The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration

Sulfide: quinone oxidoreductase (SQR) is a flavoprotein with homologues in all domains of life except plants. It plays a physiological role both in sulfide detoxification and in energy transduction. We isolated the protein from native membranes of the hyperthermophilic bacterium Aquifex aeolicus, and we determined its X-ray structure in the "as-purified,'' substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 angstrom, respectively. The structure is composed of 2 Rossmann domains and 1 attachment domain, with an overall monomeric architecture typical of disulfide oxidoreductase flavoproteins. A. aeolicus SQR is a surprisingly trimeric, periplasmic integral monotopic membrane protein that inserts about 12 angstrom into the lipidic bilayer through an amphipathic helix-turn-helix tripodal motif. The quinone is located in a channel that extends from the si side of the FAD to the membrane. The quinone ring is sandwiched between the conserved amino acids Phe-385 and Ile-346, and it is possibly protonated upon reduction via Glu-318 and/or neighboring water molecules. Sulfide polymerization occurs on the re side of FAD, where the invariant Cys-156 and Cys-347 appear to be covalently bound to polysulfur fragments. The structure suggests that FAD is covalently linked to the polypeptide in an unusual way, via a disulfide bridge between the 8-methyl group and Cys-124. The applicability of this disulfide bridge for transferring electrons from sulfide to FAD, 2 mechanisms for sulfide polymerization and channeling of the substrate, S2-, and of the product, S-n, in and out of the active site are discussed.

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20000 tons dry. The supply of healthy sporelings is key to the success of commercial cultivation of Undaria. Previous studies demonstrated that instead of the zoospore collection method, sporelings can be cultured through the use of gametophyte clones. This paper reports the experimental results on mass culture of clones and sporeling raising in commercial scale. Light had an obvious effect on growth of gametophyte clones. Under an irradiance of 80 mumol m(-2) s(-1) and favorable temperature of 22-25degreesC, mean daily growth rate may reach as high as 37%. Several celled gametophyte fragments were sprayed onto the palm rope frame. Gametogenesis occurred after 4-6 days. Juvenile sporeling growth experiments showed that nitrate and phosphate concentrations of 2.9 10(-4) mol 1(-1) and 1.7 10(-5) mol 1(-1) were sufficient to enable the sporelings to maintain a high daily growth rate. Sporelings can reach a length of 1 cm in a month. Since 1997, extension of the clone technique has been carried out in Shandong Province. Large-scale production of sporelings for commercial cultivation of 14 and 31 hectares in 1997 and 1998 had been conducted successfully.

Study on the concentration and seasonal variation of inorganic elements in 35 species of marine algae

The concentrations of five major and 28 trace elements in 35 marine algae collected along the coast of China were determined by instrumental neutron activation analysis. The concentrations of halogens, rare earth elements and many transition metal elements in marine algae are remarkably higher than those in terrestrial plants. The concentration factors for 31 elements in all collected algae were calculated, those for tri- and tetra-valent elements were higher than those of the mono- and di-valent elements in marine algae. The biogeochemical characteristics of inorganic elements in marine algae were investigated. In addition, the seasonal variation of inorganic elements in Sargassum kjellmanianum was also studied. (C) 1998 Elsevier Science B.V. All rights reserved.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular characterization of an ecdysone inducible gene E75 of Chinese shrimp Fenneropenaeus chinensis and elucidation of its role in molting by RNA interference

Ecdysone inducible gene. E75 is a primary target of ecdysone receptor (EcR). and is found to play a critical role in the molting process of arthropods In this study, a cDNA encoding the E75 of Chinese shrimp Fenneropenaeus chinensis (FcE75) was cloned using RT-PCR and RACE techniques FcE75 cDNA was 3611 bp in length with an ORF of 2394 bp. The deduced amino acid sequence of FcE75 had the highest sequence identity to E75 from a land crab Gecarcinus lateral's and E75 of the shrimp Metapenaeus crisis Quantitative real-time PCR revealed a prominently high expression of FcE75 mRNA in the whole body RNA extract of late premolt period (D3) juvenile shrimp. The role of E75 in the process of shrimp molting was investigated using the RNA interference technique Long double-stranded RNA corresponding to the FcE75 (dsE75) efficiently silenced the FcE75 transcript levels in juvenile F. chinensis. Further, injection with dsE75 completely arrested the molting process in experimental shrimp which eventually caused death Setogenic analysis of the uropods from molt-arrested shrimp, showed defective epidermal retraction, poor development of setae and new cuticle. These results indicate that E75 might be related to the molting process and is essential for proper molting and survival of shrimp This is the first report demonstrating the use of double stranded RNA to elucidate the possible role of E75 in the molting of decapod crustaceans (C) 2010 Elsevier Inc All rights reserved

Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively But its expression level did not change significantly during peptidoglycan (PGN) stimulation The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3) The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way The agglutinating activity could be inhibited by D-mannose. LPS and glucan, but not by D-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate Immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs (C) 2010 Elsevier Ltd All rights reserved

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Genetic analysis of meio- and mito-gynogenetic stocks of Paralichthys olivaceus with microsatellite markers

Twenty microsatellite markers (Po1, Po13, Po33, Po35, Po42, Po48, Po56, Po89, Po91, kop6, kop7, kop8, kop12, kop15, kop18, kop21, kop22, kop23, kop26, Po-strl) were used to assess the meiogynogenetic and mitogynogenetic stocks of the left-eyed flounder, Paralichthys olivaceus, which were derived from single pair crossing. Twelve of the 20 loci utilized showed heterozygosity in the female and were mapped in relation to their centromeres in the meiogynogenetic diploid flounder. Microsatellite-centromere map distance, calculated under the assumption of complete interference, ranged from 15.8 cM for kop22 to 50 cM for Po13, Po56 and Po89. Excluding the kop22, the heterozygosities of the rest of the loci were close to 100%, suggesting the occurrence of near complete interference on the chromosome arms that carried these loci. In the mitogynogenetic diploid flounder, each individual showed exact homozygousity and the segregation profiles did not deviate from the Mendelian 1: 1 pattern. The results indicated that there was no lethal gene linked with the loci analyzed. Such high interference accounted for the high recombination rates and large map distances. The Po13 and Po56 loci, Po91 and kop18 loci, kop15 and kop21 loci are tightly linked on the same chromosome arm in pairs.

Physical mechanism and numerical simulations of surface layer temperature inversion in tropical ocean

The one-dimensional Kraus-Turner mixed layer model improved by Liu is developed to consider the effect of salinity and the equations of temperature and salinity under the mixed layer. On this basis, the processes of growth and death of surface layer temperature inversion is numerically simulated under different environmental parameters. At the same time, the physical mechanism is preliminarily discussed combining the observations at the station of TOGA-COARE 0 degrees N, 156 degrees E. The results indicate that temperature inversion sensitively depends on the mixed layer depth, sea surface wind speed and solar shortwave radiation, etc., and appropriately meteorological and hydrological conditions often lead to the similarly periodical occurrence of this inversion phenomenon.

Ship-board measurements and estimations of air-sea fluxes in the western tropical Pacific during TOGA COARE

Direct air-sea flux measurements were made on RN Kexue #1 at 40 degrees S, 156 degrees E during the Tropical Ocean Global Atmosphere (TOGA) Coupled Ocean-Atmospheric Response Experiment (COARE) Intensive Observation Period (IOP). An array of six accelerometers was used to measure the motion of the anchored ship, and a sonic anemometer and Lyman-alpha hygrometer were used to measure the turbulent wind vector and specific humidity. The contamination of the turbulent wind components by ship motion was largely removed by an improvement of a procedure due to Shao based on the acceleration signals. The scheme of the wind correction for ship motion is briefly outlined. Results are presented from data for the best wind direction relative to the ship to minimize flow distortion effects. Both the time series and the power spectra of the sonic-measured wind components show swell-induced ship motion contamination, which is largely removed by the accelerometer correction scheme, There was less contamination in the longitudinal wind component than in the vertical and transverse components. The spectral characteristics of the surface-layer turbulence properties are compared with those from previous land and ocean results, Momentum and latent heat fluxes were calculated by eddy correlation and compared to those estimated by the inertial dissipation method and the TOGA COARE bulk formula. The estimations of wind stress determined by eddy correlation are smaller than those from the TOGA COARE bulk formula, especially for higher wind speeds, while those from the bulk formula and inertial dissipation technique are generally in agreement. The estimations of latent heal flux from the three different methods are in reasonable agreement. The effect of the correction for ship motion on latent heat fluxes is not as large as on momentum fluxes.

热喷涂锌铝涂层防腐的电化学研究

海洋环境下的钢铁设施结构的腐蚀问题一直是困扰人们的难题，而热喷涂锌铝防腐技术正是解决这一问题的重要技术之一，因此本文着重对锌铝涂层防腐进行电化学上的探讨，以期用于指导防腐的实践。对锌、铝以及三种不同组分(Zn15%Al, Zn30%Al, Zn55%Al)的合金丝制成电极进行极化曲线测试，以判别不同成分的锌铝合金的耐蚀性能, 结果表明，合金的极化特性与锌类似，合金中含铝量越高，其耐蚀性能越强。另对不同方案的热喷涂锌铝复合涂层进行了极化曲线的研究，结果表明，纯锌的腐蚀电流密度比较大，纯铝的腐蚀电流密度最小，锌铝复合层、铝镁合金、铝稀土合金的性能介于两者之间。外层为铝，内层为锌的喷涂层其极化特性与铝类似，钝化状态与铝相近。镁和稀土的加入能够改善铝的牺牲保护能力，并抑制其阴极极化过程，但没有从根本上改变铝的钝化特性。对不同方案的热喷涂锌铝复合层及其与氯化橡胶漆的覆盖层进行了156天的室内盐雾试验，以考查其耐蚀性能的优劣，并对原始试样和定期（1天，4天，8天，16天，34天，75天，156天）取出的腐蚀试样进行交流阻抗谱的测试。结果表明，单纯有机涂层的防腐能力有限，锌铝金属涂层及其与有机涂层的复合层均具有优异的防腐性能。但锌与氯化橡胶漆的配合性能较差，156天后出现较大的破裂鼓泡，而铝与有机层的配合相当得好。所有涂层方案中以喷铝并覆以有机涂层的防护方案较佳。在国内外关于锌铝复合涂层及其有机覆盖层的电化学研究比较少，因此本文在选题上具有新意。通过系统地对锌铝合金、锌铝复合层及有机涂层的防护体系进行了电化学研究，得出了满意的结论，对于指导热喷涂在实际防腐中的应用，选用最佳的防护系统，最大限度地降低腐蚀损失，具有重要意义。

黄海冷水团及邻近海域小型底栖动物多样性及其环境效应

2007年夏季对黄海冷水团及邻近海域共48个站位的小型底栖动物组成、丰度和生物量，以及环境因子进行了科考研究。所调查站位的小型底栖动物平均丰度达2194 ± 1598 inds./10cm2，其中北黄海17个站位平均丰度为3408 ± 1578 inds./10cm2，南黄海31个站位平均丰度为1529 ± 1121 inds./10cm2。调查站位平均生物量为1839 ± 1289 g dwt/10cm2，其中北黄海站位平均生物量为2760 ± 1340g dwt/10cm2，南黄海平均生物量为1335 ± 902g dwt/10cm2。在分选出的共18个小型底栖动物类群中，丰度上均以自由生线虫占绝对优势，达总量的88%，且在南（88.3%）、北黄海（87.7%）基本无差异。在生物量上，同样以自由生线虫贡献最多（42％），多毛类居次（22%），其他生物量较多的还有桡足类（13%）和甲壳类幼体（12%）。在小型底栖动物的垂直分布上，分布于沉积物表层0-2cm的小型底栖动物占79%，次表层2-5cm占17%，最底层5-8cm仅占4%。统计分析表明研究站位小型底栖动物丰度和生物量与沉积物叶绿素a、有机质含量、中值粒径显著或极显著正相关，与水深呈极显著负相关，此外小型底栖动物生物量与沉积物粉砂粘土含量显著负相关。 同年秋季搭载开放航次对黄海5个站位、东海3个站位、南海2个站位的小型底栖动物组成、丰度和生物量，以及环境因子进行了调查研究。对三个海域小型底栖动物的比较研究发现，平均丰度以黄海最高，达2132 ± 946 inds./10cm2，东海次之，为1954 ± 2047 inds./10cm2，而南海仅156 ± 56 inds./10cm2；三海域的平均生物量依次为2193 ± 1148 g dwt/10cm2、1865 ± 1555 g dwt/10cm2和212 ± 22 g dwt/10cm2。在分选出的共14个小型底栖动物类群中，丰度上均以自由生线虫占绝对优势，分别占总量的85%、89%、85%。在生物量上，黄海以自由生线虫贡献最多（33％），多毛类居次（18%）；东海二者比例相近（约37％），而南海则以多毛类占绝对优势（56％），线虫居次（25%）。在小型底栖动物的垂直分布上，三个海区差异较大：分布于沉积物表层0-2cm的小型底栖动物在黄海高达90%，东海仅46%，在南海为63%。统计分析表明，本研究站位小型底栖动物丰度与沉积物中的叶绿素a及脱镁叶绿素a含量和底温呈显著正相关，与水深呈显著负相关。该结果与本航次之后在广东湛江和海南以东的南海海域开展的908调查结果形成了鲜明对照，后者的小型底栖动物及线虫丰度与沉积物中有机质含量呈显著正相关，与水深呈显著负相关，表明近海受人类干扰影响较大。 本文利用微宇宙实验方法，来确定不同浓度梯度的Cu、Pb以及Cu/ Pb混合重金属污染物对青岛湾小型底栖动物（主要是线虫）的影响。加入污染物后，分别在1、3、7、14、21天进行取样分析。结果显示，Cu和Cu/Pb混合高浓度实验单元组的线虫丰度除在第21天有较明显减少外，在整个实验周期内基本没有变化，分析可能系高浓度Cu的固定作用从而使小型底栖动物无法腐烂降解造成的。同一时间尺度上，各重金属污染物实验单元的线虫丰度均高于（或接近于）空白对照组，较高浓度的重金属污染物实验单元的线虫丰度高于（或接近于）较低浓度重金属污染物实验单元，Cu/Pb混合低浓度实验单元的线虫丰度高于同一时间尺度Cu低浓度和Pb低浓度实验单元。推测是由于采样点的线虫群落中存在对Cu和Pb的耐受种或者“机会种”造成的。

浒苔多糖的化学组分及生物学活性的初步研究

浒苔（Enteromorpha）是青岛沿海常见的一种绿藻。2008年6月份，在我国黄海海域爆发了一场大面积浒苔“绿潮”。本文研究了青岛沿海产缘管浒苔(QE)和“绿潮”浒苔(GE)的化学组成，并对其多糖的化学组成、结构及生物学活性进行了初步研究，为进一步开发利用浒苔提供实验依据。 对两种浒苔主要化学组成进行了分析，QE除灰分含量明显高于GE的含量外，水分、碳水化合物、粗脂肪和粗蛋白含量均低于GE的。两种浒苔均含有丰富的氨基酸，其中含量最高的是天门冬氨酸和谷氨酸；必需氨基酸含量占总氨基酸含量的42.755%和44.989%。不饱和脂肪酸含量占总脂肪酸含量的34.156%和34.278%。结果表明浒苔是一种矿物质和蛋白含量高，脂肪含量低，必需氨基酸和必需脂肪酸含量高的优质食物资源。 以水提醇沉法制备浒苔多糖，QE和GE多糖分别命名为QEP和GEP，并利用琼脂糖凝胶离子交换色谱对所得多糖进行分级纯化。高效液相色谱测定单糖组成，两种多糖均为鼠李糖-木糖-葡萄糖醛酸聚合物，其中鼠李糖含量占中性糖的67.55%和65.56%，木糖占中性糖的24.61%和19.33%。离子交换色谱分级结果表明，QEP主要由两种组分，分别为鼠李糖-木糖-半乳糖-糖醛酸聚合物和鼠李糖-木糖-糖醛酸聚合物；GEP主要组分为鼠李糖-木糖-甘露糖-糖醛酸聚合物。利用红外光谱、甲基化分析和核磁共振分析多糖QEP的结构，并利用ChemNMR软件辅助分析NMR谱图，结果表明，多糖主要有(1→2,4)-和(1→4)-连接的鼠李糖残基，(1→4)-连接的木糖残基，硫酸根主要连接在鼠李糖基3位。 研究了QEP及其分级组分对超氧阴离子、羟自由基、1,1-二苯基苦基苯肼(DPPH)的清除作用和还原能力，结果表明各组分均具有一定的清除自由基能力，其中QEP各项清除能力均较强，且还原能力最强，具有良好的抗氧化活性。以透明质酸和甘油作为对照，研究了GEP和本实验室褐藻多糖硫酸酯(FPS)及其衍生物的保湿和吸湿能力，结果表明GEP、低分子量FPS，以及低分子量FPS的离子交换凝胶色谱分级产物均有较强的保湿和吸湿活性，且与天然活性保湿因子透明质酸的保湿和吸湿特征很相似。GEP相对透明质酸、低分子量FPS及其衍生物而言，样品制备简单，原材料来源丰富，是良好的保湿活性物质。