Molecular cloning and expression pattern of 14 kDa apolipoprotein in orange-spotted grouper, Epinephelus coioides

A novel fish-specific apolipoprotein (apo-14 kDa) has been recently cloned from eel and pufferfish. However, its expression pattern has not been elucidated. in this study, EcApo-14 has been screened from hypothalamic cDNA library of male orange-spotted grouper, which shows 62.9%, 51%, 46.9%, 43.2%, and 31.9% identities to Apo-14 of European flounder, pufferfish, Japanese eel, gibel carp, and grass carp, respectively. RT-PCR analysis reveals that this gene is first transcribed in neurula embryos and maintains a relatively stable expression level during the following embryogenesis. EcApo-14 transcripts are at a very high level during embryonic and early larval development in the yolk syncytial layer (YSL), and decrease in YSL and form intense staining in liver at 3 days after hatching. In adult tissues, EcApo-14 is predominantly expressed in liver and brain. The data suggested that EcApo-14 might play an important role in liver and brain morphogenesis and growth. (c) 2005 Elsevier Inc. All rights reserved.

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6 - 99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.

Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinensis

A copper/zinc superoxide dismutase (Cu/ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene of the human parasite Clonorchis sinensis have been cloned and their gene products functionally characterized. Genes Cu/ZnSOD and MnSOD encode proteins of 16 kDa and 25.4 kDa, respectively. The deduced amino acid sequences of the two genes contained highly conserved residues required for activity and secondary structure formation of Cu/ZnSOD and MnSOD, respectively, and show up to 73.7% and 75.4% identities with their counterparts in other animals. The genomic DNA sequence analysis of Cu/ZnSOD gene revealed this as an intronless gene. Inhibitor studies with purified recombinant Cu/ ZnSOD and MnSOD, both of which were functionally expressed in Escherichia coli, confirmed that they are copper/zinc and manganese-containing SOD, respectively. Immunoblots showed that both C. sinensis Cu/ZnSOD and MnSOD should be antigenic for humans, and both, especially the C. sinensis MnSOD, exhibit extensive cross-reactions with sera of patients infected by other trematodes or cestodes. RT-PCR and SOD activity staining of parasite lysates indicate that there are no significant differences in mRNA level or SOD activity for both species of SOD, indicating cytosolic Cu/ZnSOD and MnSOD might play a comparatively important role in the C. sinensis antioxidant system.

Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi

A viperin gene has been cloned from the mandarin fish (Siniperca chuatsi). From the first transcription initiation site, the mandarin fish viperin gene extends 3163 nucleotides to the end of the 3' untranslated region, and it contains six exons and five introns. The open reading frame of the viperin transcript has 1062 nucleotides which encode a 354 amino acid peptide. The amino acid sequence of mandarin fish viperin shows high identities with its homologues in teleosts and mammals except for the first 70 amino acids. A characteristic feature in the viperin promoter region was the presence of five putative ICSBP (IRF8) binding sites and one IRFI binding site. The viperin gene expressed mainly in lymphoid tissues before stimulation, but its expression can be examined in almost all the organs investigated after stimulation with virus or Poly I:C. The expression pattern and promoter sequence may be considered as the indirect evidence that the transcription of viperin is regulated by interferons or interferon induced genes. (C) 2004 Elsevier B.V. All rights reserved.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae)

The genome segments 1, 2, and 3 of the grass carp reovirus (GCRV), a tentative species assigned to genus Aquareouirus, family Reouiridae, were sequenced. The respective segments 1, 2, and 3 were 3949, 3877, and 3702 nucleotides long. Conserved moths 5' (GUUAUUU) and 3' (UUCAUC) were found at the ends of each segment. Each segment contains a single ORF and the negative strand does not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VPI might represent the viral guanyly/methyl transferase (involved in the capping process of RNA transcripts) and VP3 the NTPase/helicase (involved in the transcription and capping of viral RNAs), The highest amino acid identities (26-41%) were found with orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses, and orthoreoviruses, It should also permit to precise the taxonomic status of these different viruses. (C) 2000 Academic Press.

猪场废水短程硝化反硝化-厌氧氨氧化脱氮研究

畜禽废水是农村水环境污染的主要来源之一，其处理的难点在于脱氮。传统生物脱氮法具有能耗高、需大量外加碳源等缺点，开发低成本、高效率的新型生物脱氮技术具有重要意义。 本研究将短程硝化反硝化和厌氧氨氧化两种脱氮新技术结合，让前者为后者创造去除可降解COD、降低总氮负荷、调整pH、调整氨氮和亚硝酸盐氮浓度比例等进水条件，而后者可在无需外加碳源的条件下进一步脱氮，二者结合可成为高氨氮、低C/N废水脱氮的新途径。 试验以低碳氮比猪场废水为研究对象，首先进行了短程硝化反硝化预处理研究，同时启动并运行调控厌氧氨氧化反应器，最后以经过短程硝化反硝化预处理的猪场废水为进水，进行厌氧氨氧化脱氮考察。实验表明：（1）短程硝化反硝化作为厌氧氨氧化的预处理工序是可行的。猪场废水通过短程硝化反硝化，可以达到基本去除可生化COD、部分脱氮、控制出水氨氮和亚硝酸盐氮浓度之比在1︰1左右、pH在7.5～8.0的目的， COD和总氮平均去除率分别为64.3%、49.1%,出水可达到厌氧氨氧化反应的进水要求。（2）采用模拟废水启动厌氧氨氧化反应器，经过5个月左右的运行调控，反应器启动成功并稳定运行，最高总氮去除率为87.1%，总氮容积去除率最高达到0.14kg/m3.d；整个稳定阶段，氨氮、亚硝酸盐氮、硝酸盐氮的变化量之比为1︰1.21︰0.33。（3）经过短程硝化反硝化预处理的猪场废水厌氧氨氧化脱氮效果稳定，氨氮、亚硝酸盐氮、总氮、COD的平均去除率分别为93.0%、99.4%、84.6%、18.1%，处理效果与模拟废水处理系统相比无明显变化。（4）经过短程硝化反硝化预处理后，猪场废水中残留有机物成分在厌氧氨氧化反应过程中无显著变化，主要为酯类和烷烃类物质；残留有机物对厌氧氨氧化效果无明显影响。（5）采用PCR技术进行特殊功能菌种检测，结果表明模拟废水处理系统和猪场废水处理系统的菌群中均含有厌氧氨氧化菌和好氧硝化菌；通过blast比对，厌氧氨氧化菌扩增序列与未培养的Planctomycetales菌和Candidatus Brocadia fulgida菌16S rRNA部分序列相似性分别为95％、90%。（6）MPN法菌种计数结果显示，模拟废水处理系统和猪场废水处理系统的菌群中均含有硝化细菌、亚硝化细菌和少量反硝化菌，实验条件下的微生物系统是一个厌氧氨氧化菌与好氧硝化菌、反硝化菌共存的系统。 Poultry wastewater is one of the main source of water pollution in rural areas，and nitrogen removal is the most difficult part in treating poultry wastewater. There are some disadvantages in traditional nitrogen removal, such as high energy consumption and more additional organic carbon. It is important to develop ecolomical and efficient technologyies. Shortcut nitricfication/denitrification, as a pretreatment process, was combined with Anammox in this research, so that part of total nitrogen and most degradable COD could be removed by the former, and further nitrogen removal could be implemented by the latter. The combination of the two technologies was a new approach to treat wastewater with high ammonium and low C/N. Piggery wastewater with low C/N was treated in lab-scale experiment. Firstly, shortcut nitrification/denitrification was investigated, and Anammox reactor was started up successfully at the same time. Then piggery wastewater after pretreatment was treated by Anammox. The results showed :(1) It was feasible to take nitrification/denitrification as the pretreatment process of Anammox. By using this process, part of total nitrogen and COD were removed, the ratio of ammonium and nitrite reached around 1︰1 and the pH was about 7.8, which were favorable for Anammox. The average removal percentage of COD and total nitrogen were about 64.3% and 49.1%, respectively. (2) Simulated wastewater was used to start up Anammox reactor. The reactor was started up successfully within 5 months and stable performance was achieved. The highest nitrogen removal reached 87.1% and the biggest volumetric total nitrogen removal rate reached 0.14kg/m3.d. The average ratio of ammonium, nitrite and nitrate was 1:1.21:0.33. (3)Taking the effluent of shortcut nitrification/denitrification as the influent, the nitrogen removal efficiency of Anammox was stable, and the the average removal percentage of ammonium, nitrite, total nitrogen and COD were 93.0%, 99.4% , 84.6% and 18.1%, respectively, which had little difference with that by using simulated wastewater..(4) After pretreatment, the residual organic carbon in piggery wastewater showed no obvious change during the Anammox process, and the main organic compounds were saturated hydrocarbon and ester, which had no obvious negative effect on Anammox process.(5) By PCR technology, the existence of Anammox bacteria was confirmed and the aerobic nitrifying bacteria was found to coexist as well. The result of blast showed that the identities of Anammox bacterium to part of 16S rRNA sequence of uncultured Planctomycetales bacterium and Candidatus Brocadia fulgida bacterium were 95% and 90%, respectively.（6）By MPN method, nitrite oxidizer, ammonium oxidizer and denitrification bacteria were detected in both simulated and piggery wastewater treatment system of Anammox, and the microorganism system was composed of Anammox bacteria，aerobic bacteria and denitrification bacteria together.

BRST quantization and canonical Ward identity of the supersymmetric electromagnetic interaction system

According to the method of path integral quantization for the canonical constrained system in Becchi-Rouet-Stora-Tyutin scheme, the supersymmetric electromagnetic interaction system was quantized. Both the Hamiltonian of the supersymmetric electromagnetic interaction system in phase space and the quantization procedure were simplified. The BRST generator was constructed, and the BRST transformations of supersymmetric fields were gotten; the effective action was calculated, and the generating functional for the Green function was achieved; also, the gauge generator was constructed, and the gauge transformation of the system was obtained. Finally, the Ward-Takahashi identities based on the canonical Noether theorem were calculated, and two relations between proper vertices and propagators were obtained.

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg2+, Ca2+, Sr2+ and Ba2+ were used for,clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg2+, Ca2+ and Sr2+ are counterions. When Ba2+ is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr2+ induces the greatest structural transition.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Molecular cloning, characterization and mRNA expression of peroxiredoxin in Zhikong scallop Chlamys farreri

Peroxiredoxin V (PRX V) is known as an atypical 2-cysteine peroxiredoxin that protects the organisms against various oxidative stresses and functions in signal transduction. The cDNA of a PRX V gene (designated as CfPRX) was cloned from scallop Chlamys farreri. The full-length sequence of CfPRX cDNA was of 2,179 bp with a 564 bp open reading frame encoding a peptide of 187 amino acids. Sequence comparison showed that CfPRX shared higher identities with PRX Vs than that with other isoforms of PRX, indicating CfPRX was a member of the PRX V family. Fluorescent real-time quantitative PCR analysis revealed the presence of CfPRX transcripts in gill filaments, adductor muscle, heart, gonad, kidney and hemocytes, and the stimulation of Listonella anguillarum significantly (P < 0.01) enhanced the mRNA expression of CfPRX in hemocyte. These results indicated that CfPRX was a constitutive and inducible acute-phase protein which was involved in the immune resistance to L. anguillarum stimulation.

Two thymosin-repeated molecules with structural and functional diversity coexist in Chinese mitten crab Eriocheir sinensis

Recently, beta-thymosin-like proteins with multiple thymosin domains (defined as thymosin-repeated proteins) have been identified from invertebrate. In the present study, the cDNAs of two thymosin-repeated proteins (designated EsTRP1 and EsTRP2) were cloned from Chinese mitten crab by expressed sequence tags (EST) techniques. BLAST analysis presented three and two thymosin domains in EsTRP1 and EsTRP2, respectively, with the identities amongst the five domains varying from 47% to 100%. Both EsTRP1 and EsTRP2 shared high similarities with previously identified vertebrate beta-thynnosins and invertebrate thymosin-repeated proteins (TRPs) with the identities ranging from 43% to 78%, indicating that EsTRPs were new members of the beta-thymosin family. Real-time RT-PCR assay was adopted to determine the tissue distribution of EsTRPs and their temporal expression profile in hemocytes after pathogen stimulation and injury challenge. The expression of EsTRP1 transcript was predominantly detectable in the tissues of hemocytes, hepatopancreas and gonad with the highest expression in hemocytes, while the highest expression level of EsTRP2 was found in heart. EsTRP1 mRNA expression in hemocytes significantly increased at 3 and 48 h after Listonella anguillarum challenge, but there was no significant variation in EsTRP2 temporal expression profile. The injury challenge reduced the mRNA expression of EsTRPs, with the down-regulation of EsTRP2 expression occurred earlier than that of EsTRP1. The cDNA fragments encoding their mature peptides of EsTRP1 and EsTRP2 were recombined and expressed in Escherichia coli. The activities of recombinant proteins (rEsTRP1 and rEsTRP2) were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and lysoplate assay. rEsTRP2 could significantly accelerate the growth of human hepatocellular carcinoma cell line, but there was no significant effect of rEsTRP1 on the tumor cell proliferation. Both rEsTRP1 and rEsTRP2 did not possess the ability of killing Micrococcus luteus and L. anguillarum. The differences in the tissue distribution of mRNA transcripts, the response to pathogen stimulation and injury challenge, and the effect of recombinant proteins on human cell proliferation, indicated that there were functional diversity between the two structurally different molecules, EsTRP1 and EsTRP2. (C) 2009 Elsevier Ltd. All rights reserved.

First molluscan TNFR homologue in Zhikong scallop: Molecular characterization and expression analysis

Tumor necrosis factor receptors (TNFRs) are a superfamily of proteins characterized by the unique cysteine-rich domain (CRD) and their important roles in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The first member of the molluscan TNFR family, designated as CfTNFR, was identified from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTNFR was of 1334 bp, consisting of a 5' UTR of 17 bp, a 3'UTR of 69 by with a poly (A) tail, and an open reading frame (ORE) of 1248 by encoding a polypeptide of 415 amino acids with a theoretical isoelectric point of 8.33 and predicted molecular weight of 47.07 kDa. There were a signal peptide, a CRD, a transmembrane region and a death domain in the deduced amino acid sequence of CfTNFR, suggesting that it was a typical type 1 membrane protein. The high identities (22-40%) of CfTNFR with other TNFR superfamily members indicated that CfTNFR should be a member of TNFR superfamily, and moreover, it should be the first death domain-containing TNFR found in invertebrates. Phylogenetic analysis revealed that CfTNFR was closely related to TNFR-like proteins from Strongylocentrotus purpuratus, Drosophila melanogaster and Ciona intestinalis, and they formed a separate branch apart from vertebrate TNFRs. The spatial expression of CfTNFR transcripts in healthy and bacteria challenged scallops was examined by quantitative real-time PCR. CfTNFR transcripts could be detected in all tested tissues, including haemocytes, gonad, gill, mantle and hepatopancreas, and significantly up-regulated in the tissues of gonad, gill, mantle and hepatopancreas after Listonella anguillarum challenge, indicating that CfTNFR was constitutive and inducible acute-phase protein involved in immune defence. The present results suggested the existence of the TNFR-like molecules and TNF-TNFR system in low invertebrates, and provided new insights into the role of CfTNFR in scallop innate immune responses to invading microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32%similar to 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, A)Dnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana. (C) 2007 Published by Elsevier Inc.