RESUMEN
Decay-accelerating factor (DAF) is an essential member of the complement regulatory protein family that plays an important role in immune response and host homeostasis in mammals. However, the immune function of DAF has not been well characterized in bony fish. In this study, a complement regulatory protein named CiDAF was firstly characterized from Ctenopharyngodon idella and its potential roles were investigated in intestine following bacterial infection. Similar to mammalian DAFs, CiDAF has multiple complement control protein (CCP) functional domains, suggesting the evolutionary conservation of DAFs. CiDAF was broadly expressed in all tested tissues, with a relatively high expression level detected in the spleen and kidney. In vivo immune challenge experiments revealed that CiDAF strongly responded to bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and PAMPs (lipopolysaccharide (LPS) or muramyl dipeptide (MDP)) challenges. In vitro RNAi experiments indicated that knockdown of CiDAF could upregulate the expression of complement genes (C4b, C5 and C7) and inflammatory cytokines (TNF-α, IL-1ß and IL-8). Moreover, 2000 ng/mL of CiDAF agonist progesterone effectively alleviated LPS- or MDP-induced intestinal inflammation by regulating expression of complement factors, TLR/PepT1 pathway genes and inflammatory cytokines. Overall, these findings revealed that CiDAF may act as a negative regulator of intestinal complement pathway and immune response to bacterial challenge in grass carp.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Intestinos , Animales , Carpas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Enfermedades de los Peces/inmunología , Inmunidad Innata/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Intestinos/inmunología , Regulación de la Expresión Génica/inmunología , Filogenia , Perfilación de la Expresión Génica/veterinaria , Aeromonas hydrophila/fisiología , Secuencia de Aminoácidos , Alineación de Secuencia/veterinaria , Proteínas del Sistema Complemento/inmunologíaRESUMEN
Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α, il-1ß and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin, nk-lysin and ß-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1, tlr2, tlr4, myd88, irak4, irak1, traf6, nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1, tnfr2, tradd, caspase-8, caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin, zo-1, claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes) and genus (Proteus, Cetobacterium, Bacteroides, and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp.
RESUMEN
The aim of this study is to investigate the effect of dietary protein levels on flesh quality, oxidative stress, and autophagy status in the muscles of triploid crucian carp (Carassius carassius triploid), and the related molecular mechanisms. Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. It could be found that the whole-body ash content significantly increased in high protein level groups (p < 0.05). The 29% dietary protein level group exhibited the highest muscle moisture, although there was an inconspicuous decrease in the chewiness of the muscles when compared with the other groups. The dietary protein level influenced the content of free amino acids and nucleotides, especially the content of flavor amino acids, which exhibited an increasing tendency along with the increasing protein level, such as alanine and glutamic acid, while the flavor nucleotides showed different fluctuation trends. Moreover, the genes related to muscle development were shown to be influenced by the dietary protein level, especially the expression of MRF4, which was up-regulated with the increasing dietary protein levels. The 29% dietary protein level promoted the majority of analyzed muscle genes expression to the highest level when compared to other dietary levels, except the Myostain, whose expression reached its highest at 38% dietary protein levels. Furthermore, the effect of dietary protein levels on antioxidant signaling pathway genes were also examined. High protein levels would boost the expression of GSTα; GPX1 and GPX4α mRNA expression showed the highest level at the 32% dietary protein group. The increasing dietary protein level decreased both mRNA and protein expressions of Nrf2 by up-regulating Keap1. Autophagy-related gene expression levels reached the peak at 32% dietary protein level, as evidenced by a similar change in protein expression of FoxO1. In summary, muscle nutritional composition, antioxidative pathways, and autophagy levels were affected by the dietary protein levels. A total of 29-32% dietary protein level would be the appropriate level range to improve muscle quality and promote the antioxidant and autophagy capacity of triploid crucian carp muscles.
RESUMEN
Transforming growth factor-ß activated kinase-1 (TAK1) is an important upstream signaling molecules involved in the NF-κB signaling pathway. TAK1 interacts with TAB1 to form the TAK1-TAB1 complex, which elicits NF-κB activation through a series of cascade reactions in mammals. However, the function of TAK1 in blunt snout bream (Megalobrama amblycephala ( maTak1) and the effects of their interaction between TAK1 and TAB1 on the NF-κB activation still remains largely unknown. In the present study, maTak1 was cloned and characterized successfully based on transcriptome data. Its open reading frame is composed of 1626 nucleotides and the predicted maTAK1 protein contains 541 amino acids, which includes an N-terminal Serine/Threonine protein kinases (S/TKc) and a C-terminal coiled-coil region. Phylogenetic analysis showed that maTAK1 were clustered with those of other teleosts. MaTak1 displayed ubiquitous transcriptional expression in all the examined tissues of healthy blunt snout bream but with varied expression levels. And maTrak1 expression was dramatically enhanced in different tissues and MAF cells after LPS stimulation and A. hydrophila challenge. The result from subcellular localization analysis indicated that both maTAK1 and maTAB1 were cytoplasmic protein. The activity of NF-κB promoter could not be induced by overexpression of maTak1 or maTab1 alone, however, it could be enhanced by co-expression of maTak1 and maTab1. Co-immunoprecipitation and subcellular co-localization assay revealed that maTAK1 can combine with maTAB1 directly. The transcriptional expression level of pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) increased distinctly after the overexpression of maTak1 and maTab1. Taken together, the data obtained in this study demonstrated that the direct interaction between maTAK1 and maTAB1 might play a pivotal role in mediating host innate immune response to pathogen invasion by the production of pro-inflammatory cytokines via NF-κB signaling pathway, which might lay a solid foundation for the establishment of novel therapeutic approach to combat bacterial infection in fish.
Asunto(s)
Cipriniformes , Proteínas de Peces/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Quinasas Quinasa Quinasa PAM , FN-kappa B , Animales , Bacterias/metabolismo , Cipriniformes/genética , Cipriniformes/microbiología , Citocinas , Inmunidad Innata , Quinasas Quinasa Quinasa PAM/genética , FN-kappa B/metabolismo , Filogenia , Transducción de SeñalRESUMEN
Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (CiMAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of CiMAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , MAP Quinasa Quinasa Quinasa 4 , Aeromonas hydrophila , Animales , Carpas/genética , Carpas/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/genética , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Células HEK293 , Humanos , Inmunidad Innata/genética , Intestinos/inmunología , Intestinos/microbiología , MAP Quinasa Quinasa Quinasa 4/genética , FilogeniaRESUMEN
Mitogen-activated protein kinase kinase 6 (MKK6) is an essential component of the p38MAPK signaling pathway, which is involved in the modulation of inflammation, cell apoptosis and survival responses in mammals. However, the function of MKK6s in teleosts is still unclear. In this study, a fish MKK6 homolog (CiMKK6) was first identified from the grass carp (Ctenopharyngodon idella), a freshwater fish. CiMKK6 cDNA encodes a putative protein of 357 amino acids that contains conserved structural characteristics of the MKK6 family, including the S_TKc domain, SVAKT motif and DVD site. The deduced CiMKK6 protein exhibits high sequence homology with other reported fish MKK6s and shares the closest relationship with MKK6 from Danio rerio. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMKK6 mRNA was widely expressed in all tested tissues and stages of embryonic development. Additionally, the transcript levels of CiMKK6 in the intestine were significantly upregulated in response to bacterial muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) stimulation. Moreover, subcellular localization analysis indicated that CiMKK6 was distributed in both the cytoplasm and the nucleus of HEK293T cells. Finally, overexpression of CiMKK6 significantly enhanced the transcriptional activity of the AP-1 reporter gene in HEK293T cells. Overall, these findings may help better clarify the immune function of teleost MKK6s and provide new insight into the immune defense mechanisms of grass carp.
Asunto(s)
Proteínas Bacterianas/inmunología , Carpas/genética , Carpas/inmunología , Inmunidad Innata/genética , MAP Quinasa Quinasa 6/genética , Animales , Proteínas Bacterianas/administración & dosificación , Dipéptidos/administración & dosificación , Dipéptidos/inmunología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Células HEK293 , Humanos , MAP Quinasa Quinasa 6/metabolismo , Oligopéptidos/administración & dosificación , Oligopéptidos/inmunología , Distribución AleatoriaRESUMEN
As the fifth most common cancer and the fourth leading cause of cancer-related death in the world, gastric cancer (GC) poses a potential threat to human health. However, there is still a lack of effective means for the early screening and treatment of GC, and therefore, GC remains a difficult disease to overcome. With the continuous in-depth research on circular RNAs (circRNAs), an increasing body of evidence indicates that circRNAs play an important role in a wide variety of diseases, particularly cancer. Proliferation, invasion and metastatic spread of cancer cells are strongly associated with abnormal circRNA expression. Hence, circRNAs are considered a candidate biomarker for GC diagnosis and prognosis, and a target for cancer treatment. The focus has been on the association of GC with circRNAs, thus it is necessary to briefly review and summarize the relevant research to provide the research findings across the area to researchers, and to indicate the direction for future research. The present review provides an overview on the biogenesis and functions of circRNAs in GC, predicting their possible clinical application as ideal biomarkers and potential targets of treatment in GC.
RESUMEN
An eight-week experiment was undertaken to examine the effect of dietary hydroxyproline (Hyp) supplementation on growth performance, collagen synthesis, muscle quality of an improved triploid crucian carp (Carassius auratus Triploid) (ITCC). Six isonitrogenous (340 g/kg diet), isolipidic (60 g/kg diet) and isocaloric (17.80 MJ/kg diet) diets were formulated containing a certain amount of Hyp: 0.09% (the control group), 0.39, 0.76, 1.14, 1.53 and 1.90%. Each diet was randomly assigned to three tanks and each group was fed two times daily until apparent satiation. The results showed that growth performance and feed utilization of ITCC were significantly improved with the dietary Hyp level was increased from 0.09 to 0.76%. Crude protein, threonine and arginine content in the dorsal muscle in 0.76% hydroxyproline group were significantly higher than those in basic diet group (p < 0.05). The muscle textural characteristics increased remarkably with the amount of Hyp in the diet rising from 0.09 to 1.53% (p < 0.05). Meanwhile, the contents of type I collagen (Col I) and Pyridinium crosslink (PYD) in the muscle of fish were significantly increased by dietary Hyp (p < 0.05). The muscle fiber diameter and density of the fish were significantly increased when fed with 0.76% Hyp (p < 0.05). Furthermore, dietary supplementation with an appropriate concentration of Hyp substantially increased the expression of genes involved in collagen synthesis (col1a1, col1a2, p4hα1, p4hß, smad4, smad5, smad9, and tgf-ß) and muscle growth (igf-1, tor, myod, myf5, and myhc) (p < 0.05). In conclusion, dietary supplementation of Hyp can enhance fish growth performance, collagen production, muscle textural characteristics and muscle growth of ITCC. According to the SGR broken-line analysis, the recommended supplementation level of Hyp was 0.74% in the diet for ITCC, corresponding to 2.2% of dietary protein.
RESUMEN
Liver Kinase B1 (LKB1) is a serine/threonine kinase that can regulate energy metabolism and skeletal muscle growth. In the present study, LKB1 cDNA of triploid crucian carp (Carassius auratus) was cloned. The cDNA contains a complete open reading frame (ORF), with a length of 1326 bp, encoding 442 amino acids. Phylogenetic tree analysis showed that the LKB1 amino acid sequence of the triploid crucian carp had a high sequence similarity and identity with carp (Cyprinus carpio). Tissue expression analysis revealed that LKB1 was widely expressed in various tissues. LKB1 expressions in the brain were highest, followed by kidney and muscle. In the short-term LKB1 activator and inhibitor injection experiment, when LKB1 was activated for 72 h, expressions of myogenic differentiation (MyoD), muscle regulatory factor (MRF4), myogenic factor (MyoG) and myostatin 1 (MSTN1) were markedly elevated and the content of inosine monophosphate (IMP) in muscle was significantly increased. When LKB1 was inhibited for 72 h, expressions of MyoD, MyoG, MRF4 and MSTN1 were markedly decreased. The long-term injection experiment of the LKB1 activator revealed that, when LKB1 was activated for 15 days, its muscle fibers were significantly larger and tighter than the control group. In texture profile analysis, it showed smaller hardness and adhesion, greater elasticity and chewiness. Contrastingly, when LKB1 was inhibited for 9 days, its muscle fibers were significantly smaller, while the gap between muscle fibers was significantly larger. Texture profile analysis showed that adhesion was significantly higher than the control group. A feeding trial on triploid crucian carp showed that with dietary lysine-glutamate dipeptide concentration increasing, the expression of the LKB1 gene gradually increased and was highest when dipeptide concentration was 1.6%. These findings may provide new insights into the effects of LKB1 on fish skeletal muscle growth and muscle quality, and will provide a potential application value in improvement of aquaculture feed formula.
RESUMEN
Inosine monophosphate (IMP) is the main flavoring substance in aquatic animal, and adenosine monophosphate deaminase1 (AMPD1) gene is a key gene in IMP formation. At present, the research on the mechanism of AMPD1 regulating IMP formation in aquatic animal is still blank. In this study, in order to study the mechanism of AMPD1 regulating IMP formation in fish, the full open reading frame (ORF) of AMPD1 which was 2160bp was obtained for the first time in triploid crucian carp (Carassius auratus). It encoded 719 amino acids with a molecular mass of 82.97 kDa, and the theoretical isoelectric point value was 6.31. The homology analysis showed that the homology of triploid crucian carp and diploid Carassius auratus was the highest, up to 99%. And the phylogenetic tree showed that triploid crucian carp was grouped with diploid Carassius auratus, Culter alburnus, and Danio rerio. And real-time fluorescence quantitative results showed that AMPD1 was expressed specifically in muscle of triploid crucian carp (p < 0.05). The results of detection the localization of AMPD1 in cells indicated that the AMPD1 was mainly localized in cytoplasm and cell membrane. Further, we examined the effects of glutamate which was the promotor of IMP formation on the expression of AMPD1 and the formation of IMP in vivo and in vitro experiments, the results showed that 3% glutamate and 2 mg/ml glutamate could significantly promote AMPD1 expression and IMP formation in triploid crucian carp muscle tissue and muscle cells (p < 0.05). Then we inhibited the expression of AMPD1 in vivo and in vitro experiments, we found the formation of IMP in muscle tissue and muscle cells of triploid crucian carp all were inhibited and they affected the gene expression of AMPK-mTOR signaling pathway. The all results showed that AMPD1 mediated glutamate through AMPK-mTOR signaling pathway to regulate the formation of fish IMP.
RESUMEN
Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5' upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.
Asunto(s)
Carpas , Enfermedades de los Peces , Acetilmuramil-Alanil-Isoglutamina , Animales , Carpas/genética , Carpas/metabolismo , Proteínas de Peces , Células HEK293 , Humanos , Inmunidad Innata , Intestinos , Mamíferos/metabolismo , FN-kappa B/metabolismo , Filogenia , Factor de Transcripción AP-1/genéticaRESUMEN
The nutritional functions of tributyrin (TB) have been extensively studied, but questions remain regarding its influence on the growth of juvenile grass carp (Ctenopharyngodon idellus) and the regulation pathway to PepT1 in the intestine of grass carp. To answer the remaining questions, feeding trials, cell trials, and peritoneal injection trials were conducted in this study. The results showed that an appropriate level of TB (0.5 g/kg and 1.0 g/kg) supplementation in feed significantly promoted the growth performance of juvenile grass carp. The expressions of intestine genes (CDX2, SP1 and PepT1) related to oligopeptide transportation increased in the 0.5 g/kg TB group of feeding trials and both the 5 mM and 10 mM TB groups of the intestine cell trials, respectively. Subsequently, the injection trials of inhibitors CDX2 and SP1 demonstrated that the inhibition of CDX2 or SP1 decreased the mRNA expression of PepT1. Finally, the results of independent or combined treatments of TB and the inhibitors suggested that CDX2/SP1 mediated TB regulation on PepT1. These findings may help us to better understand the functions of TB on growth and PepT1 oligopeptide transportation, which could be modulated by dietary TB through the CDX2/SP1-PepT1 pathway in juvenile grass carp.
RESUMEN
The p38 mitogen-activated protein kinases (MAPKs) are essential cytoplasmic signal molecules of innate immune pathways that play a vital role in host immune defense responses to pathogenic challenges. In this study, two fish p38 genes (Cip38α and Cip38ß) were characterized for the first time from the grass carp Ctenopharyngodon idella. Similar to other reported p38MAPKs, both Cip38α and Cip38ß contained a conserved phosphorylation motif (Thr-Gly-Tyr, TGY) and a substrate binding site (Ala-Thr-Arg-Trp, ATRW) in the serine/threonine protein kinase (S_TKc) domain. Expression profile analysis showed that Cip38α and Cip38ß mRNAs were broadly expressed in all of the examined tissues and developmental stages of C. idella. In addition, in vivo injection experiments directly revealed that Cip38α and Cip38ß showed strong responsiveness to Aeromonas hydrophila and muramyl dipeptide (MDP) challenges, and their expression levels were significantly upregulated in the intestine of grass carp. Additionally, the MDP-induced expression levels of intestinal inflammatory cytokines (TNF-α and IL-15) and an antimicrobial peptide (ß-defensin) were significantly inhibited by the p38MAPK-specific inhibitor SB203580. Moreover, the nutritional dipeptide carnosine and Ala-Gln were found to significantly suppress the bacterial MDP-induced expression of p38MAPK pathway genes and inflammatory cytokines in the intestine of grass carp. Finally, overexpression analysis demonstrated that Cip38α and Cip38ß could act as efficient activators in the regulation of AP-1 signaling pathways through interaction with CiMKK6. Altogether, this study provided experimental evidence of the presence of a functional p38 pathway in grass carp, which revealed its involvement in the intestinal immune response to bacterial challenges in bony fish.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/inmunología , Carpas/inmunología , Carpas/microbiología , Inmunidad Innata/inmunología , Intestinos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Aeromonas hydrophila/inmunología , Animales , Citocinas/inmunología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/inmunología , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/microbiología , Intestinos/microbiología , FN-kappa B/inmunología , Transducción de Señal/inmunologíaRESUMEN
Oligopeptide transporter 1 (Pept1) is located on the brush border membrane of the intestinal epithelium and plays an important role in dipeptide and tripeptide absorption from protein digestion. In this study, we cloned and characterized the cDNA sequence of Janus kinase 2 (JAK2) from Ctenopharyngodon idella. The expression patterns of JAK2 in various tissues and developmental stages were characterized by quantitative real-time PCR (qRT-PCR). The mRNA expression levels of JAK2 and Pept1 regulated by leptin in the intestine were also analyzed in vitro and in vivo. The cDNA sequence of JAK2 is 3378 bp in length, and the mRNA of JAK2 was broadly expressed in all tissues and embryonic stages of C. idella analyzed. In addition, we found that leptin regulated expression of JAK2 and Pept1 in the intestine; Pept1 expression was down-regulated by the JAK2 inhibitor AG490 in vivo and in vitro. Furthermore, luciferase experiments showed that overexpression of the JAK2 gene significantly upregulated the activity of the Pept1 5' regulatory sequence in C. idella. In conclusion, these results may help in elucidating the regulatory effect of the leptin-mediated JAK2 pathway on intestinal Pept1 expression in C. idella and the molecular mechanism of peptide transport by the intestinal transporter Pept1 in fishes.
RESUMEN
The c-Jun NH2-terminal kinases (JNKs) are an evolutionarily conserved family of serine/threonine protein kinases that play critical roles in the pathological process in species ranging from insects to mammals. However, the function of JNKs in bacteria-induced intestinal inflammation is still poorly understood. In this study, a fish JNK (CiJNK) pathway was identified, and its potential roles in bacterial muramyl dipeptide (MDP)-induced intestinal inflammation were investigated in Ctenopharyngodon idella. The present CiJNK was found to possess a conserved dual phosphorylation motif (TPY) in a serine/threonine protein kinase (S_TKc) domain and to contain several potential immune-related transcription factor binding sites, including nuclear factor kappa B (NF-κB), activating protein 1 (AP-1), and signal transducer and activator of downstream transcription 3 (STAT3), in its 5' flanking regions. Quantitative real-time PCR results revealed that the mRNA levels of the JNK pathway genes in the intestine were significantly upregulated after challenge with a bacterial pathogen (Aeromonas hydrophila) and MDP in a time-dependent manner. Additionally, the JNK pathway was found to be involved in regulating the MDP-induced expression levels of inflammatory cytokines (IL-6, IL-8, and TNF-α) in the intestine of grass carp. Moreover, the nutritional dipeptide carnosine and Ala-Gln could effectively alleviate MDP-induced intestinal inflammation by regulating the intestinal expression of JNK pathway genes and inflammatory cytokines in grass carp. Finally, fluorescence microscopy and dual-reporter assays indicated that CiJNK could associate with CiMKK4 and CiMKK7 involved in the regulation of the AP-1 signaling pathway. Overall, these results provide the first experimental demonstration that the JNK signaling pathway is involved in the intestinal immune response to MDP challenge in C. idella, which may provide new insight into the pathogenesis of inflammatory bowel disease.
Asunto(s)
Aeromonas hydrophila/fisiología , Carpas/metabolismo , Infecciones por Bacterias Gramnegativas/metabolismo , Inflamación/metabolismo , Intestinos/inmunología , Acetilmuramil-Alanil-Isoglutamina/inmunología , Animales , Antígenos Bacterianos/inmunología , Carpas/microbiología , Citocinas/metabolismo , Proteínas de Peces/metabolismo , Mediadores de Inflamación/metabolismo , MAP Quinasa Quinasa 4/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
Mitogen-activated protein kinase kinases (MKKs) are a class of evolutionarily conserved signalling intermediates of the MAPK signalling pathway that can be activated by a diverse range of pathogenic stimuli and are crucial for the regulation of host immune defence. In this study, two fish MKK genes (CiMKK4 and CiMKK7) were first identified and characterized from grass carp (Ctenopharyngodon idella). Similar to other reported MKKs, the present CiMKK4 and CiMKK7 contained a conserved serine/threonine protein kinase (S_TKc) domain and a canonical dual phosphorylation motif. Quantitative real-time PCR results showed that CiMKK4 and CiMKK7 were broadly transcribed in all selected tissues and developmental stages of grass carp. The mRNA expression levels of CiMKK4 and CiMKK7 in the intestine were significantly induced by bacterial muramyl dipeptide (MDP) challenge in a time-dependent manner (Pâ¯<â¯0.01). Additionally, the stimulatory effects of bacterial MDP on CiMKK4 and CiMKK7 expression in the intestine were inhibited by the bioactive dipeptide ß-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln) (Pâ¯<â¯0.05). Moreover, overexpression analysis revealed that CiMKK4 and CiMKK7 were localized throughout the entire cell and could significantly enhance AP-1 reporter gene activation in HEK293T cells. Taken together, these results provide the first experimental demonstration that CiMKK4 and CiMKK7 are involved in the intestinal immune response to MDP challenge in C. idella, which may provide new insight into the bacterial-induced intestinal inflammation of bony fishes.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/inmunología , Carpas/inmunología , Intestinos/inmunología , MAP Quinasa Quinasa 4/metabolismo , MAP Quinasa Quinasa 7/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carpas/microbiología , Línea Celular , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Proteínas de Peces/genética , Células HEK293 , Humanos , Intestinos/microbiología , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 7/genética , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , Análisis de Secuencia de ADN , Transducción de Señal/inmunologíaRESUMEN
Neurotoxins are important tools to explore the structure and function relationship of different ion channels. From the venom of Chinese spider Chilobrachys jingzhao, a novel toxin, Jingzhaotoxin-IV (JZTX-IV), is isolated and characterized. It consists of 34 amino acid residues including six acidic residues clustered with negative charge (pI=4.29). The full-length cDNA of JZTX-IV encodes an 86-amino acid precursor containing a signal peptide of 21 residues, a mature peptide of 34 residues and an intervening sequence of 29 residues with terminal Lys-Gly as the signal of amidation. Under whole-cell patch clamp conditions, JZTX-IV inhibits current and slows the inactivation of sodium channels by shifting the voltage dependence of activation to more depolarized potentials on DRG neurons, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. In addition, JZTX-IV shows a slowing inactivation of sodium channel with a hyperpolarizing shift of the steady-state inactivation on acutely isolated rat cardiac cell and DRG neurons, differs from the classic site 3 toxins that do not affect the steady-state of inactivation. At high concentration, JZTX-IV has no significant effect on tetrodotoxin-resistant (TTX-R) sodium channels on rat DRG neurons and tetrodotoxin-sensitive (TTX-S) sodium channels on hippocampal neurons. Our data establish that, contrary to known toxins, JZTX-IV neither binds to the previously characterized classic site 4, nor site 3 by modifying channel gating, thus making it a novel probe of channel gating in sodium channels with potential to shed new light on this process.
Asunto(s)
Neurotoxinas/farmacología , Péptidos/farmacología , Canales de Sodio/efectos de los fármacos , Venenos de Araña/farmacología , Arañas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Ganglios Espinales/efectos de los fármacos , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/aislamiento & purificación , Técnicas de Placa-Clamp , Péptidos/química , Péptidos/genética , Péptidos/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de Proteína , Canales de Sodio/fisiología , Venenos de Araña/química , Venenos de Araña/genética , Venenos de Araña/aislamiento & purificación , Arañas/genéticaRESUMEN
Abnormal telomerase activity is implicated in cancer initiation and development. The rs2736100 T > G polymorphism in the telomerase reverse transcriptase (TERT) gene, which encodes the telomerase catalytic subunit, has been associated with increased cancer risk. We conducted a meta-analysis to more precisely assess this association. After a comprehensive literature search of the PubMed and EMBASE databases up to November 1, 2016, 61 articles with 72 studies comprising 108,248 cases and 161,472 controls were included in our meta-analysis. Studies were conducted on various cancer types. The TERT rs2736100 polymorphism was associated with increased overall cancer risk in five genetic models [homozygous model (GG vs. TT): odds ratio (OR) = 1.39, 95% confidence interval (95% CI) = 1.26-1.54, P < 0.001; heterozygous model (TG vs. TT): OR = 1.16, 95% CI = 1.11-1.23, P < 0.001; dominant model (TG + GG vs. TT): OR = 1.23, 95% CI = 1.15-1.31, P < 0.001; recessive model (GG vs. TG + TT): OR = 1.25, 95% CI = 1.16-1.35, P < 0.001; and allele contrast model (G vs. T): OR = 1.17, 95% CI = 1.12-1.23, P < 0.001]. A stratified analysis based on cancer type associated the polymorphism with elevated risk of thyroid cancer, bladder cancer, lung cancer, glioma, myeloproliferative neoplasms, and acute myeloid leukemia. Our results confirm that the TERT rs2736100 polymorphism confers increased overall cancer risk.
Asunto(s)
Biomarcadores de Tumor/genética , Predisposición Genética a la Enfermedad , Neoplasias/genética , Polimorfismo de Nucleótido Simple , Telomerasa/genética , Estudios de Casos y Controles , Humanos , Neoplasias/patología , Pronóstico , Factores de RiesgoRESUMEN
MicroRNAs (miRs), a class of small noncoding RNAs, are key gene regulators through inducing translational repression or degradation of their target genes. However, the regulatory mechanism of miR-133b underlying hepatocellular carcinoma (HCC) growth and metastasis remains largely unclear. Here we found that miR-133b was significantly downregulated in HCC tissues and cell lines. Moreover, low miR-133b levels were significantly associated with the malignant progression of HCC. LASP1, upregulated in HCC tissues and cell lines, was then identified as a novel target of miR-133b in HCC HepG2 and Hep3B cells. Moreover, the increased expression of LASP1 was associated with HCC progression. An in vitro study showed that overexpression of miR-133b inhibited the proliferation, migration, and invasion of HepG2 and Hep3B cells. Similarly, knockdown of LASP1 reduced HepG2 and Hep3B cell proliferation, migration, and invasion. Furthermore, overexpression of LASP1 attenuated the suppressive effect of miR-133b on the malignant phenotypes of HepG2 and Hep3B cells, suggesting that miR-133b may inhibit HCC growth and metastasis via targeting LASP1. In addition, overexpression of miR-133b inhibits tumor growth of HepG2 and Hep3B cells in vivo. Therefore, the miR-133b/LASP1 axis may become a potential target for the treatment of HCC.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Hepatocelular/genética , Proteínas del Citoesqueleto/genética , Proteínas con Dominio LIM/genética , Neoplasias Hepáticas/genética , MicroARNs/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinoma Hepatocelular/patología , Movimiento Celular/genética , Proliferación Celular/genética , Proteínas del Citoesqueleto/metabolismo , Células Hep G2 , Xenoinjertos , Humanos , Proteínas con Dominio LIM/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , Persona de Mediana Edad , Invasividad Neoplásica , TransfecciónRESUMEN
Cleft lip and palate transmembrane 1-like (CLPTM1L) gene rs402710 (C > T) and rs401681 (C > T) polymorphisms have been widely studied for their potential relation to cancer risk, but studies have produced conflicting results. To systematically evaluate the association between these two polymorphisms and overall cancer risk, we conducted a comprehensive meta-analysis on all relevant articles found in the PubMed and EMBASE databases published prior to May 1, 2017. There were 26 articles with 28 studies, including 30,770 cases and 34,089 controls, for the rs402710 polymorphism and 38 articles with 48 studies, including 67,849 cases and 328,226 controls, for the rs401681 polymorphism. The pooled results indicated that both rs402710 and rs401681 polymorphisms are significantly associated with decreased overall cancer risk. In our stratification analysis, a significant association of the rs402710 polymorphism with lung and bladder cancers was identified among Asian and Caucasian populations in both hospital-based and population-based studies. The rs401681 polymorphism was significantly associated with a decreased risk of lung cancer, bladder cancer, and basal cell carcinoma in Asians and in hospital-based studies. CLPTM1L gene rs402710 and rs401681 polymorphisms thus have a protective association with various types of cancer, especially lung cancer among Asians.