Electrochemical identification of intermediate forms of urea denaturation of horse heart cytochrome c

The electrochemical identification of the urea denaturation of horse heart cytochrome c in bulk solution at the 4,4'-dithiodipyridine-modified gold electrode is reported. The results are similar to the three-step transitions of equilibrium studies (Myer et al., Biochemistry, 19 (1980) 199) of urea denaturation of cytochrome c in bulk solution. This method permits a clear resolution of which of the three steps of urea denaturation is electrochemically related. In addition, by analysing the effects of urea on the structural forms of cytochrome c and on the solution properties, as well as the cyclic voltammetric responses of the protein, the individual forms of the urea denaturation of cytochrome c can be understood. The results reflect the superposition of protein denaturation on the electrode surface and in solution.

Direct electrochemical identification of an activated intermediate formed by cytochrome C with hydrogen peroxide

An activated intermediate formed from H2O2 and cytochrome C is identified by direct electrochemical measurements.

AN INTEGRATED CALCIUM-FLUORIDE CRYSTAL IR THIN-LAYER CELL AND ITS APPLICATION TO IDENTIFICATION OF ELECTROCHEMICAL REDUCTION PRODUCT OF BILIRUBIN

An integrated CaF2 crystal optically transparent infrared (ir) thin-layer cell was designed and constructed without using any soluble adhesive materials. It is suitable for both aqueous and nonaqueous systems, and can be used not only in ir but also in uv-vis studies. Excellent electrochemical and spectroelectrochemical responses were obtained in evaluating this cell by cyclic voltammetry and steady-state potential step measurements for both ir and uv-vis spectrolectrochemistry with ferri/ferrocyanide in aqueous solution, and with ferrocene/ferrocenium in organic solvent as the testing species, respectively. The newly designed ir cell was applied to investigate the electrochemical reduction process of bilirubin in situ, which provided direct information for identifying the structure of the reduction product and proposing the reaction mechanism.

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Development of SSR primers from EST sequences and their application in germplasm identification of Porphyra lines (Rhodophyta)

This paper reports the development of SSR markers from EST data and their utilization in germplasm identification of Porphyra. The publicly available EST (expressed sequence tag) sequences of Porphyra were searched from the Internet (www.kazura.or.jp/en/plant/porphyra/EST/). From a total of 20,779 obtained EST sequences, 391 SSRs (simple sequence repeats) were analysed with SSRIT software (www.gramene.org/db/searches/ssrtool). From those, 48 SSR primer-pairs were designed and tested by commonly used SSR reaction conditions using 22 Porphyra DNA samples as templates. Results showed that 41 SSR primer-pairs gave good amplification patterns. These were used to conduct SSR analyses of genetic diversity and variety identification of the 22 Porphyra lines. A dendrogram and the DNA fingerprints of the Porphyra lines were developed based on the obtained SSR data.

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. Methods and Results: A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

Identification and characterization of the AcrR/AcrAB system of a pathogenic Edwardsiella tarda strain

Edwardsielia tarda is one of the leading marine pathogens that can infect a wide range of cultured marine species. In this study, the acrR-acrAB cluster was cloned from TX1, a pathogenic E. tarda strain isolated from diseased fish. AcrR and AcrAB were found to be involved in resistance against acriflavine and methyl viologen, which positively regulate the expression of acrAB. AcrR negatively regulates its own expression and the expression of the acrAB operon, most likely by interacting with a 24-bp operator site that overlaps the putative promoter of acrA (PacrA). The repressive effect of AcrR on PacrA could be relieved by acriflavine, methyl viologen, and ethidium bromide, the presence of each of which enhanced transcription from PacrA. Interruption of the regulated expression of acrR by introducing into TX1 a plasmid that overexpresses acrR affected growth under stress conditions, AI-2 production, and bacterial virulence. In addition, mutational analyses identified a constitutively active AcrR mutant (named N215), which exhibits full repressor activity but is impaired in its ability to interact with the inducer. Overexpression of N215 produced the same kind of but moderately stronger effect on TX1 compared to that produced by overexpression of the wild-type acrR.

Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares similar to 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia colt, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular beta-agarase. E. coli DH5 alpha harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5 alpha/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5 alpha/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5 alpha/pTAET21, is an effective vaccine candidate against E. tarda infection. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides

Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp. (C) 2009 Elsevier Ltd. All rights reserved.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties

A cluster of 11 interferon (IFN) genes were identified in the Atlantic salmon genome linked to the growth hormone I gene. The genes encode three different IFN subtypes; IFNa (two genes), IFNb (four genes) and IFNc (five genes), which show 22-32% amino acid sequence identity. Expression of the fish IFNs were studied in head kidney, leukocytes or To cells after stimulation with the dsRNA poly I:C or the imidazoquinoline S-27609. In mammals, poly I:C induces IFN-beta through the RIG-I/MDA5 or the TLR3 pathway, both of which are dependent on NF-kappa B. In contrast, S-27609 induces mammalian IFN-alpha in plasmacytoid dendritic cells through the TLR7 pathway independent of NF-kappa B. The presence of an NF-kappa B site in their promoters and their strong up-regulation by poly I:C, suggest that salmon IFNa1/IFNa2 are induced through similar pathways as IFN-beta. In contrast, the apparent lack of NF-kappa B motif in the promoter and the strong upregulation by S-27609 in head kidney and leukocytes, suggest that IFNb genes are induced through a pathway similar to mammalian IFN-alpha. IFNc genes showed expression patterns different from both IFNa and IFNb. Taken together, salmon IFNa and IFNb are not orthologs of mammalian IFN-beta and IFN-alpha, respectively, but appear to utilize similar induction pathways. (C) 2008 Elsevier Ltd. All rights reserved.

An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain

Pseudomonas fluorescens is an aquaculture pathogen that can infect a number of fish species. The virulence mechanisms of aquatic P. fluorescens remain largely unknown. Many P. fluorescens strains are able to secrete an extracellular protease called AprX, yet no AprX-like proteins have been identified in pathogenic P. fluorescens associated with aquaculture. In this study, a gene encoding an AprX homologue was cloned from TSS, a pathogenic A fluorescens strain isolated from diseased fish. In TSS, AprX is secreted into the extracellular milieu, and the production of AprX is controlled by growth phase and calcium. Mutation of aprX has multiple effects, which include impaired abilities in interaction with cultured host cells, adherence to host mucus, modulation of host immune response, and dissemination and survival in host tissues and blood. Purified recombinant AprX exhibits apparent proteolytic activity, which is optimal at pH 8.0 and 50 degrees C. The protease activity of recombinant AprX is enhanced by Ca2+ and Zn2+ and reduced by Co2+. Cytotoxicity analyses showed that purified recombinant AprX has profound toxic effect on cultured fish cells. These results demonstrate that AprX is an extracellular metalloprotease that is involved in bacterial virulence. (C) 2009 Elsevier B.V. All rights reserved.