Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Motor effort alters changes of mind in sensorimotor decision making.

After committing to an action, a decision-maker can change their mind to revise the action. Such changes of mind can even occur when the stream of information that led to the action is curtailed at movement onset. This is explained by the time delays in sensory processing and motor planning which lead to a component at the end of the sensory stream that can only be processed after initiation. Such post-initiation processing can explain the pattern of changes of mind by asserting an accumulation of additional evidence to a criterion level, termed change-of-mind bound. Here we test the hypothesis that physical effort associated with the movement required to change one's mind affects the level of the change-of-mind bound and the time for post-initiation deliberation. We varied the effort required to change from one choice target to another in a reaching movement by varying the geometry of the choice targets or by applying a force field between the targets. We show that there is a reduction in the frequency of change of mind when the separation of the choice targets would require a larger excursion of the hand from the initial to the opposite choice. The reduction is best explained by an increase in the evidence required for changes of mind and a reduced time period of integration after the initial decision. Thus the criteria to revise an initial choice is sensitive to energetic costs.

Identification of differentially expressed genes from the cross-subfamily cloned embryos derived from zebrafish nuclei and rare minnow enucleated eggs

Cross-species nuclear transfer (NT) has been used to retain the genetic viability of a species near extinction. However, unlike intra-species NT, most embryos produced by cross-species NT were unable to develop to later stages due to incompatible nucleocytoplasmic interactions between the donor nuclei and the recipient cytoplasm from different species. To study the early nucleocytoplasmic interaction in cross-species NT, two laboratory fish species (zebrafish and rare minnow) from different subfamilies were used to generate cross-subfamily NT embryos in the present study. Suppression subtractive hybridization (SSH) was performed to screen out differentially expressed genes from the forward and reverse subtractive cDNA libraries. After dot blot and real-time PCR analysis, 80 of 500 randomly selective sequences were proven to be differentially expressed in the cloned embryos. Among them, 45 sequences shared high homology with 28 zebrafish known genes, and 35 sequences were corresponding to 22 novel expressed sequence tags (ESTs). Based on functional clustering and literature mining analysis, up-and down-regulated genes in the cross-subfamily cloned embryos were mostly relevant to transcription and translation initiation, cell cycle regulation, protein binding, etc. To our knowledge, this is the first report on the determination of genes involved in the early development of cross-species NT embryos of fish. (C) 2007 Elsevier Inc. All rights reserved.

Does temporal discounting explain unhealthy behavior? A systematic review and reinforcement learning perspective.

The tendency to make unhealthy choices is hypothesized to be related to an individual's temporal discount rate, the theoretical rate at which they devalue delayed rewards. Furthermore, a particular form of temporal discounting, hyperbolic discounting, has been proposed to explain why unhealthy behavior can occur despite healthy intentions. We examine these two hypotheses in turn. We first systematically review studies which investigate whether discount rates can predict unhealthy behavior. These studies reveal that high discount rates for money (and in some instances food or drug rewards) are associated with several unhealthy behaviors and markers of health status, establishing discounting as a promising predictive measure. We secondly examine whether intention-incongruent unhealthy actions are consistent with hyperbolic discounting. We conclude that intention-incongruent actions are often triggered by environmental cues or changes in motivational state, whose effects are not parameterized by hyperbolic discounting. We propose a framework for understanding these state-based effects in terms of the interplay of two distinct reinforcement learning mechanisms: a "model-based" (or goal-directed) system and a "model-free" (or habitual) system. Under this framework, while discounting of delayed health may contribute to the initiation of unhealthy behavior, with repetition, many unhealthy behaviors become habitual; if health goals then change, habitual behavior can still arise in response to environmental cues. We propose that the burgeoning development of computational models of these processes will permit further identification of health decision-making phenotypes.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Characterization of an early gene encoding for dUTPase in Rana grylio virus

dUTPase (DUT) is a ubiquitous and important enzyme responsible for regulating levels of dUTP. Here, an iridovirus DUT was identified and characterized from Rana grylio virus (RGV) which is a pathogen agent in pig frog. The DUT encodes a protein of 164aa with a predicted molecular mass of 17.4 kDa, and its transcriptional initiation site was determined by 5'RACE to start from the nucleotide A at 15 nt upstream of the initiation codon ATG. Sequence comparisons and multiple alignments suggested that RGV DUT was quite similar to other identified DUTs that function as homotrimers. Phylogenetic analysis implied that DUT horizontal transfers might have occurred between the vertebrate hosts and iridoviruses. Furthermore, its temporal expression pattern during RGV infection course was characterized by RT-PCR and Western blot analysis. It begins to transcribe and translate as early as 4 h postinfection (p.i.), and remains detectable at 48 h p.i. DUT-EGFP fusion protein was observed in the cytoplasm of pEGFP-N3-Dut transfected EPC cells. Immunofluorescence also confirmed DUT cytoplasm localization in RGV-infected cells. Using drug inhibition analysis by a de novo protein synthesis inhibitor (cycloheximide) and a viral DNA replication inhibitor (cytosine arabinofuranoside), RGV DUT was classified as an early (E) viral gene during the in vitro infection. Moreover, RGV DUT overexpression was shown that there was no effect on RGV replication by viral replication kinetics assay. (c) 2006 Published by Elsevier B.V.

Characterization of the Rana grylio virus 3 beta-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

The 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3 beta-HSD gene homolog,was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3 beta-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3 beta-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3 beta-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3 beta-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3 beta-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3 beta-HSD might be a protein involved in host-virus interaction. @ 2006 Elsevier Inc. All rights reserved.

Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli

A goose-type lysozyme (g-lysozyme) gene has been cloned from the mandarin fish (Siniperca chuatsi), with its recombinant protein expressed in Escherichia coli. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nucleotides to the end of the 3' untranslated region, and it contains 5 exons and 4 introns. The open reading frame of the glysozyme transcript has 582 nucleotides which encode a 194 amino acid peptide. The 5' flanking region of mandarin fish glysozyme gene shows several common transcriptional factor binding sites when compared with that from Japanese flounder (Paralichthys olivaceus). The recombinant mandarin fish g-lysozyme was expressed in E. coli by using pET-32a vector, and the purified recombinant g-lysozyme shows lytic activity against Micrococcus lysodeikticus. (c) 2005 Elsevier B.V All rights reserved.

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G(4)

The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G(4). From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3' region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the N-terminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G(4). The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Classical swine fever virus (CSFV) non-structural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp), a key enzyme which initiates RNA replication by a de novo mechanism without a primer and is a potential target for anti-virus therapy. We expressed the NS5B protein in Escherichia coli. The rGTP can stimulate de novo initiation of RNA synthesis and mutation of the GDD motif to Gly-Asp-Asp (GAA) abolishes the RNA synthesis. To better understand the mechanism of viral RNA synthesis in CSFV, a three-dimensional model was built by homology modeling based on the alignment with several virus RdRps. The model contains 605 residues folded in the characteristic fingers, palm and thumb domains. The fingers domain contains an N-terminal region that plays an important role in conformational change. We propose that the experimentally observed promotion of polymerase efficiency by rGTP is probably due to the conformational changes of the polymerase caused by binding the rGTP. Mutation of the GDD to GAA interferes with the interaction between the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive. (c) 2005 Elsevier B.V. All rights reserved.

Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi

A viperin gene has been cloned from the mandarin fish (Siniperca chuatsi). From the first transcription initiation site, the mandarin fish viperin gene extends 3163 nucleotides to the end of the 3' untranslated region, and it contains six exons and five introns. The open reading frame of the viperin transcript has 1062 nucleotides which encode a 354 amino acid peptide. The amino acid sequence of mandarin fish viperin shows high identities with its homologues in teleosts and mammals except for the first 70 amino acids. A characteristic feature in the viperin promoter region was the presence of five putative ICSBP (IRF8) binding sites and one IRFI binding site. The viperin gene expressed mainly in lymphoid tissues before stimulation, but its expression can be examined in almost all the organs investigated after stimulation with virus or Poly I:C. The expression pattern and promoter sequence may be considered as the indirect evidence that the transcription of viperin is regulated by interferons or interferon induced genes. (C) 2004 Elsevier B.V. All rights reserved.

Effects of feeding rate on growth performance of white sturgeon (Acipenser transmontanus) larvae

Four 1-week trials were conducted to determine the effects of feeding rates on growth performance and body proximate composition of white sturgeon larvae during each of the first 4 weeks after initiation of feeding. Feeding rates (% body weight day(-1)) were 10, 20, 30, 40, 50, and 60 for trial I; 5, 10, 15, 20, 25, and 30 for trial II; and 2.5, 5.0, 7.5, 10.5, 12.5, and 15.0 for trials III and TV Four tanks with 200 larvae each were randomly assigned to each of the six feeding rates. Average initial body weights of the larvae were 49, 94, 180, and 366 mg, respectively, for trials I-IV. The larvae were kept at 19-20 degreesC in circular tanks and fed continuously one of two commercial salmonid soft-moist feeds using automatic feeders. Proximate composition (%) of the feeds for trials I-III and IV were 13.9 and 14.9 moisture, 52.5 and 50.0 crude protein, 10.3 and 12.9 crude fat, and 8.1 and 8.7 ash, respectively. Except mortality in trial I, gain per food fed in trial III, and body ash in all trials, growth performance and body composition were significantly (P<0.05) affected by all feeding rates. Broken line analysis on specific growth rates indicated the optimum feeding rates of white sturgeon larvae to be 26%, 13%, 11%, and 6% body weight day-respectively, for weeks 1, 2, 3, and 4 after initiation of feeding. (C) 2003 Elsevier Science B.V. All rights reserved.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

植物生长与土壤水关系调控起始期

确定植物生长与土壤水关系调控起始期是可持续利用土壤水资源的基础。以柠条为研究对象,采用中子仪对黄土丘陵半干旱区撂荒地,1～5年生柠条林生长和土壤水分进行长期定位观测和分析。结果表明:1 a内,随着时间推移,柠条利用土壤水分深度从播种时的表层土壤增加到220 cm;随着林龄增加,除丰水年2年生柠条林地土壤储水量增加外,柠条利用土壤水分深度和耗水量增加,林地土壤储水量下降。在2004年生长末期,3年生柠条林地100 cm土层的土壤含水量低于萎蔫系数,4年生柠条林地土壤旱化加剧,柠条生长与土壤水关系调控起始期是第5年。此时需要调控柠条生长与土壤水关系,采取措施降低柠条水分耗水量,实现土壤水资源可持续利用。