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.

Molecular cloning, characterization and expression analysis of the PKZ gene in rare minnow Gobiocypris rarus

Double-stranded RNA-activated protein kinase (PKR) plays an important rote in interferon-induced antiviral responses, and is also involved in intracellular signaling pathways, including the apoptosis, proliferation, and transcription pathways. In the present study, a PKR-like gene was cloned and characterized from rare minnow Gobiocypris rarus. The full length of the rare minnow PKR-like (GrPKZ) cDNA is 1946 bp in Length and encodes a polypeptide of 503 amino acids with an estimated molecular mass of 57,355 Da and a predicted isoelectric point of 5.83. Analysis of the deduced amino acid sequence indicated that the mature peptide contains two Zalpha domains and one S_TKc domain, and is most similar to the crucian carp (Carassius auratus) PKR-like amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed that GrPKZ mRNA expression is at low levels in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus, GrPKZ expression was up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). Following infection with Aeromonas hydrophila, GrPKZ transcripts were induced at 24 h post-injection (P < 0.05) and returned to control levels at 120 h post-injection. These data imply that GrPKZ is involved in antiviral defense and Toll-like receptor 4 signaling pathway in bacterial infection. (C) 2008 Elsevier Ltd. All rights reserved.

Analysis of heavy metals of muscle and intestine tissue in fish - in Banan section of Chongqing from Three Gorges Reservoir, China

The present study was carried out to investigate contamination of heavy metals in 19 fish species from the Banan section of Chongqing in the Three Gorges, Yangtze River. The results showed that the mean concentrations of heavy metals were higher in intestine than muscle, except zinc in upper strata. In the fish inhabiting the upper strata, there were significant differences between mean concentrations of As, Cr, Cu and Hg in muscle and intestine (P <0.05). There were also significant differences between mean concentrations of Cr and Cu in muscle and intestine in the fish inhabiting middle strata. However, significant differences between mean concentrations of As, Cd, Hg, Pb and Zn were measured in fish inhabiting bottom strata in both intestine and muscle tissues (P <0.05). For the fish inhabiting different strata, the concentrations of As, Cd, Cr, Cu, Hg and Ph in muscle and intestine of the fish from bottom strata (BS) were higher than those in both upper strata (US) and middle strata (MS); whereas a higher concentration of Zn was measured in muscle and intestine from fish inhabiting upper strata. Mean metal concentrations were found to be higher in age 11 than those in age I in Coreius heterodon (2- and 1-year odl fish respectively). The overall results indicated that fish muscle in the Banan section were slightly contaminated by heavy metals, but did not exceed Chinese food standards.

Molecular cloning and stress-induced expression of paralichthys olivaceus heme-regulated initiation factor 2 alpha kinase

The heme-regulated initiation factor 2 alpha kinase (HRI) is acknowledged to play an important role in translational shutoff in reticulocytes in response to various cellular stresses. In this study, we report its homologous cDNA cloning and characterization from cultured flounder embryonic cells (FEC) after treatment with UV-inactivated grass carp haemorrhagic virus (GCHV). The full-length cDNA of Paralichthys olivaceus HRI homologue (PoHRI) has 2391 bp and encodes a protein of 651 amino acids. The putative PoHRI protein exhibits high identity with all members of eIF2 alpha kinase family. It contains 12 catalytic subdomains located within the C-terminus of all Ser/Thr protein kinases, a unique kinase insertion of 136 amino acids between subdomains IV and V, and a relatively conserved N-terminal domain (NTD). Upon heat shock, virus infection or Poly PC treatment, PoHRI mRNA and protein are significantly upregulated in FEC cells but show different expression patterns in response to different stresses. In healthy flounders, PoHRI displays a wide tissue distribution at both the mRNA and protein levels. These results indicate that PoHRI is a ubiquitous eIF2a kinase and might play an important role in translational control over nonheme producing FEC cells under different stresses. (c) 2006 Elsevier Ltd. All rights reserved.

alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp PCC 7120

Anabaena sp. PCC; 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43') but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Myxobolus lentisuturalis sp n. (Myxozoa : Myxobolidae), a new muscle-infecting species from the Prussian carp, Carassius gibelio from China

A new highly pathogenic muscle-infecting species of the genus Myxobolus Butschli, 1882 is described from the Prussian carp, Carassius gibelio (Bloch, 1782) using spore morphology and SSU rDNA sequence data. Phylogenetic analyses elucidated relationship of the newly described Myxobolus lentisuturalis to other Myxobolus species and supported its position of an independent species.

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio) by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5 ' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of ANNATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3 ' untranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes). RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary, spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart, kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtellogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.

Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.

A SERINE KINASE REGULATES INTRACELLULAR-LOCALIZATION OF SPLICING FACTORS IN THE CELL CYCLE

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in 'speckles' in the nucleus of interphase cells(1). It is believed that nuclear speckles act as storage sites for splicing factors while splicing occurs on nascent transcripts(2). Splicing factors redistribute in response to transcription inhibition(3,4) or viral infection(5), and nuclear speckles break down and reform as cells progress through mitosis(6). We have now identified and cloned a kinase, SRPK1, which is regulated by the cell cycle and is specific for SR proteins; this kinase is related to a Caenorhabditis elegans kinase and to the fission yeast kinase Dsk1 (ref. 7). SRPK1 specifically induces the disassembly of nuclear speckles, and a high level of SRPK1 inhibits splicing in vitro. Our results indicate that SRPK1 mag have a central role in the regulatory network for splicing, controlling the intranuclear distribution of splicing factors in interphase cells, and the reorganization of nuclear speckles during mitosis.

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation. 地址: [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Chinese Acad Sci, Inst Modern Phys, Lanzhou 730000, Peoples R China; [Li Ning; Zhang Hong; Wang Yanling; Hao Jifang] Key Lab Heavy Ion Radiat Med Gansu Prov, Lanzhou 730000, Peoples R China; [Li Ning; Wang Yanling] Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China; [Wang Xiaohu] Gansu Tumor Hosp, Dept Radiotherapy, Lanzhou 730050, Peoples R China

Microarray-based Raman spectroscopic assay for kinase inhibition by gold nanoparticle probes

In this paper, a microarray-based surface-enhanced Raman spectroscopic (SERS) assay for detection of kinase functionality and inhibition has been reported. Biotinylated anti-phosphoserinen antibodies mark the phosphorylation and inhibition events and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry, followed by silver deposition for SERS signal enhancement. The avidin conjugated fluorescein is used as SERS probe. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), its well known substrate, kemptide, and three inhibitors, H89, HA1077, and KN62 have been chosen here to establish the SERS assay. As expected, highly selective inhibition of PKA is demonstrated with the inhibitor H89 and the inhibition assay enable to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.

Microarray-based kinase inhibition assay by gold nanoparticle probes

We report on the development of a new class of kinase microarray for the detection of kinase inhibition based on marking peptide phosphorylation/biotinylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The alpha-catalytic subunit of cyclic adenosine 5'-monophosphate-dependent protein kinase (PKA), and its well-known substrate, kemptide, were used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated with the four inhibitors: H89, HA1077, mallotoxin, and KN62. Furthermore, an inhibition assay demonstrates the ability to detect kinase inhibition as well as derive IC50 (half-maximal inhibitory concentration) plots.