zebra mussels on unionids in the Great Lakes beganin 1988. By 1990, mortality of unionids was occurring at somelocations; by 1991, extant populations of unionids in westernLake Erie were nearly extirpated; by 1992, unionid populationsin the southern half of Lake St. Clair were extirpated; by 1993,unionids in widely separated geographic areas of the Great Lakesand the Mississippi River showed high mortality due to musselinfestation. All infested unionid species in the Great Lakes(23) have become infested and exhibited mortality within twoto four years after heavy infestation began. Data indicate thatmean zebra mussel densities >5,000–6,000/m² and infestationintensities >100-200/unionid in the presence of heavy zebramussel recruitment results in near total mortality of unionids.At present, all unionid species in rivers, streams, and akesthat sympatrically occur with zebra mussels have been infestedand, in many locations, negatively impacted by zebra mussels.We do not know the potential consequences of infestation onthe 297 unionid species found in North America, but believezebra mussels pose an immediate threat to the abundance anddiversity of unionids.     

Experimental Pathogenicity of Aeromonas spp. for the Zebra Mussel, Dreissena polymorpha

Experiments were conducted to determine whether species of Aeromonas were pathogenic to the zebra mussel Dreissena polymorpha. A. jandaei, A. veronii, and A. media, identified with Biolog, were originally isolated from dead zebra mussels. When inoculated into living mussels, these bacteria resulted in the mortality of the bivalves. Two additional species, A. salmonicida salmonicida (ATCC 33678) and A. hydrophila (ATCC 7966), were also demonstrated to be pathogenic to the mussels. In addition to the pathogenicity, the data also suggest that the zebra mussels may be an important reservoir for these bacteria in freshwater environments. Received: 26 March 1997 / Accepted: 7 July 1997     

The Byssal Apparatus in the Pacific Mussel,Mytilus trossulus (Bivalvia,Mytilidae), from the Sea of Japan

Biology Bulletin - In the commercial Pacific mussel species, Mytilus trossulus, from the Sea of Japan, features of the morphological structure of the byssal apparatus, byssal threads, and pedal...     

The Physiological Ecology of the Zebra Mussel, Dreissena polymorpha, in North America and Europe

SYNOPSIS. The zebra mussel, Dreissena polymorpha (Pallas), wasintroduced into North America in 1986. Initial North American(N.A.) studies suggested that physiological responses variedbetween N.A. and European populations. However, literature reviewindicates agreement on most aspects of physiological adaptationincluding: respiratory responses; hypoxia/anoxia tolerance;salinity limits; emersion tolerance; freezing resistance; environmentalpH limits; calcium limits; starvation responses; and bioenergeticpartitioning. The main differences among N.A. and European musselsappear to be elevated upper thermal limits and temperaturesfor optimum growth among N.A. populations. N.A. zebra musselsprobably originated from the northern shore of the Black Seain the warmest portion of the mussel's European range. However,most European physiological data come from northern Europe wherepopulations may be adapted to colder temperatures. Alternatively,N.A. research suggests that mussels may have a capacity forseasonal temperature acclimatization such that responses recordedin warmer N.A. waters may be different from those recorded innorthern Europe even after short-term laboratory acclimation.Studies of genetic variation and physiological response amongEuropean and N.A. D. polymorpha populations are required toelucidate the basis for physiological differentiation. Recentlyevolved D. polymorpha has poor resistance adaptations comparedto unionacean and sphaeriid bivalves with longer freshwaterfossil histories. Poor resistance adaptations make it less suitedfor stable habitats, instead, its high fecundities, early maturity,and rapid growth are adaptations to unstable habitats whereextensive resistance adaptations are of little value.     

海洋贻贝粘附蛋白类的结构与功能

海洋贻贝粘附蛋白具有高强度、高韧性和防水性,以及极强的黏附基体的功能,这与其特殊的分子结构、多巴(DOPA)介导的链间交联和与底材之间的相互作用方式有关,并且,它还具有很好的生物相容性和可降解性,是一类极具优势和潜力的生物胶黏剂.本文主要就粘附蛋白分子的结构和功能、粘附蛋白的粘附机理以及有关粘附蛋白生物粘剂等问题对其进行综述     

Cadmium Accumulation and Metallothionein Biosynthesis in Cadmium-Treated Freshwater Mussel Anodonta woodiana

This study investigated the distribution of cadmium (Cd) and the protein level of metallothionein (MT) and examined the relationship of Cd accumulation and the MT concentration in different tissues of freshwater mussel Anodonta woodiana following Cd treatment. The mussels were exposed to Cd (4.21, 8.43, 16.86, 33.72 and 67.45 mg L^-1) for 24, 48, 72 and 96 h, respectively. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analysis. We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg g^-1 (P<0.05). MT levels in the gills and mantle of the mussels increased significantly (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05). In conclusion, our results demonstrated a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment. It is suggested that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.     

Effects of Artificial Filamentous Substrate on Zebra Mussel (Dreissena polymorpha) Settlement

Though a great deal of research focuses on the range expansion and presence of adult zebra mussels, there is still a need to understand the processes of larval settlement and how that relates to adult populations. There is evidence that marine bivalves preferentially settle on filamentous substrates such as hydroid colonies and algae; however, similar studies are rare in freshwater systems. We examined the importance of filamentous substrate for the settlement of the zebra mussel (Dreissena polymorpha) larvae by deploying PVC settlement plates with and without polypropylene filaments in the Bark River for a 6-week period. Larval supply was monitored weekly. Our results suggest that artificial filaments facilitated recruitment, primarily by increasing surface area available for attachment. Mussels on artificial filaments were significantly smaller in size than mussels attached to filamentous or control plate surfaces, providing some evidence that mussels may detach from filamentous substrate after initial settlement. This study adds to our general understanding about the role of filamentous substrates in the process of larval settlement and suggests that substrates colonized by filamentous epibionts may face increased risk of fouling by zebra mussels. An erratum to this article is available at .     

Physiological Aspects of Zebra Mussel Reproduction: Maturation, Spawning, and Fertilization

SYNOPSIS. The prolific reproductive capabilities of the zebramussel, Dreissena polymorpha, have facilitated the rapid spreadand high densities of this biofouling organism since its accidentalintroduction into North America less than 10 years ago. Researchon its reproductive mechanisms and capabilities may be valuablenot only in predicting its further spread, but also in investigatingbasic mechanisms of reproduction and development and in developingnew strategies to mitigate its impact. Since zebra mussels aredioecious and fertilization occurs externally, coordinated maturation,spawning, and other mechanisms have evolved to increase theprobability of successful fertilization. The zebra mussel undergoesan annual cycle of gonadal growth and gamete maturation, culminatingin one or more spawning events in late spring or early summer.Temperature, rates of temperature change, food availability,and effects of neighboring mussels seem to be critical variablesthat determine reproductive responses. Serotonin is a biogenicamine which is implicated in spawning behavior and can reliablytrigger spawning. Serotonin is present in the gonad in neuralvaricosities that encircle groups of gametes, and specific serotonergicligands can mimic or block spawning caused by serotonin. Infemales, serotonin reinitiates meiosis causing maturation fromprophase I to metaphase I prior to spawning. Spawned oocytescontain substances that are species specific sperm chemoattractants.The sequence of binding, entry, and subsequent nuclear movementshave been observed with fluorescence and scanning microscopy.Despite their negative ecological and economic impacts, zebramussels have also provided a new and easily obtainable resourcefor studies of reproductive mechanisms.     

重组贻贝足蛋白的研究进展与应用*

贻贝足蛋白是一类通过贻贝足腺分泌的蛋白质复合物,与基质表面发生反应而产生极强的黏附作用。其在海洋环境中具有强黏附能力、可降解性和优秀的生物相容性等优点,因此常被用做生物医药黏合剂。但提取天然蛋白质受原料来源限制,且工艺烦琐导致价格高昂,阻碍了贻贝足蛋白的进一步应用发展。微生物合成的最新进展为贻贝足蛋白的产出提供了一种新思路,并且具有扩大规模生产的意义。主要综述了贻贝足蛋白的基因工程生产方法,总结了重组蛋白在黏附抗污涂层、组织工程材料等领域的应用现状。同时对其研究方向进行了展望,指出重组贻贝足蛋白的进一步发展的关键技术是解析蛋白质的构效和层级结构,在此基础上提高其异源表达水平,以获得更多生物功效性的衍生产品。     

Bioaccumulation and Partitioning of Cadmium within the Freshwater Mussel Dreissena polymorpha Pallas

Kinetics of uptake, partitioning and elimination of cadmium were investigated in experimental studies with the freshwater mussel, Dreissena polymorpha. The radioactive cadmium isotopes ¹⁰⁹Cd and ¹¹⁵Cd were used as tracers. Shells, soft parts and body fluid of the mussel exhibited considerable differences in accumulation and elimination. Accumulation factors up to more than 70,000 were calculated for the periostracum, whereas accumulation factors for the whole mussels ranging up to 3,000 were calculated. The shells bound a great deal of cadmium, but only loosely, and the metal could be readily eliminated after transfer to uncontaminated water. In contrast, no significant amounts of the cadmium incorporated in the soft parts were eliminated. The results indicate that the major portion of cadmium in the soft parts is strongly bound and cannot be eliminated by exchange processes.