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.

Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli

Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g 1(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

Functional characterization of sll0659 from Synechocystis sp PCC 6803

Synchocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether Sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types ans mutants, In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cyclization. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Evolutionary analysis of phycobiliproteins: Implications for their structural and functional relationships

Phycobiliproteins, together with linker polypeptides and various chromophores, are basic building blocks of phycobilisomes, a supramolecular complex with a light-harvesting function in cyanobacteria and red algae. Previous studies suggest that the different types of phycobiliproteins and the linker polypeptides originated from the same ancestor. Here we retrieve the phycobilisome-related genes from the well-annotated and even unfinished cyanobacteria genomes and find that many sites with elevated d(N)/d(S) ratios in different phycobiliprotein lineages are located in the chromophore-binding domain and the helical hairpin domains (X and Y). Covariation analyses also reveal that these sites are significantly correlated, showing strong evidence of the functional-structural importance of interactions among these residues. The potential selective pressure driving the diversification of phycobiliproteins may be related to the phycobiliprotein-chromophore microenvironment formation and the subunits interaction. Sites and genes identified here would provide targets for further research on the structural-functional role of these residues and energy transfer through the chromophores.

Microstructure and wear properties of laser clad Fe based amorphous composite coatings

A FeNiSiBV amorphous composite coating was developed by laser cladding of metallic powders on AISI 1020 low carbon steel substrate. The coatings were studied using X-ray diffraction, transmission electron microscopy and scanning electron microscopy. The coating reveals different microstructures along the depth of the coating. The transition zone exhibits good metallurgical bonding between the substrate and the coating. The layer consists of amorphous phase in majority and nanocrystalline phase/crystalline phase in minority. Accompanied with the nanocrystalline phase, the amorphous phase is concentrated in the middle of the coating. The crystalline phase in the coating is identified as Fe2B. A gradient distribution of the microhardness ranges from 1208 HV0.2 to 891 HV0.2 in the coating along the depth. The coating shows higher microhardness and better wear property than the substrate.

Zooplankton functional groups on the continental shelf of the yellow sea

Zooplankton plays a vital role in marine ecosystems. Variations in the zooplankton species composition, biomass, and secondary production will change the structure and function of the ecosystem. How to describe this process and make it easier to be modeled in the Yellow Sea ecosystem is the main purpose of this paper. The zooplankton functional groups approach, which is considered a good method of linking the structure of food webs and the energy flow in the ecosystems, is used to describe the main contributors of secondary produciton of the Yellow Sea ecosystem. The zooplankton can be classified into six functional groups: giant crustaceans, large copepods, small copepods, chaetognaths, medusae, and salps. The giant crustaceans, large copepods, and small copepods groups, which are the main food resources for fish, are defined depending on the size spectrum. Medusae and chaetognaths are the two gelatinous carnivorous groups, which compete with fish for food. The salps group, acting as passive filter-feeders, competes with other species feeding on phytoplankton, but their energy could not be efficiently transferred to higher trophic levels. From the viewpoint of biomass, which is the basis of the food web, and feeding activities, the contributions of each functional group to the ecosystem were evaluated; the seasonal variations, geographical distribution patterns, and species composition of each functional group were analyzed. The average zooplankton biomass was 2.1 g dry wt m(-2) in spring, to which the giant crustaceans, large copepods, and small copepods contributed 19, 44, and 26%, respectively. High biomasses of the large copepods and small copepods were distributed at the coastal waters, while the giant crustaceans were mainly located at offshore area. In summer, the mean biomass was 3.1 g dry wt m(-2), which was mostly contributed by the giant crustaceans (73%), and high biomasses of the giant crustaceans, large copepods, and small copepods were all distributed in the central part of the Yellow Sea. During autumn, the mean biomass was 1.8 g dry wt m(-2), which was similarly constituted by the giant crustaceans, large copepods, and small copepods (36, 33, and 23%, respectively), and high biomasses of the giant crustaceans and large copepods occurred in the central part of the Yellow Sea, while the small copepods were mainly located at offshore stations. The giant crustaceans and large copepods dominated the zooplankton biomass (2.9 g dry wt m(-2)) in winter, contributing respectively 57 and 27%, and they, as well as the small copepods, were all mainly located in the central part of the Yellow Sea. The chaetognaths group was mainly located in the northern part of the Yellow Sea during all seasons, but contributed less to the biomass compared with the other groups. The medusae and salps groups were distributed unevenly, with sporadic dynamics, mainly along the coastline and at the northern part of the Yellow Sea. No more than 10 species belonging to the respective functional groups dominated the zooplankton biomass and controlled the dynamics of the zooplankton community. The clear picture of the seasonal and spatial variations of each zooplankton functional group makes the complicated Yellow Sea ecosystem easier to be understood and modeled. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of spark discharge on the anodic coatings on magnesium alloy

Spark discharge was the representative phenomenon of Micro-arc oxidation (MAO) method distinguished from other electrochemical oxidation methods. Under the spark discharge treatment, characteristics of the anodic layer were significantly changed. To investigate the influences of the spark discharge, a piece of magnesium alloy AZ91D specimen was partly treated by MAO method in alkaline silicate solution. And the microstructure, element distributions as well as the surface potential distributions of the specimen were studied by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and scanning Kelvin probe (SKP) technique. As a result of intensive spark discharge treatment, porous external layer with dense internal layer were formed on Mg alloy surface. At the same time, the depositions of OH- and SiO32- ions were accelerated, which resulted in the enrichment of element oxygen and silicon at the spark discharge region. Moreover, due to the compact internal layer, the intensive spark discharge region exhibited more positive potentials with respect to other regions, which meant this region could restrain the ejection of electron and provide effective protection to the substrate. In addition, it was found that oxygen played a vital role in determining the intensity and size of sparks, and abundant oxygen resulted in intensive and larger sparks. (c) 2005 Elsevier B.V. All rights reserved.

Characteristics of anodic coatings oxidized to different voltage on AZ91D Mg alloy by micro-arc oxidization technique

With increasing applied voltage, three types of anodic coatings, passive film, micro-spark ceramic coating and spark ceramic coating were made by micro-arc oxidization (MAO) technique on AZ91D magnesium alloy in alkali-silicate solution. The structure, composition characteristics and the electrochemical properties of coatings were also studied with SEM, XRD and EIS (electrochemical impedance spectroscopy) technique, respectively. It is found that the electrochemical properties are closely related to the structure and composition characteristics of the anodic coatings. At the same time, the characteristics of the three types of anodic coatings differ significantly, among them, the micro-spark ceramic coating, prepared in the voltage range of 170similar to220V exhibits compact, homogeneous structure and highest corrosion-resistance.

Corrosion behaviour of hot dip zinc and zinc-aluminium coatings on steel in seawater

A comparative investigation of hot dip Zn-25Al alloy, Zn-55Al-Si and Zn coatings on steel was performed with attention to their corrosion performance in seawater. The results of 2-year exposure testing of these at Zhoushan test site are reported here. In tidal and immersion environments, Zn-25Al alloy coating is several times more durable than zinc coating of double thickness. At long exposure times, corrosion rate for the Zn-25Al alloy coating remains indistinguishable from that for the Zn-55Al-Si coating of similar thickness in tidal zone, and is two to three times lower than the latter in immersion zone. The decrease in tensile strength suggested that galvanized and Zn-55Al-Si coated steel suffer intense pitting corrosion in immersion zone. The electrochemical tests showed that all these coatings provide cathodic protection to the substrate metal; the galvanic potentials are equal to - 1,050, - 1,025 and - 880 mV (SCE) for zinc, Zn-25Al alloy and Zn-55Al-Si coating, respectively, which are adequate to keep the steel inside the immunity region. It is believed that the superior performance of the Zn-25Al alloy coating is due to its optimal combination of the uniform corrosion resistance and pitting corrosion resistance. The inferior corrosion performance by comparison of the Zn coating mainly results from its larger dissolution rate, while the failure of the Zn-55Al-Si coating is probably related to its higher susceptibility to pitting corrosion in seawater.

Effect of Process Conditions on Microstructure and Corrosion Resistance of Cold-Sprayed Ti Coatings

Ti and Ti alloys can be applied to steels as a protective coating in view of its excellent resistance to corrosive environment. Cold spraying, as a new coating technique, has potential advantages in fabrication of Ti coating in comparison with conventional thermal spraying techniques. In this study, Ti coatings were prepared on carbon steel substrates by cold spraying via controlling the process conditions. The microstructure of coatings was observed by SEM. The porosity of coatings was estimated by image analysis and the bond strength was tested for comparison of the process conditions. Potentiodynamic polarization and open-circuit potential (OCP) measurements were performed to understand the corrosion behavior of the coatings. The SEM examination shows that the coatings become more compact with the increases of pressure and temperature of driving gas. The potentiodynamic polarization curves indicate that the coating which has lower porosity has lower corrosion current. The polarization and OCP measurement reveal that cold-sprayed Ti coating can provide favorable protection to carbon steel substrate. The polishing treatment of coating surface polishes the rough outer layer including the small pores as well as decreases the actual surface area of the coating, leading to the considerable improvement of corrosion resistance.

Nicotinic acid as a nontoxic corrosion inhibitor for hot dipped Zn and Zn-Al alloy coatings on steels in diluted hydrochloric acid

The inhibition effect of nicotinic acid for corrosion of hot dipped Zn and Zn-Al alloy coatings in diluted hydrochloric acid was investigated using quantum chemistry analysis, weight loss test, electrochemical measurement, and scanning electronic microscope (SEM) analysis. Quantum chemistry calculation results showed that nicotinic acid possessed planar structure with a number of active centers, and the populations of the Mulliken charge, the highest occupied molecular orbital (HOMO), and the lowest unoccupied molecular orbital (LUMO) were found mainly focused around oxygen and nitrogen atoms, and the cyclic of the benzene as well. The results of weight loss test and electrochemical measurement indicated that inhibition efficiency (IE%) increased with inhibitor concentration, and the highest inhibition efficiency was up to 96.7%. The corrosion inhibition of these coatings was discussed in terms of blocking the electrode reaction by adsorption of the molecules at the active centers on the electrode surface. It was found that the adsorption of nicotinic acid on coating surface followed Langmuir adsorption isotherm with single molecular layer, and nicotinic acid adsorbed on the coating surface probably by chemisorption. Nicotinic acid, therefore, can act as a good nontoxic corrosion inhibitor for hot dipped Zn and Zn-Al alloy coatings in diluted hydrochloric acid solution. (c) 2007 Elsevier Ltd. All rights reserved.