Effect of dietary linolenic acid/linoleic acid ratio on growth performance, hepatic fatty acid profiles and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco

The present study aimed to evaluate the effect of dietary linolenic acid (LNA)linoleic acid (LA) ratio on growth performance, hepatic fatty acid profile and intermediary metabolism of juvenile yellow catfish Pelteobagrus fulvidraco. Six isonitrogenous and isolipidic diets were formulated to contain incremental levels of LNA from 0 to 5% at the expense of corn oil (rich in LA), resulting in six dietary treatments with LNA to LA ratios ranging from 0.35 to 14.64. The experiment continued for 7 weeks. Best growth and feed intake were obtained in the fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). In contrast, feed conversion ratio was the lowest for fish fed the diets containing the LNA/LA ratios of 1.17 and 2.12 (P<0.05). Dietary LNA to LA ratios significantly influenced viscerosomatic index and hepatosomatic index (P<0.05), but not condition factor (P>0.05). Body composition was also significantly influenced by dietary LNA to LA ratios (P<0.05). Generally, liver FA compositions reflected dietary FA profiles. Declining LA and increasing LNA contents in liver were observed with the increasing dietary LNA/LA ratios (P<0.05). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increased with the increasing LNA to LA ratios, suggesting that yellow catfish could elongate and desaturate C18 polyunsaturated fatty acids into highly unsaturated fatty acids. As a consequence, the n-6 fatty acids (FA) declined, and total n-3 FA and n-3/n-6 ratios increased with the dietary ratios of LNA/LA (P<0.05). Dietary LNA to LA ratios significantly influenced several enzymatic activities involved in liver intermediary metabolism (P<0.05), such as lipoprotein lipase, hepatic lipase, pyruvate kinase, succinate dehydrogenase, malic dehydrogenase and lactate dehydrogenase, suggesting that dietary LNA/LA ratios had significant effects on nutrient metabolism in the liver. To our knowledge this is the first demonstration of the effects of dietary LNA to LA ratios on the enzymatic activities of liver in fish, which provides information on diet quality and utilization, and can also be used as an indicator of the nutritional status of this fish. (C) 2009 Elsevier B.V. All rights reserved.

Effect of high dietary starch levels on the growth performance, blood chemistry and body composition of gibel carp (Carassius auratus var. gibelio)

An 8-week growth trial was carried out in a semi-recirculation system to investigate the effect of high dietary starch levels on the growth performance, blood chemistry, starch utilization and body composition of gibel carp (Carassius auratus var. gibelio). Five isonitrogenous and isocarloric experimental diets were formulated to contain different starch levels (24%, 28%, 32%, 36% and 40% respectively). Triplicate groups of fish (24 fish per tank with an average body weight, of 8.5 g) were assigned to each diet. The results showed that dietary carbohydrate levels significantly affected the growth performance, hepatopancreatic lipid content, pyruvate kinase (PK) activity and whole-body lipid content. Growth performance, body crude lipid and plasma glucose concentrations showed a decreasing trend with an increase in dietary starch from 24% to 40%. Pyruvate kinase activities and hepatopancreatic lipid content showed an increasing trend with the dietary starch increasing from 24% to 32%, and then a decreasing trend with the dietary starch increasing from 32% to 40%. No significant difference in the hepatopancreatic hexokinase (HK) activity, plasma triglyceride contents, body crude protein, ash and calcium (Ca) and phosphorus (P) contents was observed between different treatments. In conclusion, higher dietary starch levels (32-40%) significantly (P < 0.05) decreased the growth of gibel carp in the present study.

Meat and bone meal replacement in diets for juvenile gibel carp (Carassius auratus gibelio): effects on growth performance, phosphorus and nitrogen loading

A 11-week growth trial was conducted in a flow-through system with juvenile gibel carp Carassius auratus gibelio to evaluate the effects of gradual replacement of fish meal (FM) by meat and bone meal (MBM) on growth performance, phosphorus (P) and nitrogen (N) loading. Six isonitrogenous (crude protein: 410 g kg(-1)) and isoenergetic (gross energy: 18 kJ g(-1)) diets were formulated. FM was used as the control protein. In the other five diets, 20, 40, 60, 80 and 100% FM protein was substituted with MBM20, MBM40, MBM60, MBM80, MBM100, respectively. Total P content in the diets ranged from 16.0 to 28.3 g kg(-1) and the available P was 5.0-6.6 g kg(-1). The results showed that the best growth was achieved with fish fed on the control diet and MBM20. Final body weight, weight gain, feed efficiency, protein retention efficiency and energy retention efficiency decreased with increased dietary MBM. No significant differences were found in the feeding rate and hepatosomatic index between the groups. Apparent digestibility coefficient (ADC) of dry matter, protein and P decreased with increase in dietary MBM, while there were no significant differences in the ADC of energy. P and N retention decreased linearly while P and N loading increased linearly with the increased dietary MBM levels. No significant differences were observed in the activity of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase, as well as pyruvate kinase in liver or in serum. Total superoxide dismutase activity in MBM20 was significantly higher than that of MBM100.

Effect of dietary carbohydrate sources on growth performance and utilization for gibel carp (Carassius auratus gibelio) and Chinese longsnout catfish (Leiocassis longirostris Gunther)

The nutritional function of monosaccharides, disaccharides and polysaccharides for omnivorous gibel carp and carnivorous Chinese longsnout catfish were investigated and the ability of these two species to utilize carbohydrates was compared. For each species, triplicate groups of fish were assigned to each of five groups of isoenergetic and isonitrogenous experimental diets with different carbohydrate sources: glucose, sucrose, dextrin, soluble starch (acid-modified starch) and alpha-cellulose. The carbohydrates were included at 60 g kg(-1) in Chinese longsnout catfish diets and at 200 g kg(-1) in gibel carp diets. A growth trial was carried out in a recirculation system at 27.8 +/- 1.9 degrees C for 8 weeks. The results showed that fish with different food habits showed difference in the utilization of carbohydrate sources. For gibel carp, better specific growth rate (SGR) and feed efficiency (FE) were observed in fish fed diets containing soluble starch and cellulose, but for Chinese longsnout catfish, better SGR and FE were observed in fish fed diets containing dextrin and sucrose. Apparent digestibility coefficient of dry matter (ADC(d)) and apparent digestibility coefficient of energy (ADC(e)) were significantly affected by dietary carbohydrate sources in gibel carp. ADC(d) and ADC(e) significantly decreased as dietary carbohydrate complexity increased in Chinese longsnout catfish except that glucose diet had medium ADC(d) and ADC(e). In both species, no significant difference of apparent digestibility coefficient of protein was observed between different carbohydrate sources. Dietary carbohydrate sources significantly affected body composition, and liver phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) activities also varied according to dietary carbohydrate complexity. Fish with different food habits showed different abilities to synthesize liver glycogen, and the liver glycogen content in gibel carp was significantly higher than in Chinese longsnout catfish. The influence of carbohydrate source on gluconeogenesis and lipogenesis was also different in the two fish species.

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2 alpha kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2 alpha kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2 alpha kinases might play similar roles and that PoPKR is the predominant kinase. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Characterization of the porcine differentially expressed PDK4 gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

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 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.

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.