Mitochondrial thioredoxin-2 from Manila clam (Ruditapes philippinarum) is a potent antioxidant enzyme involved in antibacterial response

Umasuthan, N.,Saranya Revathy, K.,Lee, Y.,Whang, I.,Lee, J. Academic Press 2012 Fish & shellfish immunology Vol.32 No.4

Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561bp) consists of a 498bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic <SUP>89</SUP>WCGPC<SUP>93</SUP> catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams.

A C1 inhibitor ortholog from rock bream (Oplegnathus fasciatus): Molecular perspectives of a central regulator in terms of its genomic arrangement, transcriptional profiles and anti-protease activities of recombinant peptide

Umasuthan, N.,Bathige, S.D.N.K.,Revathy, K.S.,Wickramaarachchi, W.D.N.,Wan, Q.,Whang, I.,Kim, E.,Park, M.A.,Park, H.C.,Lee, J. Pergamon Press ; Elsevier Science 2014 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.42 No.2

C1 inhibitor (C1Inh), a member of serpin superfamily, is a crucial regulator of the activation of various plasmatic cascades associated with immunity and inflammation. This study describes the identification and characterization of a C1Inh gene from rock bream Oplegnathus fasciatus (OfC1Inh) at structural, expressional and functional levels. The cDNA-(2245bp) and corresponding gDNA-sequences (5.2kbp) of OfC1Inh were isolated from rock bream transcriptome- and BAC-libraries, respectively. Predicted amino acid sequence of OfC1Inh revealed a two-domain architecture composed of an N-terminal region with two Ig-like domains and a C-terminal region with a serpin domain. Tertiary model of OfC1Inh disclosed its active site topology. In the multi-exonic genomic arrangement of OfC1Inh, it consisted of eleven exons disjoined by ten introns as observed in few other fish homologs. Our comparative analysis indicated that the teleostean C1Inhs were distinct from their non-teleostean vertebrate counterparts in terms of their (1) extended N-terminal domains, (2) evolutionary divergence and (3) exon-intron distribution. The OfC1Inh had a TATA-deficient promoter with a putative initiator element, and two tandemly arranged downstream promoter elements. Several components associated with the immune and inflammatory transcriptional activation were also predicted to exist in 5' flanking region of OfC1Inh. The exclusive mRNA levels in liver and moderate levels in extra-hepatic tissues intimated the diversified importance of OfC1Inh in rock bream physiology. We also provide an evidence for the involvement of OfC1Inh in immune balance, based on its modulated transcription upon different PAMP (lipopolysaccharide and poly I:C)- or pathogen (Streptococcus iniae and rock bream irido virus)-challenges. A recombinantly expressed fusion protein [(r)OfC1Inh] was employed in demonstrating the anti-protease function of OfC1Inh. The (r)OfC1Inh exhibited detectable inhibitory activity against C1 esterase and thrombin, where the anti-C1 esterase role was shown to be potentiated by heparin. Taken together, the results of this study provide the first line of evidence for the possible involvement of a teleostean C1Inh in fish immunity, based on its expressional response(s) and inhibitory properties against two enzymes involved in biological cascades.

A manganese superoxide dismutase with potent antioxidant activity identified from Oplegnathus fasciatus: Genomic structure and transcriptional characterization

Umasuthan, N.,Saranya Revathy, K.,Bathige, S.D.N.K.,Lim, B.S.,Park, M.A.,Whang, I.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.34 No.1

In this study, we describe the identification and characterization of manganese superoxide dismutase, an important antioxidant enzyme acting as the chief reactive oxygen species (ROS) scavenger, from rock bream Oplegnathus fasciatus (Of-mMnSOD) at genomic- and transcriptional-levels as well as the biological activity of recombinant protein. The Of-mMnSOD protein portrayed distinct MnSOD family features including signature motifs, metal association sites and the typical active site topology. It was also predicted to be localized in mitochondrial matrix. The Of-mMnSOD had a quinquepartite genome organization encompassing five exons interrupted by four introns. Comparison of its sequence and gene structure with that of other lineages emphasized its strong conservation among different vertebrates. The Of-mMnSOD was ubiquitously transcribed in different rock bream tissues with higher levels in blood cells and metabolically active tissues. Transcription of Of-mMnSOD was kinetically modulated in response to investigational challenges using mitogens (lipopolysaccharide and poly I:C) and live-pathogens (Edwardsiella tarda and rock bream irido virus) in blood cells and liver tissue. The purified recombinant Of-mMnSOD possessed potential antioxidant capacity and actively survived over a range of pH (7.5-11) and temperature (15-40 <SUP>o</SUP>C) conditions. Collectively, findings of this study suggest that Of-mMnSOD combats against oxidative stress and cellular damages induced by mitogen/pathogen-mediated inflammation, by detoxifying harmful ROS (O<SUB>2</SUB><SUP>@?-</SUP>) in rock bream.

Molecular genomic- and transcriptional-aspects of a teleost TRAF6 homolog: Possible involvement in immune responses of Oplegnathus fasciatus against pathogens

Umasuthan, N.,Bathige, S.D.N.K.,Revathy, K.S.,Nam, B.H.,Choi, C.Y.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.42 No.1

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial docking molecule for TNFR superfamily and Interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. As an adaptor protein in pathogen-induced signaling cascades, TRAF6 modulates both adaptive- and innate-immunity. In order to understand the immune responses of teleost TRAF6, Oplegnathus fasciatus TRAF6-like gene (OfTRAF6) was identified and characterized. Genomic length of OfTRAF6 (4 kb), obtained by means of a genomic BAC library, spanned seven exons which represented a putative coding sequence of 1716 bp and encoded 571 amino acids (aa) with an estimated molecular weight of 64 kDa. This putative protein demonstrated the classical tetra-domain architecture composed of a zinc finger RING-type profile, two zinc finger TRAF-type profiles, a coiled-coil region and a MATH domain. While the sequence similarity with human TRAF6 was 66.5%, OfTRAF6 shared a higher overall similarity with teleost homologs (~75-92%). Phylogeny of TRAF-family was examined and TRAF6-subfamily appeared to be the precursor of other subfamilies. In addition, the clustering pattern confirmed that OfTRAF6 is a novel member of TRAF6subfamily. Based on comparative genomic analysis, we found that vertebrate TRAF6 exhibits two distinct structures in teleost and tetrapod lineages. An intron-loss event has probably occurred in TRAF6 gene during the evolution of tetrapods from teleosts. Inspection of putative OfTRAF6 promoter revealed the presence of several immune responsive transcription factor binding sites. Real-time qPCR assay detected OfTRAF6 transcripts in eleven juvenile fish tissues with higher levels in peripheral blood cells followed by liver. Putative role of OfTRAF6 in response to flagellin, LPS, poly I:C, pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and rock bream iridovirus (RBIV) was profiled in different tissues and OfTRAF6 revealed up-regulated transcript levels. Altogether, these findings implicate that OfTRAF6 is not only involved in flagellin-induced signaling cascade, but also contributes to the antibacterial- and antiviral-responses.

Cytosolic thioredoxin from Ruditapes philippinarum: Molecular cloning, characterization, expression and DNA protection activity of the recombinant protein

Revathy, K.S.,Umasuthan, N.,Lee, Y.,Whang, I.,Kim, H.C.,Lee, J. Pergamon Press ; Elsevier Science Ltd ; Pergamon 2012 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.36 No.1

Thioredoxin (TRx) is a small redox protein that plays significant roles in protection against oxidative stress and in cell homeostasis by maintaining oxidized proteins in a reduced state. Here, we describe the isolation and characterization of a full-length TRx cDNA sequence from manila clam, Ruditapes philippinarum and named it as RpTRx. The full length sequence consists of 1416bp with an open reading frame of 318bp encoding for 106 amino acids. RpTRx protein harbors evolutionarily-conserved TRx active site <SUP>32</SUP>WCGPC<SUP>36</SUP>. Phylogenetic analysis revealed a close proximity of RpTRx with the orthologue in Japanese scallop, Chlamys farreri. RpTRx was found to be constitutively expressed in hemocyte, gill, mantle, foot and siphon indicating a general role in physiological processes in various tissues. With regard to a potential role in immune responses, the RpTRx mRNA was found to be up-regulated in hemocytes after bacterial (Vibrio tapetis) and lipopolysaccharide (LPS) challenge at 3h post-infection (p.i.); a wavering increase was observed up to 96h p.i. for LPS challenge and 48h p.i. for bacterial challenge. Thus, RpTRx may function as an intracellular antioxidant to protect the cells against ROS induced by LPS and bacterial challenges. Indeed, when recombinant RpTRx protein (rRpTRx) was over-expressed in Escherichiacoli Rosetta gami<SUP>TM</SUP> (DE3) cells, it was able to scavenge free radicals and protect super-coiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. In summary, RpTRx plays an essential role in cellular defense and maintenance of homeostasis in the manila clam.

Application of Pythagorean Fuzzy G∗ Compact Space to Identify the Preferred City to Live in India

N. B. Gnanachristy,G. K. Revathi 한국지능시스템학회 2023 INTERNATIONAL JOURNAL of FUZZY LOGIC and INTELLIGE Vol.23 No.2

The compactness in a Pythagorean fuzzy topological space generalizes the notion of a subsetof a closed space. In this study, a Pythagorean fuzzy G∗compact space was defined and itsproperties were investigated. Further application of the Pythagorean fuzzy G∗compact spaceis anticipated for examining preferred cities in India using real-time data by considering thetemperatures of the cities.

Molecular aspects, genomic arrangement and immune responsive mRNA expression profiles of two CXC chemokine receptor homologs (CXCR1 and CXCR2) from rock bream, Oplegnathus fasciatus

Umasuthan, N.,Wan, Q.,Revathy, K.S.,Whang, I.,Noh, J.K.,Kim, S.,Park, M.A.,Lee, J. Academic Press 2014 FISH AND SHELLFISH IMMUNOLOGY Vol.40 No.1

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5'-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream.

An Optimal Agent Based (Oab) Architecture for Web Service Discovery

Suganya. D,Revathy.A,R.G. Suresh Kumar,N. Moganarangan,D.Madhavan 보안공학연구지원센터 2016 International Journal of Database Theory and Appli Vol.9 No.3

Service Discovery is done based on Keyword Match, Simple Semantic Description Services, and Rich Semantic Description Service. Web Services raise the web to a new level by integrating computational aspects. Web Services are accessed by computer programs but these still need help by humans. Web-services should be described in a formal, semantic way, so programs can find, compose and invoke them automatically. Existing system discover the service based on QoS (Quality of Service). It uses the Agent to discover the service, but normalization & classification are not done by Agent here. Existing system are using the selection algorithm, which works in association with QoS value, based on consumer requirements. So In our proposed system we proposed the Normalized, Classification, & Ranking of the QoS parameter based on minimization maximization criteria using agent based architecture. We rank the services according to their QoS levels and their services matching.

Al-doped ZnS layers synthesized by solution growth method

K. Nagamani,N. Revathi,P. Prathap,Y. Lingappa,K.T. Ramakrishna Reddy 한국물리학회 2012 Current Applied Physics Vol.12 No.2

ZnS is one of the potential candidates as a window/buffer layer for solar photovoltaic applications. Aldoped ZnS nanocrystalline films were grown by a simple and economic process, chemical solution growth method. The layers were prepared for different Al-dopant concentrations that vary in the range, 0e10 at. %. The effect of Al-doping on the composition, structure, optical, electrical and photoluminescence properties of the synthesized layers was determined using appropriate techniques. The elemental composition of a typical sample with 6 at. % ‘Al’ in ZnS was Zn ¼ 44.9 at. %, S ¼ 49.8 at. % and Al ¼ 5.3 at. %. The films were nanocrystalline in nature and showed (111) plane of ZnS as the preferred orientation for all the doping concentrations. The layers with 6 at. % of Al showed a crystallite size of w9 nm. The FTIR studies confirmed the presence of ZnS in the layers. The layers showed an average transmittance of w75% in the visible region. The change of photoluminescence behaviour with dopant concentration was also studied. The electrical resistivity was considerably decreased from 107 Ucm to 103 Ucm with Al-doping. The detailed analysis of results will be presented and discussed.

Two NF-κB inhibitor-alpha (IκBα) genes from rock bream (Oplegnathus fasciatus): Molecular characterization, genomic organization and mRNA expression analysis after immune stimulation

Lee, Y.,Umasuthan, N.,Whang, I.,Revathy, K.S.,Lee, S.,De Zoysa, M.,Oh, C.,Kang, D.H.,Noh, J.K.,Lee, J. Academic Press 2014 FISH AND SHELLFISH IMMUNOLOGY Vol.41 No.2

IκBα is a member of IκB family, which sequesters NF-κB in an inactivate form in the cytoplasm and blocks the translocation of NF-κB to nucleus. The IκBα paralogs of rock bream (OfIκBα-A and OfIκBα-B) encoded IκBα proteins with typical features including, highly conserved IκB degradation motif, six ankyrin repeats and a PEST sequence. However, their amino acid identity and similarity were only 55.6 and 69.7%, respectively suggesting that these two genes could be the two different isoforms of IκBα. The number and size of the exons of OfIκBα-A and OfIκBα-B were conserved well with all the compared vertebrate species, although they have significantly different genomic sizes. Phylogenetic analysis revealed that OfIκBα-A and OfIκBα-B proteins cluster with IκBα family members; however, they were grouped with different subclades in IκBα family. Tissue specific expression of OfIκBα mRNA was constitutively detected in all the tested tissues, and they showed the higher transcription level in heart, liver, gill and peripheral blood cells, respectively. The injection of flagellin stimulated the mRNA expression of OfIκBα paralogs in head kidney and intestine. Moreover, the OfIκBα mRNA expression in gill and liver was significantly up-regulated by LPS, poly I:C and Edwardsiella tarda challenges. The transcription of OfIκBα was up-regulated in early-phase of injection and then rapidly restored. These results suggest that the OfIκBα paralogs might be involved in rapid immune responsive reactions in rock bream against bacterial and viral pathogens.