Other articles by Chi-Yao Chang on PubMed

• Expression of Ayu (Plecoglossus Altivelis) Pit-1 in Escherichia Coli: Its Purification and Immunohistochemical Detection Using Monoclonal Antibody Protein Expression and Purification. Mar, 2002  |   Pubmed ID: 11858725 The pituitary-specific transcription factor Pit-1 belongs to the family of POU-domain proteins and is known to play an important role in the differentiation of pituitary cells. Here we report the complete nucleotide sequence of cDNA encoding Pit-1 from the brackish water fish, ayu (Plecoglossus altivelis). Nucleotide sequence analysis of 1910 bp of ayu Pit-1 cDNA revealed an open reading frame of 1074 bp that encodes a protein of 358 amino acids containing a POU-specific domain, POU homeodomain, and an STA (Ser/Thr-rich activation) transactivation domain. We inserted the coding region of Pit-1 cDNA, obtained by PCR, into a pET-20b(+) plasmid to produce recombinant Pit-1 in Escherichia coli BL21 (DE3) pLysS cells. Upon induction with isopropyl beta-D-thiogalactopyranoside, Pit-1 was expressed and accumulated as inclusion bodies in E. coli. The protein was then purified in one step by affinity chromatography on a nickel-nitrilotriacetic acid agarose column under denaturing conditions. This method yielded 0.7 mg of highly pure and stable protein per 200 ml of bacterial culture. A band of 40 kDa, resolved as recombinant ayu Pit-1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, agrees well with the molecular mass calculated from the translated cDNA sequence. The purified recombinant Pit-1 was confirmed in vitro through Western blot analysis, using its monoclonal antibody. This monoclonal antibody detected Pit-1 in the nuclei of ayu developing pituitary by immunohistochemical reaction. It serves as a good reagent for the detection of ayu Pit-1 in situ.
• Molecular Cloning and Developmental Expression of Zinc Finger Transcription Factor MTF-1 Gene in Zebrafish, Danio Rerio Biochemical and Biophysical Research Communications. Mar, 2002  |   Pubmed ID: 11866436 Metal-responsive transcription factor, MTF-1 is a zinc finger protein, shown to be essential for embryonic development. Homozygous knockout mouse embryos for MTF-1 die in utero at day 14 of gestation, due to liver decay. In the present study, we report the complete nucleotide sequence of cDNA encoding zebrafish MTF-1 and the amino acid sequence similarity with that of mouse, human, fish and Drosophila. The size of the zebrafish MTF-1 cDNA is 3,379 bp and the coding region (1,779 bp) encodes a polypeptide of 593 amino acids. The putative zinc finger and transactivation domains comprised by zebrafish MTF-1 were also determined. The zebrafish MTF-1 shows high identity of 97, 93, 93 and 67% in the DNA binding zinc finger domain and 51, 44, 48 and 20% overall identity with fugu, human, mouse and Drosophila, respectively. RT-PCR results show the maternal expression of MTF-1 transcripts. The pattern of MTF-1 gene expression during embryonic and early larval development was studied by whole-mount in situ hybridization using DIG-labeled anti-sense RNA probe. Stronger and ubiquitous expression was observed during the embryonic stages whereas, specific expression, especially in the neural parts, was observed throughout the stages studied after hatching.
• Structural Restoration of Inactive Recombinant Fish Growth Hormones by Chemical Chaperonin and Solvent Restraint Approaches Protein Engineering. May, 2002  |   Pubmed ID: 12034864 Recombinant proteins may undergo conformational distortion, leading to aggregation and loss of function, when they are expressed in heterologous systems. The structural and functional restoration of such inactive proteins is highly desirable. We have over-expressed recombinant growth hormones from the fish ayu (Plecoglossus altivelis) and yellow grouper (Epinephelus awoara) by a pET expression system. Both recombinant proteins accumulate as insoluble form in Escherichia coli. We refolded these inactive proteins into the active form using a stepwise refolding process with a dilute denaturing agent as a steric blocker and chemical chaperonin. Optical characterization showed that stable folding intermediates with a helical conformation can be detected in the molten globule state. Moreover, the function of restored recombinant growth hormones was demonstrated by its ability to stimulate proliferation in zebrafish liver cells.
• In Vitro Efficiency of Intra- and Extracellular Immunization with Mouse Anti-YGNNV Antibody Against Yellow Grouper Nervous Necrosis Virus Vaccine. Aug, 2002  |   Pubmed ID: 12163274 Mouse monoclonal antibody (MAb-18) against yellow grouper nervous necrosis virus (YGNNV) coat protein was developed and assayed for its neutralization ability. In the present study, we cloned and sequenced the cDNAs encoding heavy (gamma) and light (kappa) chains by constructing a cDNA library of the MAb-18 hybridoma. Three expression vectors, pCMV-NNV-18H (gamma), pCMV-NNV-18L (kappa), and pCMV-NNV-18HL (both gamma and kappa chains) were constructed and successfully expressed in grouper brain (GB) cells. Western blotting results indicated the secretion of antibody in to the medium with successful folding. Extracellular antibodies secreted by the pCMV-NNV-18HL transfected GB cells, neutralized well with YGNNV and showed the highest neutralization index (log(10) of NI) value 4. A significant reduction of titre (99.9%) was observed, when the intracellularly immunized GB cells were propagated with the YGNNV. These preliminary demonstrations suggest the immunoprophylactic use of plasmid constructs encoding the genes of mouse MAbs and the possibility of producing transgenic pathogen-free spawners and larvae, which contain freely available MAbs against pathogen in the circulation.
• Characterization of Transactivation Domain and Developmental Expression of Pituitary Specific Transcription Factor, Pit-1 of Ayu (Plecoglossus Altivelis) General and Comparative Endocrinology. Jul, 2002  |   Pubmed ID: 12225774 Pit-1 is a pituitary-specific transcription factor, which regulates the expression of growth hormone, prolactin, and thyroid stimulating hormone-beta genes. We previously reported the expression of a Pit-1 gene from ayu (Plecoglossus altivelis), which is an important cultivated food fish in Taiwan and Japan. Comparison of ayu Pit-1 with that of salmon, turkey, and rodent, revealed that the Pit-1 structure is highly conserved through vertebrates, especially in POU-specific and POU-homeo domains. The variation among fish, bird, and mammal are mainly found in transactivation domain by alternative splicing and initiation. Three insertions were found. The gamma-insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The beta-insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit-1 beta(*) and mammalian Pit-1 beta, respectively. An additional similarity was noticed between fish and bird, as both of them contain 7 a.a. insert that is not present in mammalian Pit-1. By site directed mutagenesis, we demonstrated that the beta, gamma, and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The ayu Pit-1 protein was found to be expressed specifically in pituitary gland, and its mRNA was first detected at embryonic day 4, significantly increased at embryonic day 5, then sustained to time of hatching at day 8.
• Adenohypophysis Formation in the Zebrafish and Its Dependence on Sonic Hedgehog Developmental Biology. Feb, 2003  |   Pubmed ID: 12606280 Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.
• Cloning and Characterization of Metallothionein Gene in Ayu Plecoglossus Altivelis Aquatic Toxicology (Amsterdam, Netherlands). Feb, 2004  |   Pubmed ID: 15036867 Metallothionein (MT) has been used widely as a potential molecular marker to detect the deleterious effects of heavy metals in aquatic ecosystem. Here we exposed ayu, Plecoglossus altivelis, to zinc (Zn) and tested the distribution as well as the induction of MT in various tissues such as liver, kidney, intestine and stomach. MT induction was significant in liver tissue, followed by kidney and intestine, whereas no induction was detected in stomach. The gene encoding ayu MT was successfully cloned and characterized. Complete nucleotide sequencing and analysis of the 4.5 kb DNA fragment containing the ayu MT gene revealed that the gene has three exons interrupted by two introns, a 5'-flanking region of about 2.5 kb and about 1.6 kb of 3'-flanking region. In grouper heart and kidney cells, the 2.5 kb promoter containing eight metal responsive elements (MREs), two hepatic nuclear factor 5 responsive elements (HNF5REs) and one cAMP responsive element (CRE) had the highest reporter activity.
• Expression of Metallothionein Gene During Embryonic and Early Larval Development in Zebrafish Aquatic Toxicology (Amsterdam, Netherlands). Aug, 2004  |   Pubmed ID: 15276328 Metallothionein (Mt) has been considered as a molecular marker of metal pollution in aquatic ecosystems. Less is known about the expression of mt gene during embryogenesis. Here, we report the cloning, sequencing, and the expression pattern of mt gene during developmental stages in zebrafish. The zebrafish embryogenesis when takes place in a medium containing a dosage of 1000 microM zinc resulted in high mortality, indicating the deleterious effect of zinc on development. The zebrafish mt gene consists of three exons encoding 60 amino acids with 20 conserved cysteine residues. RT-PCR result indicates the maternal contribution of Mt transcripts. Using digoxigenin (DIG)-labeled anti-sense RNA probe, whole-mount in situ hybridization was performed to observe the expression pattern of zebrafish mt gene during embryonic and early larval stages. Stronger as well as ubiquitous expression of mt gene during early embryonic stages narrowed to specific expression after hatching. The mt promoter region contains seven copies of putative metal-responsive elements (MREs), which are shown to be important for the high level activity by deletion analysis. The expression of mt gene during embryogenesis implies its significant role on development.
• Identification and Characterization of a Novel Gene of Grouper Iridovirus Encoding a Purine Nucleoside Phosphorylase The Journal of General Virology. Oct, 2004  |   Pubmed ID: 15448350 Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. It catalyses the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to free bases and (2'-deoxy)ribose 1-phosphates. Here, a novel piscine viral PNP gene that was identified from grouper iridovirus (GIV), a causative agent of an epizootic fish disease, is reported. This putative GIV PNP gene encodes a protein of 285 aa with a predicted molecular mass of 30 332 Da and shows high similarity to the human PNP gene. Northern and Western blot analyses of GIV-infected grouper kidney (GK) cells revealed that PNP expression increased in cells with time from 6 h post-infection. Immunocytochemistry localized GIV PNP in the cytoplasm of GIV-infected host cells. PNP-EGFP fusion protein was also observed in the cytoplasm of PNP-EGFP reporter construct-transfected GK and HeLa cells. From HPLC analysis, the recombinant GIV PNP protein was shown to catalyse the reversible phosphorolysis of purine nucleosides and could accept guanosine, inosine and adenosine as substrates. In conclusion, this is the first report of a viral PNP with enzymic activity.
• Complete Genome Sequence of the Grouper Iridovirus and Comparison of Genomic Organization with Those of Other Iridoviruses Journal of Virology. Feb, 2005  |   Pubmed ID: 15681403 The complete DNA sequence of grouper iridovirus (GIV) was determined using a whole-genome shotgun approach on virion DNA. The circular form genome was 139,793 bp in length with a 49% G + C content. It contained 120 predicted open reading frames (ORFs) with coding capacities ranging from 62 to 1,268 amino acids. A total of 21% (25 of 120) of GIV ORFs are conserved in the other five sequenced iridovirus genomes, including DNA replication, transcription, nucleotide metabolism, protein modification, viral structure, and virus-host interaction genes. The whole-genome nucleotide pairwise comparison showed that GIV virus was partially colinear with counterparts of previously sequenced ranaviruses (ATV and TFV). Besides, sequence analysis revealed that GIV possesses several unique features which are different from those of other complete sequenced iridovirus genomes: (i) GIV is the first ranavirus-like virus which has been sequenced completely and which infects fish other than amphibians, (ii) GIV is the only vertebrate iridovirus without CpG sequence methylation and lacking DNA methyltransferase, (iii) GIV contains a purine nucleoside phosphorylase gene which is not found in other iridoviruses or in any other viruses, (iv) GIV contains 17 sets of repeat sequence, with basic unit sizes ranging from 9 to 63 bp, dispersed throughout the whole genome. These distinctive features of GIV further extend our understanding of molecular events taking place between ranavirus and its hosts and the iridovirus evolution.
• Characterization of Serum Immunoglobulin M of Grouper and CDNA Cloning of Its Heavy Chain Veterinary Immunology and Immunopathology. Feb, 2006  |   Pubmed ID: 16199094 Immunoglobulin M (IgM) from the whole serum of grouper fish, Epinephelus coioides was purified by affinity chromatography using protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed that the relative molecular masses (Mr) of the equimolar heavy and light chains of IgM were 78,000 and 27,000, respectively. The cDNAs encoding IgM heavy chain comprising its variable (VH) and constant (CH) regions have been cloned and sequenced from a grouper kidney cDNA library by antibody screening method. Five VH (130-142 amino acids) and four CH (450-454 amino acids) families were identified. The variable and constant regions were conserved with their putative domains. All the four constant region domains (CH1-CH2-CH3-CH4) contained each three conserved cysteine residues, which are considered to form the inter- and intra-chain disulfide bridges. There were three carbohydrate acceptor sites in the constant region. In general, the pattern of IgM gene organization seems to resemble that of other teleosts. Moreover, the CH genes in grouper IgM occur as multifamily as reported in Atlantic salmon and common carp.
• Inhibition of Nervous Necrosis Virus Propagation by Fish Mx Proteins Biochemical and Biophysical Research Communications. Dec, 2006  |   Pubmed ID: 17069756 Mx proteins are interferon induced, antiviral proteins, expressed in response to treatment with double stranded RNA or virus infection. Here we report the cloning, sequencing, and antiviral property of three forms of Mx genes, MxI, MxII, and MxIII from grouper (Epinephelus coioides). Multiple comparison of grouper Mx amino acid sequences revealed the conservation of Mx putative GTP-binding domain, dynamin family signature, and leucine zipper motif. We have established a new cell line, grouper brain 3 (GB3), and prepared stable clones expressing Flag-epitope tagged grouper MxI, MxII, and MxIII. Immunostaining shows that all the three grouper Mx proteins are localized in the cytoplasm. To examine the antiviral activity of grouper Mx proteins, these stable clones were infected by a nodavirus, yellow grouper nervous necrosis virus (YGNNV), and the results showed that all the three Mx isoforms have the efficiency of reducing the titre of virus from 10- to 100-fold. Moreover, through immunocytochemistry we demonstrated that Mx protein can inhibit the YGNNV propagation in GB3 cells. Taken together, this study demonstrates that grouper Mx proteins have efficient inhibitory activity against nodavirus, the most endangered virus of fish, and this information would be helpful to design effective DNA vaccines that can confer an early non-specific antiviral protection.
• Microscopy-based Mass Measurement of a Single Whole Virus in a Cylindrical Ion Trap Angewandte Chemie (International Ed. in English). Dec, 2006  |   Pubmed ID: 17146754
• Expression Pattern of Metallothionein, MTF-1 Nuclear Translocation, and Its Dna-binding Activity in Zebrafish (Danio Rerio) Induced by Zinc and Cadmium Environmental Toxicology and Chemistry. Jan, 2007  |   Pubmed ID: 17269467 Metallothionein is a small (6-kDa), cysteine-rich protein expressed by a six-zinc finger protein called metal-responsive element-binding transcription factor-1 (MTF-1) in response to Zn and Cd. Our previous reports have shown the basal expression of metallothionein (mt) and MTF-I (mtf-1) genes in embryo and early larval stages of zebrafish (Danio rerio). In the present study, we investigated the mt expression in zebrafish early larvae induced by exposure to Cd and Zn (48, 72, 96, and 120 h postfertilization). Whole-mount in situ hybridization showed that Zn induced mt expression in the olfactory pit, cerebellum, ceratobranchials, liver, chloride cells, and neuromasts of the lateral line. Cadmium also induced mt expression in all the above regions except the cerebellum. Using fluorescence techniques, we have shown that Zn and Cd mediate cytoplasmic and nuclear translocation of MTF- 1-enhanced green fluorescent protein fusion protein in zebrafish liver cell line. The MTF-1 protein was produced recombinantly by inserting zebrafish mtf-1 cDNA (1.8 kb) into pET-20b(+) expression vector and expressing in Escherichia coli BL21 (DE3) pLysS host strain competent cell on induction with isopropyl-beta-D-thiogalactopyranoside. The protein was then purified by affinity chromatography on a nickel-nitrilotriacetic acid column. Electrophoretic mobility shift assay revealed binding of the recombinant MTF-1 in response to Zn and Cd at the putative metal-responsive elements (MREs) in the promoter region of the mt gene. Taken together, these results suggest that Zn and Cd are efficiently involved with mt expression induced in zebrafish embryos and with MTF-1 nuclear translocation and that this induction is achieved through the activation of MTF-1 binding at the MREs.
• Analysis of Codon Usage Bias and Base Compositional Constraints in Iridovirus Genomes Virus Research. Jun, 2007  |   Pubmed ID: 17434639 The codon usage bias and the base composition variations in the available 12 complete iridovirus genome sequences have been investigated. We re-evaluated the number of open reading frames (ORFs) in each published iridovirus genome and analyzed its correlation against the genome size. The result shows that there is a direct relationship between the number of ORFs and the genome size. The codon usage patterns of these iridoviruses are found to be phylogenetically conserved. A significant variation in the base content among the 12 iridovirus genomes has been observed, with G+C content ranges widely from 27 to 55%. Moreover, the preferential use of bases in codons is different among higher and lower G+C content genomes. A preferential codon usage among viral genomes is also noticed. Effective number of codon (Enc) plot reveals that the G+C compositional constraint is the main factor that determines the codon usage bias. Relative synonymous codon usage analysis of methyltransferase containing as well as lacking viruses suggests that the codon usage is not influenced by the methylation-mediated mutation. In addition, the comparison of the codon usage of iridovirus hosts and the iridovirus genomes reveals that the host tRNA pool may be responsible for the base compositional constraint. This study represents the most comprehensive analysis to date for iridovirus codon usage patterns.
• Iridovirus Bcl-2 Protein Inhibits Apoptosis in the Early Stage of Viral Infection Apoptosis : an International Journal on Programmed Cell Death. Jan, 2008  |   Pubmed ID: 17955372 The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis. However, a DNA fragmentation assay of UV-exposed GK cells after GIV infection revealed an inhibition of apoptosis. The UV- or heat-inactivated GIV failed to inhibit apoptosis, implying that a gene or protein of the viral particle might contribute to an apoptosis inhibitory function. The DNA ladder assay for GIV-infected GK cells after UV irradiation confirmed that apoptosis inhibition was an early process which occurred as early as 5 min post-infection. A GIV-Bcl sequence comparison showed distant sequence similarities to that of human and four viruses; however, all possessed the putative Bcl-2 homology (BH) domains of BH1, BH2, BH3, and BH4, as well as a transmembrane domain. Northern blot hybridization showed that GIV-Bcl transcription began at 2 h post-infection, and the mRNA level significantly increased in the presence of cycloheximide or aphidicolin, indicating that this GIV-Bcl is an immediate-early gene. This was consistent with the Western blot results, which also revealed that the virion carries the Bcl protein. We observed the localization of GIV-Bcl on the mitochondrial membrane and other defined intracellular areas. By immunostaining, it was proven that GIV-Bcl-expressing cells effectively inhibited apoptosis. Taken together, these results demonstrate that GIV inhibits the promotion of apoptosis by GK cells, which is mediated by the immediate early expressed viral Bcl gene.
• Dietary Sodium Alginate Administration Affects Fingerling Growth and Resistance to Streptococcus Sp. and Iridovirus, and Juvenile Non-specific Immune Responses of the Orange-spotted Grouper, Epinephelus Coioides Fish & Shellfish Immunology. Jul, 2008  |   Pubmed ID: 18485740 The percent weight gain (PWG) and feeding efficiency (FE) of fingerling orange-spotted grouper, Epinephelus coioides, fed diets containing sodium alginate at 1.0 and 2.0 g kg(-1) were calculated on the 2nd, 4th, 6th, and 8th weeks after feeding. Survival rates of the fingerling grouper against Streptococcus sp. and an iridovirus, and non-specific immune parameters such as alternative complement activity (ACH50), lysozyme activity, natural haemagglutination activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity of juvenile grouper were also determined when the fish were fed diets containing sodium alginate at 0.5, 1.0, or 2.0 g kg(-1). The PWG and FE of fish were better when the fish were fed diets containing sodium alginate at 1.0, and 1.0 and 2.0 g kg(-1), respectively. The PWG and FE of fish fed the 0, 1.0 and 2.0 g kg(-1) sodium alginate-containing diets after 8 weeks were 271.0%, 454.4% and 327.8%, and 0.61, 0.72 and 0.68, respectively. Fish fed a diet containing sodium alginate at the level of 2.0 g kg(-1) had a significantly higher survival rate than those fed the control diet after challenge with Streptococcus sp. and an iridovirus causing an increase of survival rate by 25.0% and 16.7%, respectively, compared to the control group. The ACH(50) level of fish fed the sodium alginate-containing diets at 2.0 g kg(-1) was significantly higher than those fed the 1.0 g kg(-1) sodium alginate diet and control diet after 12 days, and had increased to 1.9-fold, compared to those fed the control diet. The lysozyme activity, phagocytic activity, respiratory bursts, and SOD level of fish fed the sodium alginate-containing diets at 1.0 and 2.0 g kg(-1) were significantly higher than those fed the control diet after 12 days, and had increased to 1.97- and 1.68-fold, 1.35- and 1.50-fold, 1.63- and 1.81-fold, and 1.23- and 1.31-fold, respectively, compared to those fed the control diet. We therefore recommend dietary sodium alginate administration at 1.0 and 2.0 g kg(-1), respectively, to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus.
• Differential Display of Grouper Iridovirus-infected Grouper Cells by Immunostaining Biochemical and Biophysical Research Communications. Aug, 2008  |   Pubmed ID: 18519026 Grouper iridovirus (GIV) is one of the most devastating infectious pathogens of aquaculture fish. When infecting a susceptible cell line, such as GK-2, GIV causes antigenic changes in host cellular proteins. To understand the host gene expression characteristics after viral infection, we developed an immunostaining method to screen differentially expressed genes of fish cells in response to GIV infection using phage display complementary DNA libraries. In total, 66 genes were identified from grouper kidney and brain cell lines. These genes are related to replication, transcription, translation, immunity, apoptosis, structure proteins, metabolism, energy, protein modification, and homeostasis. Four dynamic antigenic patterns were observed among these immunocloned genes upon GIV infection. Microarray analysis further confirmed the transcriptional patterns of 80% of the identified genes. This immunostaining screening method provides insights into a host's cellular protein response to viral infection on a translational basis.
• Assessment of the Tumorigenesis and Drug Susceptibility of Three New Canine Mammary Tumor Cell Lines Research in Veterinary Science. Apr, 2010  |   Pubmed ID: 19748110 Three canine mammary tumor (CMT) cell lines, namely DE-E, DE-F and DE-SF, have been established from a surgically excised specimen of a malignant mammary tumor. These CMT cell lines have been cultured for over 200 passages. The cell doubling time was estimated to be approximately 30 h for all three cell lines. DE-E, DE-F and DE-SF were epithelial, fibroblast and spindle fibroblast in morphology, respectively. Under electron microscope, DE-F and DE-SF cells displayed a higher nucleus/cytoplasm ratio as compared with DE-E. Variation in chromosome number was also observed in the three cell lines. In addition to the morphological characteristics, these cell lines displayed differential patterns of several known mammary tumor cell markers. Following xenotransplantation of the CMT cells into nude mice, DE-F and DE-SF developed tumors within 2 weeks, whereas DE-E failed to develop any visible tumor up to 8 weeks after injection. Lastly, the CMT cell lines exhibited differential chemoresistance to several anti-tumor drugs, including melatonin, cyclosporine A, tamoxifen and indole, suggesting that these cell lines can be used as a comparative experimental model for the tumorigenesis of mammary carcinomas and a valuable tool for anti-cancer drug screening.
• Structure of Grouper Iridovirus Purine Nucleoside Phosphorylase Acta Crystallographica. Section D, Biological Crystallography. Feb, 2010  |   Pubmed ID: 20124695 Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 A resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 A, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an alpha/beta structure with a nine-stranded mixed beta-barrel flanked by a total of nine alpha-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.
• Transcriptional Analysis of Orange-spotted Grouper Reacting to Experimental Grouper Iridovirus Infection Developmental and Comparative Immunology. Jun, 2012  |   Pubmed ID: 22504162 Disease caused by grouper iridovirus (GIV) has resulted in economic losses due to high mortality in grouper culture. Thirty-eight up- and 48 down-regulated known entities have been identified using a GIV-infected grouper kidney cDNA microarray chip. Further quantitative validation was executed in the head-kidney and spleen for 24 candidate genes and 7 immune factors following GIV inoculation. Significant induction with various patterns could be seen in 30 tested genes in the spleen. However, only 23 genes had induction in the head-kidney and meanwhile 5 genes showed reduction. Transcriptional expression profiles of selected genes in response to lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (PIC) were also established to compare with the GIV-stimulated expression. The results indicated that the responses of most genes facing GIV invasion have more similarities to PIC treatment than LPS. Seven genes are thought to be interferon-related factors: RNA helicase DHX58, ISG15, viperin, HECT E3 ligase (HECT), CD9, urokinase plasminogen activator surface receptor (PLAUR) and Mx-1. Following immunization with inactivated GIV, significant induction could be seen in DHX58, viperin, IL-1β, IL-8, COX-2, HECT, PLAUR, IgM, Mx-1, very large inducible GTPase-1 (VLIG1) and TNF-α in the head-kidney or spleen, and the latter 6 genes also had a gradual increasing pattern by a boosting immunization. These factors might play important roles in adaptive antiviral protection. Thus, we have characterized the temporal response patterns of virus responsive genes and have also identified several potential immune markers to further investigate host antiviral defense mechanisms.
• Molecular Characterization and Expression Analysis of Four Isotypes of Immunoglobulin Light Chain Genes in Orange-spotted Grouper, Epinephelus Coioides Developmental and Comparative Immunology. Mar, 2013  |   Pubmed ID: 23186640 To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.
• Identification of the Proteins Required for Fatty Acid Desaturation in Zebrafish (Danio Rerio) Biochemical and Biophysical Research Communications. Nov, 2013  |   Pubmed ID: 24103751 Zebrafish Δ-5/Δ-6 fatty acid desaturase (Z-FADS) catalyzes the cascade synthesis of long-chain polyunsaturated fatty acids (PUFAs), thereby playing a pivotal role in several biological processes. In the current study, we report that the Z-FADS protein exists in close proximity to certain cytochrome b5 reductases (CYB5R2 and 3) and elongases (ELOVL2, 4, 5 and 7) on the endoplasmic reticulum, as determined using fluorescence microscopy and fluorescence resonance energy transfer. HeLa cells co-transfected with zebrafish fads and elovl2, 4, and 5 produced docosahexaenoic acid (DHA), as detected by gas chromatography. In addition, immunofluorescence cytochemistry and Western blot data revealed that Z-FADS is present in the mitochondria of HeLa cells. Collectively, our results implicate that Z-FADS, the sole fatty acid desaturase ever been identified in zebrafish, can serve as a universal fatty acid desaturase during lipogenesis.
• Controlled Oxidation of Aliphatic CH Bonds in Metallo-monooxygenases: Mechanistic Insights Derived from Studies on Deuterated and Fluorinated Hydrocarbons Journal of Inorganic Biochemistry. May, 2014  |   Pubmed ID: 24629413 The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions.
• MicroRNA Regulation of DNA Repair Gene Expression in 4-aminobiphenyl-treated HepG2 Cells Toxicology. Aug, 2014  |   Pubmed ID: 24857880 We examined the role of miRNAs in DNA damage response in HepG2 cells following exposure to 4-aminobiphenyl (4-ABP). The arylamine 4-ABP is a human carcinogen. Using the Comet assay, we showed that 4-ABP (18.75-300μM) induces DNA damage in HepG2 cells after 24h. DNA damage signaling pathway-based PCR arrays were used to investigate expression changes in genes involved in DNA damage response. Results showed down-regulation of 16 DNA repair-related genes in 4-ABP-treated cells. Among them, the expression of selected six genes (UNG, LIG1, EXO1, XRCC2, PCNA, and FANCG) from different DNA repair pathways was decreased with quantitative real-time PCR (qRT-PCR). In parallel, using the miRNA array, we reported that the expression of 27 miRNAs in 4-ABP-treated cells was at least 3-fold higher than that in the control group. Of these differential 27 miRNAs, the most significant expression of miRNA-513a-5p and miRNA-630 was further validated by qRT-PCR, and was predicted to be implicated in the deregulation of FANCG and RAD18 genes, respectively, via bioinformatic analysis. Both FANCG and RAD18 proteins were found to be down-regulated in 4-ABP-treated cells. In addition, overexpression and knockdown of miRNA-513a-5p and miRNA-630 reduced and increased the expression of FANCG and RAD18 proteins, respectively. Based on the above results, we indicated that miRNA-513a-5p and miRNA-630 could play a role in the suppression of DNA repair genes, and eventually lead to DNA damage.
• Characterization of Protein Marker Expression, Tumorigenicity, and Doxorubicin Chemoresistance in Two New Canine Mammary Tumor Cell Lines BMC Veterinary Research. Sep, 2014  |   Pubmed ID: 25267010 Canine mammary tumors (CMTs) are the most common type of cancer found in female dogs. Establishment and evaluation of tumor cell lines can facilitate investigations of the biological mechanisms of cancer. Different cell models are used to investigate genetic, epigenetic, and cellular pathways, cancer progression, and cancer therapeutics. Establishment of new cell models will greatly facilitate research in this field. In the present study, we established and characterized two new CMT cell lines derived from a single CMT.
• Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing MAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection PloS One. 2015  |   Pubmed ID: 25938761 Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.
• Iridovirus CARD Protein Inhibits Apoptosis Through Intrinsic and Extrinsic Pathways PloS One. 2015  |   Pubmed ID: 26047333 Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.