Publications by authors named "Chi-Yao Chang"

34 Publications

The acute effects of whole body vibration stimulus warm-up on skill-related physical capabilities in volleyball players.

Sci Rep 2021 Mar 10;11(1):5606. Epub 2021 Mar 10.

Office of Physical Education, Chung Yuan Christian University, Taoyüan, Taiwan, ROC.

Whole body vibration (WBV) has been suggested to improve athletes' neuromuscular strength and power. This study investigated the effect of single WBV stimulation on volleyball-specific performance. The participants were 20 elite male volleyball players who performed a 1-min warm-up exercise on a vibration platform at a frequency of 30 Hz and peak-to-peak displacement of 2 mm. After the warm-up exercise, the participants performed a blocking agility test (BAT), 10-m sprinting test, agility T-test, and counter movement jump test. We compared the participants' performance at four time points (Pretest, Post 0, Post 1, and Post 2). The results revealed that the participants' BAT performance and maximum rate of force development improved significantly 1 min after the vibration stimulation (p < 0.01). The WBV (frequency of 30-Hz, peak-to-peak displacement of 2 mm) intervention significantly improved the volleyball-specific defensive performance and speed strength of the participants. Accordingly, by undergoing WBV as a form of warm-up exercise, the technique and physical fitness of volleyball players can be improved.
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http://dx.doi.org/10.1038/s41598-021-85158-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946948PMC
March 2021

The Applications of Landing Strategies in Badminton Footwork Training on a Backhand Side Lateral Jump Smash.

J Hum Kinet 2020 Jul 21;73:19-31. Epub 2020 Jul 21.

Graduate Institute of Sports Science, National Taiwan Sport University, Taoyuan, Taiwan.

Previous research in badminton has associated unilateral landings following overhead strokes with the occurrence of knee injuries. Smashing involves tensing the abdomen muscles while swinging the racket rapidly and maintaining one's balance while performing coordinated movements and steps; this process puts stress on the player's lower limbs. However, few studies have compared the effects of different stroke training while performing various types of badminton strokes. This study investigated the influence of different stroke training on the smash action of badminton players. Three stroke training conditions were considered: shadow, target striking, and smashing. Sixteen male experienced badminton players were recruited for this study. One-way repeated-measures ANOVA with Bonferroni correction was used to identify the differences. At the initial contact with the ground, the knee flexion and knee valgus angles under the smash condition were significantly higher than target and shadow conditions. Under the smash condition, hip abduction was significantly higher than under the target and shadow conditions. Moreover, the hip abduction under the target condition was significantly higher than under the shadow condition. At the maximum knee flexion, the hip abduction under the smash and target conditions was significantly higher than under the shadow condition. Regarding the time from the moment of initial contact to the peak of vertical ground reaction force it was shorter under the smash condition than the target and shadow conditions. The vertical ground reaction force was higher under the smash condition than under the target and shadow conditions. The 50 ms impulse was higher under the smash condition than under the target and shadow conditions. The main findings of this study are that under the smash condition, the motion in the frontal plane increased, which produced higher loads on the joints in the lower limbs. Player performed the same footwork under the three conditions, but the landing strategies differed because of unique swing motions and techniques. The condition under which a player hits a shot to a target area can affect the landing. The results of this study suggest that target practice is more effective for improving the landing technique employed during actual shots than shadow practice.
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http://dx.doi.org/10.2478/hukin-2020-0002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386134PMC
July 2020

Influences of Lateral Jump Smash Actions in Different Situations on the Lower Extremity Load of Badminton Players.

J Sports Sci Med 2020 06 1;19(2):264-270. Epub 2020 May 1.

Office of Physical Education, Chung Yuan Christian University, Taoyuan, Taiwan, R.O.C.

Badminton atypical actions and hitting movements often occur during the game; therefore, many special footwork methods have been developed to facilitate the rapid movements required to hit the shuttlecock, including quick turning and jumping and quick directional change movements. Studies have shown that the majority of badminton sport injuries occur in the lower extremity joints of athletes. The purpose of this study was to investigate the influences of hitting motion and unanticipated hitting direction on landing mechanics after backhand lateral jump smashing and landing to analyze joint stiffness and torque changes in three lower extremity joints. Recruited sixteen badminton athletes.The capture frequency of the Vicon Motion System (300Hz), Kistler force platform (1500Hz) and Vicon Nexus Version 1.8.5 software were used simultaneously to capture the kinematic and kinetic parameter of backhand side lateral jump smash footwork. The swing actions were divided into two situations, shadow (footwork and racket swinging practice without targets) and hitting (footwork and stroke shuttlecock) actions, whereas the directions were divided into directional and non-directional. Two-way repeated measures ANOVA with the LSD correction was used to compare the differences among the four conditions. The significance level was set to = 0.05. Results shown that, at the peak of torque, the ankle plantar flexion of the non-directional shadow (p < 0.05) were greater than that of directional shadow (p < 0.05); meantime, ankle torque change of non-directional shadow (p < 0.05) and directional hitting (p < 0.05) was lower than that of non-directional hitting, but the non-directional hitting was larger compared to non-directional shadow (p < 0.05) at the maximum vertical GRF. The hip extension at peak of torque of directional hitting were larger than that of non-directional shadow (p < 0.05). The shadow actions hip flexion angle was larger than that of directional hitting at initial contact, but the non-directional hitting hip abduction was has the significant difference among all the conditioning. The hip flexion angle of non-directional shadow was larger than that of directional hitting (p < 0.05), the hip abduction angle of the non-directional hitting was greater than that of non-directional shadow (p < 0.05) at the peak VGRF. Elite badminton players execute different training movements; the joint stiffness was in the same state. In the hitting actions has greater ankle and hip joint torque than shadow actions. The badminton player was change joint range of motion to adjust lower limbs stiffness.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196745PMC
June 2020

Beneficial effects of a negative ion patch on eccentric exercise-induced muscle damage, inflammation, and exercise performance in badminton athletes.

Chin J Physiol 2020 Jan-Feb;63(1):35-42

Department of Exercise and Health Science, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan.

Complementary and alternative medicines (CAMs) are widely applied and accepted for therapeutic purposes because of their numerous benefits. Negative ion treatment belongs to one of the critical categories defined by the National Center for CAM, with such treatment capable of air purification and ameliorating emotional disorders (e.g., depression and seasonal affective disorder). Negative ions can be produced naturally and also by a material with activated energy. Exercise-induced muscle damage (EIMD) often occurs due to inadequate warm up, high-intensity exercise, overload, and inappropriate posture, especially for high-intensive competition. Few studies have investigated the effects of negative ion treatment on muscular injury in the sports science field. In the current study, we enrolled badminton athletes and induced muscle damage in them through eccentric exercise in the form of a high-intensity squat program. We evaluated the effects of negative ion patches of different intensities at three points (preexercise, postexercise, and recovery) by analyzing physiological indexes (tumor necrosis factor [TNF]-α, interleukin [IL]-6, IL-10, creatine kinase [CK], and lactate dehydrogenase [LDH] levels) and performing a functional assessment (a countermovement jump [CMJ] test). We found that a high-intensity negative ion patch could significantly reduce the levels of TNF-α, an injury-associated inflammatory cytokine, and related markers (CK and LDH). In addition, muscular overload-caused fatigue could be also ameliorated, as indicated by the functional CMJ test result, and related muscular characteristics (tone and stiffness) could be effectively improved. Thus, the negative ion treatment could effectively improve physiological adaption and muscular fatigue recovery after EIMD in the current study. The negative ion patch treatment can be further integrated into a taping system to synergistically fulfill exercise-induced damage protection and functional elevation. However, the effects of this treatment require further experimental validation.
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http://dx.doi.org/10.4103/CJP.CJP_33_19DOI Listing
February 2020

The Acute Effects of Whole-Body Vibration on Fencers' Special Abilities.

Percept Mot Skills 2019 Oct 26;126(5):973-985. Epub 2019 Jul 26.

2 Office of Physical Education, Chung Yuan Christian University, Taoyuan.

The purpose of this research was to study the effects of a whole-body vibration (WBV) warm-up for improving fencers' performance on variables derived from a lunge reaction test, the 10-meter sprint, and the countermovement jump. We compared fencer performances at four time intervals: (a) preintervention, (b) immediately postintervention, (c) 1-minute postintervention, and (d) 2-minute postintervention. Study participants were 16 male fencers. The vibration frequency was 30 Hz, and its amplitude was two mm. After each WBV session, participants significantly improved their performance on all measures at both one and two minutes after the intervention. Specifically, lunge reaction tests scores improved by 5.50% and 7.34%, respectively, relative to preintevention testing ( < .01), peak power output improved by 4.94% and 11.52%, respectively ( < .05), and maximum rate of force development improved by 13.41% and 18.38%, respectively ( < .01). Acute WBV (frequency = 30 Hz, peak-to-peak amplitude of two mm) induced neuromuscular activation and improved lunge reaction scores, agility, and power.
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http://dx.doi.org/10.1177/0031512519863573DOI Listing
October 2019

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope.

J Vis Exp 2017 03 24(121). Epub 2017 Mar 24.

Institute of Cellular and Organismic Biology, Academia Sinica;

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.
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http://dx.doi.org/10.3791/55417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408986PMC
March 2017

Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways.

PLoS One 2015 5;10(6):e0129071. Epub 2015 Jun 5.

Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan; Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129071PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457926PMC
March 2016

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 4;10(5):e0126121. Epub 2015 May 4.

Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126121PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418827PMC
April 2016

Characterization of protein marker expression, tumorigenicity, and doxorubicin chemoresistance in two new canine mammary tumor cell lines.

BMC Vet Res 2014 Sep 30;10:229. Epub 2014 Sep 30.

Background: 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.

Results: We established two cell lines from a single malignant CMT specimen: DTK-E and DTK-SME. Morphologically, the DTK-E cells were large, flat, and epithelial-like, whereas DTK-SME cells were round and epithelial-like. Doubling times were 24 h for DTK-E and 18 h for DTK-SME. On western blots, both cell lines expressed cytokeratin AE1, vimentin, cytokeratin 7 (CK7), and heat shock protein 27 (HSP27). Moreover, investigation of chemoresistance revealed that DTK-SME was more resistant to doxorubicin-induced apoptosis than DTK-E was. After xenotransplantation, both DTK-E and DTK-SME tumors appeared within 14 days, but the average size of DTK-SME tumors was greater than that of DTK-E tumors after 56 days.

Conclusion: We established two new cell lines from a single CMT, which exhibit significant diversity in cell morphology, protein marker expression, tumorigenicity, and chemoresistance. The results of this study revealed that the DTK-SME cell line was more resistant to doxorubicin-induced apoptosis and exhibited higher tumorigenicity in vivo than the DTK-E cell line. We anticipate that the two novel CMT cell lines established in this study will be useful for investigating the tumorigenesis of mammary carcinomas and for screening anticancer drugs.
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http://dx.doi.org/10.1186/s12917-014-0229-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189743PMC
September 2014

MicroRNA regulation of DNA repair gene expression in 4-aminobiphenyl-treated HepG2 cells.

Toxicology 2014 Aug 23;322:69-77. Epub 2014 May 23.

Department of Life Sciences, National Central University, Jhongli, Taiwan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tox.2014.05.003DOI Listing
August 2014

Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

J Inorg Biochem 2014 May 21;134:118-33. Epub 2014 Feb 21.

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan; Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jinorgbio.2014.02.005DOI Listing
May 2014

Identification of the proteins required for fatty acid desaturation in zebrafish (Danio rerio).

Biochem Biophys Res Commun 2013 Nov 5;440(4):671-6. Epub 2013 Oct 5.

Institute of Fisheries Science, National Taiwan University, Taipei 10617, Taiwan; Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan; Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan.

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.
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http://dx.doi.org/10.1016/j.bbrc.2013.09.127DOI Listing
November 2013

Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

Dev Comp Immunol 2013 Mar 24;39(3):169-79. Epub 2012 Nov 24.

Laboratory of Molecular Genetics, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, ROC.

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.
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http://dx.doi.org/10.1016/j.dci.2012.11.003DOI Listing
March 2013

Transcriptional analysis of orange-spotted grouper reacting to experimental grouper iridovirus infection.

Dev Comp Immunol 2012 Jun 12;37(2):233-42. Epub 2012 Apr 12.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan, ROC.

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.
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http://dx.doi.org/10.1016/j.dci.2012.04.002DOI Listing
June 2012

Structure of grouper iridovirus purine nucleoside phosphorylase.

Acta Crystallogr D Biol Crystallogr 2010 Feb 22;66(Pt 2):155-62. Epub 2010 Jan 22.

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.

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.
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http://dx.doi.org/10.1107/S0907444909048276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2815667PMC
February 2010

Assessment of the tumorigenesis and drug susceptibility of three new canine mammary tumor cell lines.

Res Vet Sci 2010 Apr 11;88(2):285-93. Epub 2009 Sep 11.

Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan.

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.
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http://dx.doi.org/10.1016/j.rvsc.2009.08.006DOI Listing
April 2010

Differential display of grouper iridovirus-infected grouper cells by immunostaining.

Biochem Biophys Res Commun 2008 Aug 2;372(4):674-80. Epub 2008 Jun 2.

Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan, ROC.

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.
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http://dx.doi.org/10.1016/j.bbrc.2008.05.126DOI Listing
August 2008

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 Immunol 2008 Jul 3;25(1-2):19-27. Epub 2007 Dec 3.

Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan.

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.
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http://dx.doi.org/10.1016/j.fsi.2007.11.011DOI Listing
July 2008

Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection.

Apoptosis 2008 Jan;13(1):165-76

Molecular Genetics Laboratory, Rm. 336, Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road Section 2, Nankang, Taipei, Taiwan, ROC.

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.
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http://dx.doi.org/10.1007/s10495-007-0152-yDOI Listing
January 2008

Analysis of codon usage bias and base compositional constraints in iridovirus genomes.

Virus Res 2007 Jun 16;126(1-2):196-206. Epub 2007 Apr 16.

Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan, ROC.

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.
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http://dx.doi.org/10.1016/j.virusres.2007.03.001DOI Listing
June 2007

Expression pattern of metallothionein, MTF-1 nuclear translocation, and its dna-binding activity in zebrafish (Danio rerio) induced by zinc and cadmium.

Environ Toxicol Chem 2007 Jan;26(1):110-7

Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan.

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.
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http://dx.doi.org/10.1897/06-153r.1DOI Listing
January 2007

Microscopy-based mass measurement of a single whole virus in a cylindrical ion trap.

Angew Chem Int Ed Engl 2006 Dec;45(48):8131-4

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan.

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http://dx.doi.org/10.1002/anie.200603839DOI Listing
December 2006

Inhibition of nervous necrosis virus propagation by fish Mx proteins.

Biochem Biophys Res Commun 2006 Dec 19;351(2):534-9. Epub 2006 Oct 19.

Molecular Genetics Laboratory, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.

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.
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http://dx.doi.org/10.1016/j.bbrc.2006.10.063DOI Listing
December 2006

Characterization of serum immunoglobulin M of grouper and cDNA cloning of its heavy chain.

Vet Immunol Immunopathol 2006 Feb 30;109(3-4):255-65. Epub 2005 Sep 30.

Department of Food Science, National Kinmen Institute of Technology, Kinmen, Taiwan.

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.
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http://dx.doi.org/10.1016/j.vetimm.2005.08.029DOI Listing
February 2006

Complete genome sequence of the grouper iridovirus and comparison of genomic organization with those of other iridoviruses.

J Virol 2005 Feb;79(4):2010-23

Graduate Scholl of Life Science, Ntional Defense Medical Center, Tapei, Taiwan, Republic of China.

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.
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http://dx.doi.org/10.1128/JVI.79.4.2010-2023.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC546566PMC
February 2005

Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase.

J Gen Virol 2004 Oct;85(Pt 10):2883-2892

Institute of Zoology, Academia Sinica, Taipei 115, Taiwan, Republic of China.

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.
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http://dx.doi.org/10.1099/vir.0.80249-0DOI Listing
October 2004

Expression of metallothionein gene during embryonic and early larval development in zebrafish.

Aquat Toxicol 2004 Aug;69(3):215-27

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan.

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.
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http://dx.doi.org/10.1016/j.aquatox.2004.05.004DOI Listing
August 2004

Cloning and characterization of metallothionein gene in ayu Plecoglossus altivelis.

Aquat Toxicol 2004 Feb;66(2):111-24

Molecular Genetics Laboratory, 336, Institute of Zoology (44), Academia Sinica, 128, Academia Road Section 2, NanKang, Taipei 11529, Taiwan, ROC.

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.
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http://dx.doi.org/10.1016/j.aquatox.2003.06.003DOI Listing
February 2004

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog.

Dev Biol 2003 Feb;254(1):36-49

Max-Planck Institute for Immunobiology, Stuebeweg 51, D-79108, Freiburg, Germany.

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.
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http://dx.doi.org/10.1016/s0012-1606(02)00124-0DOI Listing
February 2003

Characterization of transactivation domain and developmental expression of pituitary specific transcription factor, Pit-1 of ayu (Plecoglossus altivelis).

Gen Comp Endocrinol 2002 Jul;127(3):307-13

Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, ROC.

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.
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http://dx.doi.org/10.1016/s0016-6480(02)00057-6DOI Listing
July 2002