Nervous Necrosis Virus Coat Protein Mediates Host Translation Shutoff through Nuclear Translocalization and Degradation of Polyadenylate Binding Protein.

Nervous necrosis virus (NNV) belongs to the Betanodavirus genus of the Nodaviridae family and is the main cause of viral nervous necrosis disease in marine fish larvae and juveniles worldwide. The NNV virion contains two positive-sense, single-stranded RNA genomes, which encode RNA-dependent RNA polymerase, coat protein, and B2 protein. Interestingly, NNV infection can shut off host translation in orange-spotted grouper (Epinephelus coioides) brain cells; however, the detailed mechanisms of this action remain unknown. In this study, we discovered that the host translation factor, polyadenylate binding protein (PABP), is a key target during NNV takeover of host translation machinery. Additionally, ectopic expression of NNV coat protein is sufficient to trigger nuclear translocalization and degradation of PABP, followed by translation shutoff. A direct interaction between NNV coat protein and PABP was demonstrated, and this binding requires the NNV coat protein N-terminal shell domain and PABP proline-rich linker region. Notably, we also showed that degradation of PABP during later stages of infection is mediated by the ubiquitin-proteasome pathway. Thus, our study reveals that the NNV coat protein hijacks host PABP, causing its relocalization to the nucleus and promoting its degradation to stimulate host translation shutoff. IMPORTANCE Globally, more than 200 species of aquacultured and wild marine fish are susceptible to NNV infection. Devastating outbreaks of this virus have been responsible for massive economic damage in the aquaculture industry, but the molecular mechanisms by which NNV affects its host remain largely unclear. In this study, we show that NNV hijacks translation in host brain cells, with the viral coat protein binding to host PABP to promote its nuclear translocalization and degradation. This previously unknown mechanism of NNV-induced host translation shutoff greatly enhances the understanding of NNV pathogenesis and provides useful insights and novel tools for development of NNV treatments, such as the use of orange-spotted grouper brain cells as an in vitro model system.

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

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.

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

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 a = 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.

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

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.

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

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.

Effect of Load Carriage Lifestyle on Kinematics and Kinetics of Gait.

Backpacks are commonly worn by many people for multiple purposes. This study investigated the effects of habitual wearing of backpacks on lower limb kinematics and kinetics. Fourteen participants were recruited for analysis. All participants performed four randomly assigned scenarios, including running and walking at speeds of 3.5 and 1.5 m/s, respectively, with and without load carriage. The motion analysis system and force plate were used to investigate the lower limb kinematics and kinetics. A paired sample t-test was performed for statistical measurement with a significance level of α = .05. The results indicated that active force, breaking force, impact peak, loading rate, active peak, maximum braking, hip flexion, and hip range of motion were substantially higher under load carriage conditions than under walking condition, however, time to peak was lower. Conversely, during load carriage running, active force, braking impulse, time to peak, ankle plantarflexion, and ankle range of motion were all higher than those during running. Carrying a backpack weighing 10% of the body weight induced different foot strike patterns at both speeds; during load carriage walking, the hip tended to flex more; whereas, during load carriage running, the ankle tended to flex more. In conclusion, human body seems to adopt different gait strategies during load carriage walking and running. That is, the hip strategy is used during walking, while the ankle strategy is used during running.

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

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.

Structure of grouper iridovirus purine nucleoside phosphorylase.

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.

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

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.

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

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 (p < .01), peak power output improved by 4.94% and 11.52%, respectively (p < .05), and maximum rate of force development improved by 13.41% and 18.38%, respectively (p < .01). Acute WBV (frequency = 30 Hz, peak-to-peak amplitude of two mm) induced neuromuscular activation and improved lunge reaction scores, agility, and power.

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

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.

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

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.

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

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.

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

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.

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

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.

Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection.

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.

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.

Cloning and characterization of metallothionein gene in ayu Plecoglossus altivelis.

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.

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.

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

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.