Fasudil reduces ß-amyloid levels and neuronal apoptosis in APP/PS1 transgenic mice via inhibition of the Nogo-A/NgR/RhoA signaling axis.

Recent studies have shown that Nogo-A and the Nogo-A receptor affect ß-amyloid metabolism and the downstream Rho GTP enzyme signaling pathway, which may affect the levels of ß-amyloid and tau. Nogo-A may play a key role in the pathogenesis of Alzheimer's disease. However, the underlying molecular mechanisms of Fasudil treatment in Alzheimer's disease are not yet clear. Our results have found that Fasudil treatment for two months substantially ameliorated behavioral deficits, diminished ß-amyloid plaque and tau protein pathology, and alleviated neuronal apoptosis in APP/PS1 transgenic mice. More importantly, two well-established markers for synaptic function, growth-associated protein 43 and synaptophysin, were upregulated after Fasudil treatment. Finally, the levels of Nogo-A, Nogo-A receptor complex NgR/p75NTR/LINGO-1 and the downstream Rho/Rho kinase signaling pathway were significantly reduced. These findings suggest that Fasudil exerts its neuroprotective function in Alzheimer's disease by inhibiting the Nogo-A/NgR1/RhoA signaling pathway.

NADPH oxidase 2 mediates cardiac sympathetic denervation and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced cardiomyopathy.

Doxorubicin has been used extensively as a potent anticancer agent, but its clinical use is limited by its cardiotoxicity. However, the underlying mechanisms remain to be fully elucidated. In this study, we tested whether NADPH oxidase 2 (Nox2) mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced heart failure. Nox2 knockout (KO) and wild-type (WT) mice were randomly assigned to receive a single injection of doxorubicin (15 mg/kg, i.p.) or saline. WT doxorubicin mice exhibited the decreases in survival rate, left ventricular (LV) wall thickness and LV fractional shortening and the increase in the lung wet-to-dry weight ratio 1 week after the injections. These alterations were attenuated in Nox2 KO doxorubicin mice. In WT doxorubicin mice, myocardial oxidative stress was increased, myocardial noradrenergic nerve fibers were reduced, myocardial expression of PGP9.5, GAP43, tyrosine hydroxylase and norepinephrine transporter was decreased, and these changes were prevented in Nox2 KO doxorubicin mice. Myocyte autophagy was increased and myocyte size was decreased in WT doxorubicin mice, but not in Nox2 KO doxorubicin mice. Nox2 mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy-both of which contribute to cardiac atrophy and failure after doxorubicin treatment.

Fasudil alleviates cerebral ischemia­reperfusion injury by inhibiting inflammation and improving neurotrophic factor expression in rats.

The Rho kinase inhibitor fasudil exerts neuroprotective effects. We previously showed that fasudil can regulate M1/M2 microglia polarization and inhibit neuroinflammation. Here, the therapeutic effect of fasudil on cerebral ischemia­reperfusion (I/R) injury was investigated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model in Sprague­Dawley rats. The effect of fasudil on the phenotype of microglia and neurotrophic factors in the I/R brain and its potential molecular mechanism was also explored. It was found that fasudil ameliorated neurological deficits, neuronal apoptosis, and inflammatory response in rats with cerebral I/R injury. Fasudil also promoted the polarization of microglia into the M2 phenotype, in turn promoting the secretion of neurotrophic factors. Furthermore, fasudil significantly inhibited the expression of TLR4 and NF­κB. These findings suggest that fasudil could inhibit the neuroinflammatory response and reduce brain injury after I/R injury by regulating the shift of microglia from an inflammatory M1 phenotype to an anti­inflammatory M2 phenotype, which may be related to the regulation of the TLR4/ NF­κB signal pathway.

[Acupuncture ameliorates neurological function by suppressing microglia polarization and inflammatory response after cerebral ischemia in rats].

OBJECTIVE: To observe the effect of acupuncture on microglia polarization and inflammatory reaction in rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. METHODS: Thirty male SD rats were randomly divided into sham operation, model, and acupuncture groups, with 10 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery (MCAO) for 1 h, followed by reperfusion. After modeling, rats in the acupuncture group received manual acupuncture stimulation of "Dazhui" (GV14), "Baihui"(GV20), "Shuigou" (GV26), bilateral "Zusanli" (ST36) and "Fengchi" (GB20) by twirling the needles rapidly for 10 s/acupoint every 10 min, with the needles retained for 20 min. The treatment was conducted once daily for successive 7 days. The neurological function was evaluated according to Longa's method. The state of CIRI was observed after Nissl staining, and the expression levels of Iba-1, iNOS, Arg1, BDNF, GDNF and NeuN in the ischemic cortex tissue were detected by immunofluorescence staining. The contents of TNF-α, IL-6 and IL-10 in the ischemic tissue were assayed by ELISA. The protein expression levels of BDNF, GDNF, TLR4, MyD88 and NF-κB in the ischemic tissues were detected by Western blot. RESULTS: The neurological deficit score on the 24 h and 7th day was considerably higher in the model group than in the sham operation group (P<0.01), and evidently lower on the 7th day in the acupuncture group than in the model group (P<0.01). The number of NeuN positive cells，the area of immunofluorescence dual labelling of Arg1, BDNF and GDNF positive staining, IL-10 content, BDNF and GDNF protein expressions were significantly decreased (P<0.01), and the immunofluorescence dual labelling area of Iba-1 and iNOS, TNF-α and IL-6 contents, the pretein expression levels of TLR4, MyD88 and NF-κB considerably increased (P<0.01) in the model group relevant to the sham operation group. In contrast to the model group, the acupuncture group had a significant increase in the number of NeuN positive cells, the immunofluorescence dual labelling area of Arg1, BDNF and GDNF positive staining, IL-10 content, and BDNF and GDNF protein expressions (P<0.05, P<0.01), and an evident decrease in Iba-1 and iNOS positive staining, contents of TNF-α and IL-6, and the protein expression levels of TLR4, MyD88 and NF-κB (P<0.01, P<0.05). Nissl staining showed a marked reduction in the number of neurons, the nucleus pyknosis and nissl bodies and loose arrangement of the neuronal cells in the model group, which was relatively milder in the acupuncture group. CONCLUSION: Acupuncture intervention can improve neurological function in CIRI rats, which may be related to its effects in regulating the polarization of microglia, reducing inflammatory reaction and increasing the secretion of neurotrophic factors in the brain, inhibiting TLR4/MyD88/NF-κB signaling pathway.

The efficacy and safety of neoadjuvant nimotuzumab for gastric cancer: A meta-analysis of randomized controlled studies.

INTRODUCTION: The efficacy of neoadjuvant nimotuzumab for gastric cancer remained controversial. We conducted a systematic review and meta-analysis to explore the efficacy of neoadjuvant nimotuzumab plus chemotherapy vs chemotherapy for gastric cancer. METHODS: We have searched PubMed, EMbase, Web of science, EBSCO, and Cochrane library databases through May 2019, and included randomized controlled trials assessing the efficacy of neoadjuvant nimotuzumab plus chemotherapy vs chemotherapy for gastric cancer. This meta-analysis was performed using the random-effect model. RESULTS: Four randomized controlled trials were included in the meta-analysis. There were 128 patients included in intervention group and 131 patients included in control group. Overall, compared with chemotherapy for gastric cancer, neoadjuvant nimotuzumab plus chemotherapy showed no substantial influence on response rate (risk ratio [RR] = 1.22; 95% CI = 0.78-1.89; P = .38), disease control rate (RR = 2.22; 95% confidence interval [CI] = 0.32-15.40; P = .42), rash (RR = 1.26; 95% CI = 0.96-1.66; P = .10), neutropenia (RR = 1.26; 95% CI = 0.96-1.66; P = .10), anemia (RR = 1.08; 95% CI = 0.62-1.89; P = .78), or nausea (RR = 1.19; 95% CI = 0.96-1.48; P = .12), but might improve the incidence of vomiting (RR = 1.60; 95% CI = 1.03-2.50; P = .04). CONCLUSIONS: Neoadjuvant nimotuzumab might provide no additional benefits to the treatment of gastric cancer.

Knockdown and overexpression of Unc-45b result in defective myofibril organization in skeletal muscles of zebrafish embryos.

BACKGROUND: Unc-45 is a myosin chaperone and a Hsp90 co-chaperone that plays a key role in muscle development. Genetic and biochemical studies in C. elegans have demonstrated that Unc-45 facilitates the process of myosin folding and assembly in body wall muscles. Loss or overexpression of Unc-45 in C. elegans results in defective myofibril organization. In the zebrafish Danio rerio, unc-45b, a homolog of C. elegans unc-45, is expressed in both skeletal and cardiac muscles. Earlier studies indicate that mutation or knockdown of unc-45b expression in zebrafish results in a phenotype characterized by a loss of both thick and thin filament organization in skeletal and cardiac muscle. The effects of unc-45b knockdown on other sarcomeric structures and the phenotype of Unc-45b overexpression, however, are poorly understood in vertebrates. RESULTS: Both knockdown and overexpression provide useful tools to study gene function during animal development. Using such methods, we characterized the role of Unc-45b in myofibril assembly of skeletal muscle in Danio rerio. We showed that, in addition to thick and thin filament defects, knockdown of unc-45b expression disrupted sarcomere organization in M-lines and Z-lines of skeletal muscles in zebrafish embryos. Western blotting analysis showed that myosin protein levels were significantly decreased in unc-45b knockdown embryos. Similarly, embryos overexpressing Unc-45b also exhibited severely disorganized myosin thick filaments. Disruption of thick filament organization by Unc-45b overexpression depends on the C-terminal UCS domain in Unc-45b required for interaction with myosin. Deletion of the C-terminal UCS domain abolished the disruptive activity of Unc-45b in myosin thick filament organization. In contrast, deletion of the N-terminal TPR domain required for binding with Hsp90α had no effect. CONCLUSION: Collectively, these studies indicate that the expression levels of Unc-45b must be precisely regulated to ensure normal myofibril organization. Loss or overexpression of Unc-45b leads to defective myofibril organization.

Olive flounder (Paralichthys olivaceus) myogenic regulatory factor 4 and its muscle-specific promoter activity.

Myogenic regulatory factor 4 (MRF4) is a basic helix-loop-helix (bHLH) transcription factor that plays crucial roles in myoblast differentiation and maturation. Here, we report the isolation of the olive flounder (Paralichthys olivaceus) mrf4 gene and the spatiotemporal analysis of its expression patterns. Sequence analysis indicated that flounder mrf4 shared a similar structure with other vertebrate MRF4, including the conserved bHLH domain. Flounder mrf4 contains 3 exons and 2 introns. Sequence alignment and phylogenetic analysis showed that it was highly homologous with Salmo salar, Danio rerio, Takifugu rubripes, and Tetraodon nigroviridis mrf4. Flounder mrf4 was first expressed in the medial region of somites that give rise to slow muscles, and later spread to the lateral region of somites that give rise to fast muscles. Mrf4 transcript levels decreased significantly in mature somites in the trunk region, and expression could only be detected in the caudal somites, consistent with the timing of somite maturation. Transient expression analysis showed that the 506 bp flounder mrf4 promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos.

Protection of chickens from Newcastle disease and infectious laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle disease virus and gB gene of infectious laryngotracheitis virus.

A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization.

Transcriptome Analysis of Epithelioma Papulosum Cyprini Cells Infected by Reovirus Isolated from Allogynogenetic Silver Crucian Carp.

The present study aimed to identify differentially expressed genes (DEGs) and major signal transduction pathways that were related to the immune response of epithelioma papulosum cyprinid (EPC) cells to reoviruses isolated from allogynogenetic silver crucian carp. The study also lays a theoretical foundation for the pathogenesis and immunity of the reovirus, which is helpful to the breeding of cyprinids fish. Reovirus infected and uninfected EPC cells were analyzed by using a new-generation high-throughput sequencing technology. DEGs were identified, annotated, and classified, and the signal pathways involved in the response to reovirus infection were identified by using bioinformatics tool. The data were assembled into 92,101 contigs with an average length of 835.24 bp and an N50 value of 1432 nt. Differential expression analysis of all the genes identified 3316 DEGs at a false discovery rate (FDR) of <0.01 and a fold-change of ≥3, of which 1691 were upregulated genes, 1625 were downregulated, and about 305 were immune-related genes. Gene Ontology (GO) enrichment analysis resulted in the annotation of 3941 GO terms, including 2719 biological processes (37,810 unigenes), 376 cell components (7943 unigenes), and 846 molecular functions (11,750 unigenes). KEGG metabolic pathway analysis matched the DEGs from pre-and post-infection EPC cells to 193 pathways, of which 35 were immune-related, including the Toll-like receptor, cytokine-cytokine receptor interaction, and the JAK-STAT signaling pathways.

Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression.

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos.

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis.

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus).

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.

Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor.

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MRF4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder (Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine.

[An analysis of the clinical and radiological features of bronchioloalveolar carcinoma].

OBJECTIVE: To describe the clinical and radiological features of bronchioloalveolar carcinoma. METHODS: Data of 1 050 inpatients of lung cancer, including 50 cases of pathology-proven bronchioloalveolar carcinoma, diagnosed in our hospital between 1993 and 2003, were retrospectively reviewed. The clinical information of all the patients and the HRCT findings of 31 cases of bronchioloalveolar carcinoma were analyzed. RESULTS: There was a female predominance (60%) in the 50 patients with bronchioloalveolar carcinoma. Cough was the most common presenting symptom (20/50). Twenty-four patients sought medical attention because of abnormal chest X-rays, most of which were of nodular type at the early stage. Twenty patients were completely asymptomatic at presentation. Out of the 31 cases with HRCT, eight were of the miliary type. "Bubble-like lucency" and calcifications inside nodules were present in 7 cases respectively out of the 11 cases of the nodular type. There were 9 cases each with low attenuation consolidation, pseudocavities, reticular shadowing and ground-glass opacities. The signs of "crazy paving" and "CT angiogram sign" (distinct vasculature at the background of low-attenuated consolidation at the peak of contrast scanning) were found in 2 cases. CONCLUSIONS: Bronchioloalveolar carcinoma accounted for 4.76% of lung carcinoma in our series, with a female predominance. High prevalence of asymptomatic patients at presentation and unusual long course should prompt regular chest X-ray examination. The characteristics of HRCT findings are very helpful in its diagnosis and differential diagnosis.

Comparison of growth characteristics between skeletal muscle satellite cell lines from diploid and triploid olive flounder Paralichthys olivaceus.

OBJECTIVES: According to myosatellite cell lines (MSCs) established in vitro from diploid and triploid flounder, we compared the characters of growth and differentiation of their MSCs. The results would be useful for learning the muscle development mechanism in teleosts. MATERIALS AND METHODS: The skeletal muscle cells from the diploid and triploid olive flounder Paralichthys olivaceus were isolated and cultured in vitro, respectively, and the cells were characterized at the morphology and molecular level; meanwhile, the performance of these cells' proliferation and differentiation were analyzed. RESULTS: Two new skeletal muscle cell lines (POMSCS(2n) and POMSCS(3n)) from diploid and triploid flounder have been respectively subcultured for 67 times and 66 times. The cultured cells were mostly spindle-like mononuclear cells. They have normal flounder diploid karyotype (2n=48t) and triploid karyotype (3n=72t), respectively. Muscle satellite cell gene marker (pax7b) and myogenic cell protein marker (Desmin) were all expressed in cells of two cell lines. Both of the cells could differentiate into the large polynucleated muscle fibre cells, and immunofluorescence reactions of myosin heavy chain (MyHC) were positive. There were more cells of POMSCS(3n) to differentiate into the muscle fibre cells than that of POMSCS(2n). However, POMSCS(2n) cells proliferated more rapidly than those of POMSCS(3n) (P < 0.05). The significant fluorescent signals were observed in both POMSCS(2n) and POMSCS(3n) cells after transfected with pEGFP-N3 reporter plasmid. CONCLUSIONS: The two cell lines have been established and characterized as MSCs. We suppose that it might be the differentiation capacity, rather than the proliferation activity of MSCs to play a key role in the better growth of triploid ones than diploid. Both cell lines will become the ideal tools to learn the mechanism of fish MSCs proliferation, differentiation and regeneration during muscle development in the future.

Expression of Bblhx3, a LIM-homeobox gene, in the development of amphioxus Branchiostoma belcheri tsingtauense.

Amphioxus Bblhx3 was identified as a LIM-homeobox gene expressed in gastrulae. Structural analysis suggested that it is a member of lhx3 but not of lhx1 gene group. Whole mount in situ hybridization revealed that expression of Bblhx3 was initiated at the early gastrula stage and continued at least until 10-day larvae. Expression of Bblhx3 first appeared in the vegetal and future dorsal area in initial gastrulae and became restricted to the endoderm during gastrulation. In neurulae and early larvae, Bblhx3 was expressed in the developing neural tube, the notochord and preoral pit lineage. In 10-day larvae, Bblhx3 was expressed only in the preoral pit. This expression pattern is apparently distinct from that of vertebrate lhx3 genes that are not expressed during gastrulation.

Four viral proteins of white spot syndrome virus (WSSV) that attach to shrimp cell membranes.

White spot syndrome virus (WSSV) is a major shrimp pathogen that has a widespread negative affect on shrimp production in Asia and the Americas. It is known that WSSV infects shrimp cells through viral attachment proteins (VAP) that bind with shrimp cell receptors. However, the identity of both WSSV VAP and shrimp cell receptors remains unclear. We used digoxigenin (DIG)-labeled shrimp hemocyte and gill cell membranes to bind to WSSV proteins immobilized on nitrocellulose membranes, and 4 putative WSSV VAP (37 kDa, 39 kDa and 2 above 97 kDa) were identified. Mass spectrometric analysis identified the 37 kDa putative VAP as the product of WSSV gene VP281.

[Advances in Detection Technologies of in vivo Expression of Bacterial Virulence Gene.].

The interactions between bacterial pathogens and their hosts is complex. To further our understanding ofathe pathogenesisaof bacterial pathogens, it is necessary to identify bacterial virulence genes that are specifically induced in vivo during infection and probe their regulation in vivo. Toward this end, several technologies, such as in vivo expression technology (IVET), signature-tagged mutagenesis (STM), differential fluorescence induction (DFI), genomic analysis and mapping by in vitro transposition (GAMBIT) and in vivo induced antigen technology (IVIAT), have been developed. The purpose of this reviewais to update the reader on the many advances of these technologies, and to discuss their advantages and disadvantages.

Analyzing cold tolerance mechanism in transgenic zebrafish (Danio rerio).

Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13 °C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28 °C. After 2 h at 13 °C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P<0.05). At 13 °C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28 °C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish.

Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus).

During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5'-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.