Structure,organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3′ and 5′ RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5′-terminal untranslated region (UTR), a 355 bp 3′-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74–96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.     

Increased liver protein and mRNA expression of natural killer cell-enhancing factor B (NKEF-B) in ayu (Plecoglossus altivelis) after Aeromonas hydrophila infection

Natural killer cell-enhancing factor (NKEF) may mediate cellular responses to proinflammatory molecules. The liver proteins of Aeromonas hydrophila-infected ayu (Plecoglossus altivelis) and healthy control fish were analyzed by 2DE. A protein, which increased significantly in diseased fish, was identified as NKEF-B by MALDI-TOF-MS. A full-length cDNA clone of this protein was subsequently isolated. It contains 1092 bp with an open reading frame of 591 bp, coding for 197 amino acids with MW 21.9 kDa and pI 6.38, values similar to those determined by 2DE. Ayu NKEF-B had highest similarity (93.1% amino acid identity) to those of carp and zebrafish. Phylogenetic analysis showed that ayu NKEF-B falls into the fish NKEF-B cluster and is most closely related to that of carp and zebrafish. It was determined that ayu NKEF-B mRNA expression was significantly increased in many tissues at the early stage of bacterial infection. In conclusion, the increased NKEF-B mRNA and protein expression in ayu were closely associated with A. hydrophila infection.     

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with β-glucan supplements

The objective of the present study was to determine the action of β-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. β-glucan (MacroGard^®), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4 × 10⁸ bacteria per fish and were sampled at time 0 and 6 h, 12 h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1β, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with β-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6 h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed β-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1β and il6 in infected fish fed β-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected β-glucan group. In conclusion, a diet supplemented with β-glucan (MacroGard^®) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.     

A manganese superoxide dismutase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis

Apolipoproteins are carrier proteins that bind to lipids to form lipoprotein particles and have been shown to play an important role in lipid metabolism. In this study, a full-length cDNA for apolipoprotein E, named AsapoE, was cloned from the Chinese sturgeon (Acipenser sinensis). This cDNA sequence is 1289 bp in length, and codes for a polypeptide of 274 amino acid residues, which is 45% and 42% identical to that of the rainbow trout and zebrafish, respectively, and 39%, 30%, and 29% identical to frog, mouse, and human respectively. The predicted AsApoE protein has a conserved amphipathic α-helix region with the potential to bind to lipids. RT-PCR analysis reveals that AsapoE is expressed in all tissues examined with a preferential expression in the kidney and liver. During the embryo development stage, AsapoE mRNA is low but still detectable at gastrula stage embryos; then AsapoE mRNAs reach a higher level in muscle contraction stage embryos, this relatively stable expression persists during the following embryogenic stages and declines 1 day after hatching. These results will serve as a basis for comparative studies on vertebrate apoE genes.     

Molecular characterization of trypsinogens and development of trypsinogen gene expression and tryptic activities in grass carp (Ctenopharyngodon idellus) and topmouth culter (Culter alburnus)

This study examined the gene structures and expression of trypsinogens, as well as the trypsin activities of the grass carp Ctenopharyngodon idellus (herbivorous) and the topmouth culter Culter alburnus (carnivorous), which are commercially important freshwater species of the family Cyprinidae in China. Isolated full-length trypsinogen cDNA clones were 869 bp and 857 bp. The deduced amino acid sequences were 242 aa and 247 aa long, both containing the highly conserved residues essential for serine protease catalytic and conformational maintenance. The results from isoelectric and phylogenetic analyses suggest that grass carp trypsinogen is grouped with teleost trypsinogen group I, while topmouth culter trypsinogen is grouped with group II. The expression pattern of trypsinogen mRNA was similar between these two species, appearing 2 days post-hatching (dph) and reaching peaks at 11 and 23 dph. The trypsin-specific activities in both species were detected 2 dph and reached the major peaks at 8 dph, however the minor peaks were observed at 20 dph in the grass carp and 17 dph in the topmouth culter. The trypsin-specific activity was significantly higher in the grass carp than in the topmouth culter, which may be attributed to the nature of their different nutritional habits.     

Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway

The cytoplasmic helicase protein RIG-I (retinoic acid-inducible gene I) and downstream signaling molecules, MAVS (mitochondrial antiviral signaling protein), TRAF3 (TNF-receptor-associated factor 3) and TBK1 (TANK-binding kinase 1), have significant roles in the recognition of cytoplasmic 5'-triphosphate ssRNA and short dsRNA, and phosphorylation of IRF-3 (interferon regulatory factor 3) and IRF-7 which is responsible for the induction of type I interferons (IFN). In the present study, the full-length cDNAs of RIG-I, MAVS, TRAF3 and TBK1 were cloned and identified in common carp (Cyprinus carpio L.). The deduced protein of carp RIG-I is of 946 aa (amino acids), consisting of two CARDs (caspase-recruitment domain), a DEXDc (DExD/H box-containing domain), a HELICc (helicase superfamily c-terminal domain) and a RD (regulatory domain). Carp MAVS is of 585 aa, containing a CARD, a proline-rich region and a TM (transmembrane domain). Carp TRAF3 encodes a protein of 573 aa, including a RING (really interesting new gene), two TRAF-type zinc fingers, a coiled coil and a MATH-TRAF3 (meprin and TRAF homology) domain. Carp TBK1 is of 727 aa and contains a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Carp RIG-I, MAVS, TRAF3 and TBK1 mRNAs are ubiquitously expressed in all tissues examined. In response to SVCV infection, carp RIG-I and MAVS mRNAs were up-regulated at different levels in spleen, head kidney and intestine tissues at different time points. Similarly, both carp IRF-3 and IRF-7 mRNAs were significantly up-regulated in the detected tissues. Especially in intestine, the IRF-3 and IRF-7 mRNAs of carp increased and reached 25.3-fold (at 3 dpi) and 224.7-fold (at 5 dpi). Noteworthily, a significant growth of carp TRAF3 and TBK1 mRNA was also mainly found in intestine (7.0-fold and 11.3-fold at 5 dpi, respectively). These data implied that the expression profiles of IRF-3/-7 mRNAs in carp correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK, and carp RIG-I and MAVS may be involved in antiviral responses through the RIG-I viral recognition signaling pathway in a TRAF3/TBK1-dependent manner.     

Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells

AimsMonocyte chemotactic protein-1 (MCP-1) plays an important role in recruiting monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of MCP-1.Main methodsThe effect of indoxyl sulfate on the expression of MCP-1 was determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS).Key findingsDN + IS, DH, and DH + IS rats showed significantly increased mRNA expression of MCP-1 in the kidneys compared with DN rats. DH + IS rats tended to show increased mRNA expression of MCP-1 in the kidneys compared with DH rats. Immunohistochemistry demonstrated the stimulatory effects of indoxyl sulfate on MCP-1 expression and monocyte/macrophage infiltration in the kidneys. Indoxyl sulfate upregulated mRNA and protein expression of MCP-1 in HK-2 cells. Indoxyl sulfate induced activation of ERK, p38, and JNK as well as of NF-κB and p53 in HK-2 cells. An antioxidant, and inhibitors of NF-κB, p53, ERK pathway (MEK1/2), and JNK suppressed indoxyl sulfate-induced mRNA expression of MCP-1 in HK-2 cells.SignificanceIndoxyl sulfate upregulates renal expression of MCP-1 through production of reactive oxygen species (ROS), and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of MCP-1 in the kidneys.     

Molecular isolation and characterisation of carp transforming growth factor beta 1 from activated leucocytes

The transforming growth factor (TGF beta) family of proteins are a set of pleiotropic secreted signalling molecules with unique and potent immunoregulatory properties. In this study the molecular cloning of carp TGF beta 1 is reported. A partial cDNA of the TGF beta protein was initially identified from a cDNA pool, obtained by subtracting the cDNAs from Con A-induced carp head kidney leucocytes from uninduced carp head kidney leucocyte cDNA. The entire coding sequence was assembled by sequencing both ends of the cDNA clone by using an anchored PCR reaction. Sequence analysis revealed an ORF encoding a protein of 376 amino acids, containing the similar unique pattern of conserved cysteines (seven out of nine) in the cysteine knot structure which exists in all known TGF beta proteins. Compared with other animal TGF beta s, the cDNA clone shows approximately 59-42, 40-38 and 37-36% amino acid identity with TGF beta 1, TGF beta 3 and TGF beta 2 respectively. Carp TGF beta 1 is expressed at low levels in carp head kidney, spleen, egg and liver, whereas its messenger RNA level is increased after activation of the head kidney leucocytes with Con A. Sequence analysis and pattern of expression suggests that this is the carp TGF beta 1.     

Sphingomyelin synthase 2 over-expression induces expression of aortic inflammatory biomarkers and decreases circulating EPCs in ApoE KO mice

AimsThis study sought to assess the effect of sphingomyelin synthase 2 (SMS2) over-expression on plaque component and endothelial dysfunction in atherosclerosis.Main methodsWe generated recombinant adenovirus vectors containing human SMS2 cDNA (AdV-SMS2) or control gene GFP cDNA (AdV-GFP). Both AdVs were injected (i.v.) into ApoE KO mice to establish SMS2 over-expressing and control mice models, respectively. The mice were fed a high fat diet for 30 days. We then examined their plasma lipid levels, expression levels of aortic inflammatory biomarkers critical for the plaque's stability, and numbers of peripheral endothelial progenitor cells (EPC).Key findingsCompared with the control mice, SMS2 over-expression had significantly (1) increased aortic matrix metalloproteinase-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), tissue factor (TF) and cyclooxygenase-2 (COX-2) mRNA levels (1.9-fold, 2.2-fold, 2.6-fold and 3.2-fold, respectively, P < 0.01) and protein levels (2.2-fold, 1.9-fold, 1.9-fold and 2.1-fold, respectively, P < 0.01); (2) increased MMP-2, COX-2 in situ expression in aortic root (2.6-fold and 2.3-fold, respectively, P < 0.01); (3) decreased aortic COX-1 mRNA levels (65%, P < 0.01) and protein levels (64%, P < 0.01); and (4) decreased CD34/KDR-positive cells (33%, P < 0.01), circulating angiogenic cells (CACs) (50%, P < 0.05), and colony forming units (CFUs) (40%, P < 0.05) in circulation.SignificanceSMS2 over-expression was probably associated with increased expression of aortic inflammatory biomarkers, as well as decreased numbers of CD34/KDR-positive cells, CACs and CFUs in circulation. Therefore, SMS2 over-expression might correlate with endothelial dysfunction and aggravate atherosclerotic plaque instability in ApoE KO mice.     

赤眼鳟Toll样受体3基因cDNA全长克隆及表达分析

为探究赤眼鳟（Squaliobarbus curriculus）Toll样受体3（Toll-like receptor 3,ScTLR3）基因是否参与抗病毒免疫反应,实验运用RACE技术,克隆得到ScTLR3基因cDNA全长序列,并进行了生物信息学分析;通过Real-Time qPCR技术,检测了ScTLR3 mRNA在健康赤眼鳟10个组织中的分布以及感染草鱼呼肠孤病毒（GCRV）后肝脏、脾脏、体肾和头肾中的表达特征。结果表明:ScTLR3基因cDNA序列全长4043 bp,包括5-非编码区（UTR）216 bp,开放阅读框（ORF）2715 bp和3-UTR 1112 bp;ScTLR3共编码904个氨基酸残基,推导的蛋白分子量102.67 kD,理论等电点8.76;SMART结构域预测显示,ScTLR3由N端的信号肽（SP）、富亮氨酸结构域（LRRs）、跨膜结构域（TM）和C端的Toll/白介素-1受体结构域（TIR）组成。实时荧光定量结果显示,ScTLR3mRNA在检测的各组织中均有表达,肝脏中的相对表达量极显著高于其他组织（P0.01）;感染GCRV后,肝脏、脾脏、体肾和头肾组织中ScTLR3 mRNA均上调表达,肝脏、脾脏和体肾组织中的相对表达量在24h达到峰值,分别为对照组的5倍、7倍和6倍。研究表明,ScTLR3具有TLRs家族基因的典型结构特征,并能被GCRV诱导表达,推测其在赤眼鳟抗GCRV入侵免疫反应中发挥了重要作用。     

Cloning and characterization of a fish specific gelsolin family gene,ScinL, in olive flounder (Paralichthys olivaceus)

Scinderin like (ScinL) gene is a unique gelsolin family gene found only in fish. In this study ScinL gene was cloned in olive flounder for the first time and characterized its expression and function. Flounder ScinL cDNA consists of 2911 nucleotides encoding a putative protein of 720 amino acids (79.4 kDa). In phylogenetic analysis, flounder ScinL is closely related to ScinL of zebra fish, anableps, and fugu with the similarity of 51–72%. Fish ScinLs are positioned between gelsolin and scinderin of other species. Flounder ScinL protein has the highly conserved actin and PIP2 binding sites, Ca^2 + coordination site, and a C-terminal latch helix preventing the activation of ScinL protein in the absence of Ca^2 +. Putative binding sites for NFAT and AP-1 were found in 5′ flanking region. Constitutive ScinL expression was found in most organs and the expression level was higher in gill, head kidney, trunk kidney, spleen and skin than muscle, stomach, intestine and brain. In Q-PCR analysis ScinL and CYP1A1 gene expression were significantly upregulated by BaP in head kidney in vivo and in vitro, and in macrophage cells. Upregulated ScinL expression by BaP was blocked by EGTA, indicating a calcium dependent regulation of ScinL expression.     

The evidence of apelin has the bidirectional effects on feeding regulation in Siberian sturgeon (Acipenser baerii)

Apelin is a peptide, mainly produced in the brain, which participates in several physiologic effects. However, knowledge about the mechanism of appetite regulation in teleosts, including the role of apelin is not well understood. The aim of this study is to explore the effect of feeding status on the expression of apelin mRNA in the whole brain and the effects of injection of apelin on food intake in Siberian sturgeon (Acipenser baerii). In this study, we first cloned the apelin cDNA sequence of the Siberian sturgeon. We obtained a 1046-bp cDNA fragment, including a 237-bp open reading frame (ORF) that encoded 78 amino acids. Apelin was widely distributed in 11 tissues related to feeding regulation, with the highest expression in thewhole brain, followed by the spleen and trunk kidney. In addition, we measured the effects of periprandial (preprandial and postprandial) change, fasting and re-feeding on apelin mRNA expression in whole brain. The level of apelin mRNA was significantly decreased 1 h after feeding. The results of the fasting experiment showed that the expression of apelin mRNA in the brain was significantly reduced after 1 day of fasting but consistently increased throughout the 15-day food deprivation period. When the 15-day fasted fish were re-fed, apelin mRNA expression in the brain was significantly increased as compared to that of the control. These results suggest that apelin may play a bidirectional role in the regulation of food intake in the Siberian sturgeon. In order to further examine the effect of apelin on feeding regulation in Siberian sturgeons, acute and chronic intraperitoneal (i.p.) injection experiments were performed and food intakes were recorded. Results showed that acute i.p. injection of apelin-13 reduced food intake, however, chronic i.p. injection apelin-13 increased the food intake for 7 days in Siberian sturgeons. In conclusion, our results show that apelin has a bidirectional effect on feeding regulation in Siberian sturgeons by acting as a satiety factor in short-term feeding regulation and a starvation factor in long-term feeding regulation.     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.     

Oral Angiotensin-(1–7) prevented obesity and hepatic inflammation by inhibition of resistin/TLR4/MAPK/NF-κB in rats fed with high-fat diet

Obesity is characterized by a pro-inflammatory state commonly associated with type 2 diabetes and fat-liver disease. In the last few years, different studies pointed out the role of Angiotensin (Ang)-(1–7) in the metabolic regulation. The aim of the present study was to evaluate the effect of oral-administration of Ang-(1–7) in metabolism and inflammatory state of high-fat feed rats. Twenty-four male Sprague Dawley rats were randomized into three groups: High Fat Diet (HFD); Standard Diet (ST); High Fat Diet + Angiotensin-(1–7) [HFD + Ang-(1–7)]. Glycemic profile was evaluated by glucose tolerance and insulin sensitivity tests, plasmatic glucose and insulin. Cholesterol, HDL and triglycerides analyses presented lipidic profile. RT-PCR evaluated mRNA expression to ACE, ACE2, resistin, TLR4, IL-6, TNF-α and NF-κB genes. The main results showed that oral Ang-(1–7) decreased body weight and abdominal fat-mass. In addition, HFD + Ang-(1–7) treated rats presented enhanced glucose tolerance, insulin-sensitivity and decreased plasma-insulin levels, as well as a significant decrease in circulating lipid levels. These alterations were accompanied by a marked decreased expression of resistin, TLR4, ACE and increased ACE2 expression in liver. Furthermore, Ang-(1–7) decreases phosphorylation of MAPK and increases NF-κB expression. These alterations diminished expression of interleukin-6 and TNF-α, ameliorate inflammatory state in liver. In summary, the present study showed that oral-treatment with Ang-(1–7) in high-fat feed rats improved metabolism down-regulating resistin/TLR4/NF-κB-pathway.     

TNF-α -308 genotypes are associated with TNF-α and TGF-β₁ mRNA expression in blood leucocytes of humans

AimTumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the ?308 TNF-α genotypes.MethodsQuantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2^?ΔΔCT method. Detection of the ?308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.ResultsThe relative TNF-α mRNA expression revealed significant differences between the TNF-α ?308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α ?308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.ConclusionOur findings suggest that the G-allele of TNF-α ?308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.