The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Hugues Parrinello - One of the best experts on this subject based on the ideXlab platform.
-
Sex chromosome and sex locus characterization in Goldfish, Carassius auratus (Linnaeus, 1758)
BMC Genomics, 2020Co-Authors: Ming Wen, Elodie Jouanno, Romain Feron, Christophe Klopp, Qiaowei Pan, Amaury Herpin, Justine Guguin, Cédric Cabau, Margot Zahm, Hugues ParrinelloAbstract:Background - Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. Here we used sequencing approaches to better characterize sex determination and sex-chromosomes in an experimental strain of Goldfish. Results - Our results confirmed that sex determination in Goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18 °C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (~ 11.7 Mb) on Goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the coding DNA sequence of the Goldfish amh gene. Conclusions - These results show that our Goldfish strain has a relatively large sex locus on LG22, which is likely the Y chromosome of this experimental population. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers, which will be important tools for future research on sex determination in our experimental Goldfish population. However, additional work would be needed to explore whether this sex locus is conserved in other populations of Goldfish.
-
Sex chromosome and sex locus characterization in the Goldfish, Carassius auratus
2019Co-Authors: Ming Wen, Elodie Jouanno, Romain Feron, Christophe Klopp, Qiaowei Pan, Amaury Herpin, Justine Guguin, Cédric Cabau, Margot Zahm, Hugues ParrinelloAbstract:Background: Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. We used sequencing approaches to better characterize sex determination and sex-chromosomes in Goldfish. Results: Our results confirmed that sex determination in Goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18°C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (∼11.7 Mb) on Goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the Goldfish amh gene or its 5 kb proximal promoter sequence. Conclusions: These results show that Goldfish have a relatively large sex locus on LG22, which is likely the Goldfish Y chromosome. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers in Goldfish, which will be important for future research on sex determination and aquaculture applications in this species.
Miodrag Belosevic - One of the best experts on this subject based on the ideXlab platform.
-
recombinant Goldfish thrombopoietin up regulates expression of genes involved in thrombocyte development and synergizes with kit ligand a to promote progenitor cell proliferation and colony formation
Developmental and Comparative Immunology, 2015Co-Authors: Fumihiko Katakura, Barbara A Katzenback, Miodrag BelosevicAbstract:Thrombopoietin (TPO) is the principal regulator of thrombopoiesis and promotes the proliferation, differentiation and maturation of megakaryocytic progenitor cells in mammals. In this study we report on the molecular and functional characterization of Goldfish TPO. Quantitative expression analysis of Goldfish tpo revealed the highest mRNA levels in heart, followed by spleen, liver, brain, intestine and kidney tissues. Significant decrease of tpo and c-mpl expressions in Goldfish primary kidney macrophage (PKM) cultures, as progenitor to macrophage development progressed, indicates that TPO is not involved in monopoiesis. Recombinant Goldfish TPO (rgTPO) alone did not induce significant proliferation of progenitor cells, but TPO in cooperation with recombinant Goldfish kit ligand A (rgKITLA) supported proliferation of progenitor cells in a dose-dependent manner. In response to rgTPO or a combination of rgTPO and rgKITLA, the mRNA levels of thrombopoietic markers cd41 and c-mpl as well as thrombo/erythropoietic transcription factors gata1 and lmo2 in sorted progenitor cells were up-regulated, while the mRNA levels of granulopoietic markers (cebpα and gcsfr) and the lymphoid transcription factor gata3 were down-regulated. Furthermore, rgTPO and rgKITLA synergistically stimulated thrombocytic colony-formation. Our results demonstrate that Goldfish TPO has similar functions to mammalian TPO as a regulator of thrombopoiesis, and suggests a highly conserved molecular mechanism of thrombocyte development throughout evolution of vertebrates.
-
characterization and functional analysis of Goldfish carassius auratus l interleukin 10
Molecular Immunology, 2011Co-Authors: Leon Grayfer, Jordan W Hodgkinson, Steven J Hitchen, Miodrag BelosevicAbstract:This is the first report of comprehensive functional analysis of an interleukin-10 in bony fish. Quantitative expression analysis of Goldfish IL-10 revealed the greatest mRNA levels in the spleen tissues, peripheral blood leukocytes and granulocytes. The stimulation of cells with recombinant Goldfish (rg) TNFα2 significantly reduced IL-10 mRNA levels in granulocytes and monocytes of the Goldfish. To functionally assess the Goldfish IL-10, we generated a recombinant form of the molecule (rgIL-10). The rgIL-10 substantially reduced the expression of TNFα1, TNFα2, IL-1β1, IL-10, CXCL-8, and NADPH oxidase component, p47phox in monocytes activated with heat-killed Aeromonas salmonicida and reduced the expression of IFNγ in A. salmonicida-activated splenocytes. Pre-treatment of monocytes with rgIL-10 resulted in substantial reduction of the ROI response of the A. salmonicida or rgIFNγ-primed monocytes. The rgIL-10 bound to Goldfish monocytes and induced phosphorylation and nuclear translocation of Stat3. The rgIL-10 also induced rapid and robust increase in the mRNA levels of the Goldfish monocyte SOCS-3. Our results indicate that the function of IL-10 is highly conserved through evolution.
-
comparison of macrophage antimicrobial responses induced by type ii interferons of the Goldfish carassius auratus l
Journal of Biological Chemistry, 2010Co-Authors: Leon Grayfer, Erick Garcia Garcia, Miodrag BelosevicAbstract:Unlike mammals, bony fish have two type II interferons, IFNγ and IFNγrel, whose pro-inflammatory functions have not been fully characterized. To elucidate the distinct roles of these type II interferons of bony fish, we examined the effects of recombinant Goldfish (rg) IFNγ and IFNγrel on the macrophage antimicrobial responses, immune gene expression, and their signaling pathways. Our findings indicate that rgIFNγ and rgIFNγrel possess unique capacities to mediate each of the above processes. Q-PCR analysis revealed similar expression of both cytokines in tissues and immune cell populations of the Goldfish, although IFNγ mRNA levels were generally higher in most tissues and cell types. Whereas rgIFNγ had long-lasting effects on the priming of Goldfish monocyte ROI production, the rgIFNγrel had relatively short-lived ROI priming potential and eventually down-regulated the priming of ROI production induced by rgIFNγ or rgTNFα2. Whereas rgIFNγ induced relatively modest phagocytic and nitric oxide responses of Goldfish macrophages, rgIFNγrel induced significantly higher phagocytosis, iNOSA and iNOSB gene expression and nitric oxide production compared with rgIFNγ. The rgIFNγ and rgIFNγrel induced different gene expression profiles in Goldfish monocytes. These differences included significantly higher induction of TNFα2, CXCL8, ceruloplasmin, and interferon regulatory factor (IRFs) expression after activation of monocytes with rgIFNγrel. The rgIFNγrel was more abundant in whole cell lysates compared with rgIFNγ. Both cytokines induced the phosphorylation of Stat1, while the nuclear localization of Stat1 was only observed following treatment of monocytes with rgIFNγ. Our findings suggest the presence of functional segregation of the induction of macrophage antimicrobial functions by type II interferons of bony fish.
-
Molecular characterization of novel interferon gamma receptor 1 isoforms in zebrafish (Danio rerio) and Goldfish (Carassius auratus L.)
Molecular immunology, 2009Co-Authors: Leon Grayfer, Miodrag BelosevicAbstract:Interferon gamma (IFNγ) is a highly pleotropic pro-inflammatory and anti-viral cytokine that mediates its effects by binding to a receptor complex composed of interferon gamma receptors 1 and 2 (IFNGR1 and IFNGR2). Using gene synteny analysis, we identified a distinct isoform of the zebrafish IFNGR1. The two zebrafish IFNGR1 called here IFNGR1-1 and IFNGR1-2 were used to identify the respective cDNA sequences of the Goldfish IFNGR1-1 and IFNGR1-2. Analysis of protein sequences revealed that all fish IFNGR1 species have potential JAK1 and STAT1 docking sites. Phylogenetically, teleost IFNGR1 proteins grouped separately from those of higher vertebrates. Q-PCR analysis revealed that while the constitutive mRNA levels of the two zebrafish IFNGR1 isoforms were comparable in different tissues examined, the Goldfish IFNGR1-1 tissue expression was substantially higher than that of IFNGR1-2. Q-PCR analysis of Goldfish immune cell populations revealed highest expression of both receptor isoforms in monocytes. Incubation of Goldfish macrophages with recombinant Goldfish IFNγ2 (rgIFNγ2) up-regulated expression of both IFNGR1-1 and IFNGR1-2, while treatment of cells with rgTNFα2 only increased the expression of IFNGR1-1. Treatment with rgTGFβ resulted in more modest increases in expression of both receptor isoforms only after prolonged treatment. In vitro binding studies indicated that rgIFNGR1-1 bound to rgIFNγ1 but not rgIFNγ2, while the rgIFNGR1-2 bound to rgIFNγ2. Thus, unlike mammals that have a single IFNGR1, cyprinid fish have two distinct IFNGR1 isoforms that preferentially bind corresponding ligands, IFNγ1 and IFNγ2, respectively, suggesting that the type II interferon system of these fish species is distinct from that of higher vertebrates.
-
molecular characterization expression and functional analysis of Goldfish carassius aurutus l interferon gamma
Developmental and Comparative Immunology, 2009Co-Authors: Leon Grayfer, Miodrag BelosevicAbstract:Abstract We report on the characterization and functional analysis of IFNγ of the Goldfish. Quantitative analysis indicated the highest expression of Goldfish IFNγ in the spleen, with lower expressions in brain, gill, kidney, heart, intestine and muscle. An increase in IFNγ expression was observed in kidney leukocytes following stimulation with PHA, PolyI:C and during mixed leukocyte reaction. IFNγ was expressed in different Goldfish immune cell populations and this expression was upregulated in several of these populations following treatment of cells with recombinant Goldfish TNFα-2. A recombinant form of Goldfish IFNγ (rgIFNγ) was produced and functionally analyzed. The rgIFNγ primed Goldfish macrophages and neutrophils for enhanced respiratory burst responses and increased the phagocytic and nitric oxide responses of macrophages. Furthermore, treatment of Goldfish macrophages with rgIFNγ induced increased expression of several proinflammatory genes including TNFα-1, TNFα-2, IL-β-1, IL-β-2, IL-12-p35, IL-12-p40, RSAD-2 (=viparin), CXCL-8 (=IL-8), CCL-1, iNOS A and B and IFNγ, and decreased the expression of TLR-3, while the expression of TGFβ remained unchanged. Our results indicate that IFNγ is a central proinflammatory cytokine of teleosts.
Vance L Trudeau - One of the best experts on this subject based on the ideXlab platform.
-
Neuronal regeneration in the Goldfish telencephalon following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) insult
Canadian Science Publishing, 2018Co-Authors: Maddie J. Venables, Lei Xing, Connor C. Edington, Vance L. Trudeau, Vance L TrudeauAbstract:The constitutive regenerative ability of the Goldfish central nervous system makes them an excellent model organism to study neurogenesis. Intraperitoneal injection of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to deplete tyrosine hydroxylase-positive neurons in the adult Goldfish telencephalon. We report novel information on the ability of the Goldfish to regenerate (∼3–4 d post-MPTP insult) damaged neurons in telencephalic tissue by observing the rapid incorporation of bromodeoxyuridine into newly generated cells, which precedes the recovery of motor function in MPTP-treated animals. Specifically, the telencephalon area telencephali pars dorsalis in female Goldfish, which is associated with fish motor activity, regenerates following MPTP toxicity. The remarkable ability of Goldfish to rapidly regenerate damaged neurons provides insight into their use as model organisms to study neuroregenerative abilities within a few days following injury. We provide evidence that Goldfish are able to regenerate neurons in ∼3–4 d to both replenish and recover baseline catecholaminergic levels, thus enabling the fish to reestablish basic activities such as swimming. The study of neuron regeneration in the damaged Goldfish brain will increase our understanding of vertebrate neurogenesis and regeneration processes following central nervous system injury
-
endocrine disrupting effects of waterborne fluoxetine exposure on the reproductive axis of female Goldfish carassius auratus
Comparative Biochemistry and Physiology C-toxicology & Pharmacology, 2017Co-Authors: Jan A Mennigen, Jacob M Zamora, John P Chang, Vance L TrudeauAbstract:Abstract Evidence suggests that pharmaceuticals and personal care products reach urban watersheds, bioconcentrate in fish, and potentially disrupt physiological homeostasis. These impairments often affect hormone functions. Selective serotonin reuptake inhibitors (SRRIs) are increasingly studied with regards to their endocrine disrupting effects on teleost physiological processes, including reproduction. To examine whether FLX effects on the endocrine regulation of reproductive physiology in Goldfish are sex-specific, we exposed sexually recrudescent female Goldfish to two waterborne concentrations of FLX (0.54 μg/L and 54 μg/L) using an experimental design previously used for sexually mature male Goldfish. To evaluate possible endocrine disrupting effects, we quantified the gonadosomatic index, circulating hormone concentrations (luteinizing hormone, LH; growth hormone, GH; 17-β estradiol, E2; and testosterone, T), and the expression of isotocin and vasotocin in the telencephalon, gonadotropin subunits and GH in the pituitary, and gonadotropin receptors, GH receptors, and aromatase in the ovary. Female Goldfish exposed to 0.54 μg/L FLX exhibited a significant decrease in circulating E2, and conversely, a significant increase in circulating LH and ovarian aromatase mRNA levels, suggesting disruption of E2-mediated feedback on LH release. These results, when compared with those previously observed in males, reveal that waterborne exposure to environmentally relevant levels of FLX sex-specifically disrupts the reproductive endocrine axis in Goldfish, characterized by a decrease in E2 in females, and conversely, estrogen-like effects in males. These data emphasize the importance of studying the effect of endocrine disrupting chemicals on both sexes.
-
proteolytic processing and differential distribution of secretogranin ii in Goldfish
General and Comparative Endocrinology, 2006Co-Authors: E Zhao, Ajoy Basak, Kate Crump, Vance L TrudeauAbstract:Abstract Secretoneurin (SN) is a 33–34 amino acid neuropeptide derived by endoproteolysis of secretogranin-II (SgII), a chromogranin. A multi-antigenic strategy was used to generate a rabbit polyclonal Goldfish SN antiserum that was characterized for Western blot analysis. In the Goldfish pituitary two intermediate proteins containing SN and likely processed from the 69.6-kDa SgII precursor were detected. No immunoreactive proteins were observed in the Goldfish interrenal, ovary, cerebellum, and telencephalon whereas SgII mRNA was expressed in all these tissues. Immunoreactive levels of the ∼57 kDa product were higher in the pars distalis (PD) than in the neurointermediate lobe (NIL). The abundance of the ∼57 kDa protein indicates that this SgII-product containing the SN sequence is a major stored form in secretory granules of the Goldfish pituitary. High expression and processing of SN in the hypothalamus and pituitary suggest important roles for SgII-derived peptides in neuroendocrine tissues.
-
the human lipid regulator gemfibrozil bioconcentrates and reduces testosterone in the Goldfish carassius auratus
Aquatic Toxicology, 2005Co-Authors: Caroline Mimeault, Thomas W Moon, A J Woodhouse, X S Miao, Chris D Metcalfe, Vance L TrudeauAbstract:Human and veterinarian pharmaceuticals have been detected in the aquatic environment for a number of years, but the potential for biological effects in exposed aquatic organism is only now being reported. The lipid regulator, gemfibrozil (GEM) is detected at microg/L concentrations in domestic wastewater and ng/L concentrations in surface waters. We investigated the uptake of GEM in Goldfish (Carassius auratus) over a 96 h time period by measuring GEM in blood plasma using LC-MS/MS. Results indicated that GEM can be taken up from water through the gills. In Goldfish exposed to GEM by a single intraperitoneal injection, concentrations of GEM in the blood plasma declined rapidly over 96 h post-injection, with a half-life estimated at approximately 19 h. Exposure of Goldfish to waterborne GEM at an environmentally relevant concentration over 14 days resulted in a plasma bioconcentration factor of 113. In Goldfish exposed to aqueous concentrations of GEM for 96 h or 14 days, plasma testosterone (T) was reduced by over 50% in fish from all treatments. As a possible mechanistic explanation for the observed reduction in T, levels of steroid acute regulatory (StAR) protein transcript in Goldfish testes were assessed by RT-PCR. StAR protein is involved in the transport of cholesterol from the outer to the inner mitochondrial membrane for transformation by the first enzyme in steroidogenesis. After exposure to GEM for 96 h, a 50% decrease in StAR mRNA levels was observed in Goldfish. Gonadal StAR mRNA levels were not affected in the 14 days exposure, indicating that the observed decreases in plasma testosterone were not solely due to impaired delivery of cholesterol to the inner mitochondrial membrane. Our results demonstrate that exposure to environmental levels of GEM leads to bioconcentration of the drug in plasma and the potential for endocrine disruption in fish.
Ming Wen - One of the best experts on this subject based on the ideXlab platform.
-
Sex chromosome and sex locus characterization in Goldfish, Carassius auratus (Linnaeus, 1758)
BMC Genomics, 2020Co-Authors: Ming Wen, Elodie Jouanno, Romain Feron, Christophe Klopp, Qiaowei Pan, Amaury Herpin, Justine Guguin, Cédric Cabau, Margot Zahm, Hugues ParrinelloAbstract:Background - Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. Here we used sequencing approaches to better characterize sex determination and sex-chromosomes in an experimental strain of Goldfish. Results - Our results confirmed that sex determination in Goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18 °C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (~ 11.7 Mb) on Goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the coding DNA sequence of the Goldfish amh gene. Conclusions - These results show that our Goldfish strain has a relatively large sex locus on LG22, which is likely the Y chromosome of this experimental population. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers, which will be important tools for future research on sex determination in our experimental Goldfish population. However, additional work would be needed to explore whether this sex locus is conserved in other populations of Goldfish.
-
Sex chromosome and sex locus characterization in the Goldfish, Carassius auratus
2019Co-Authors: Ming Wen, Elodie Jouanno, Romain Feron, Christophe Klopp, Qiaowei Pan, Amaury Herpin, Justine Guguin, Cédric Cabau, Margot Zahm, Hugues ParrinelloAbstract:Background: Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. We used sequencing approaches to better characterize sex determination and sex-chromosomes in Goldfish. Results: Our results confirmed that sex determination in Goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18°C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (∼11.7 Mb) on Goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the Goldfish amh gene or its 5 kb proximal promoter sequence. Conclusions: These results show that Goldfish have a relatively large sex locus on LG22, which is likely the Goldfish Y chromosome. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers in Goldfish, which will be important for future research on sex determination and aquaculture applications in this species.
Leon Grayfer - One of the best experts on this subject based on the ideXlab platform.
-
characterization and functional analysis of Goldfish carassius auratus l interleukin 10
Molecular Immunology, 2011Co-Authors: Leon Grayfer, Jordan W Hodgkinson, Steven J Hitchen, Miodrag BelosevicAbstract:This is the first report of comprehensive functional analysis of an interleukin-10 in bony fish. Quantitative expression analysis of Goldfish IL-10 revealed the greatest mRNA levels in the spleen tissues, peripheral blood leukocytes and granulocytes. The stimulation of cells with recombinant Goldfish (rg) TNFα2 significantly reduced IL-10 mRNA levels in granulocytes and monocytes of the Goldfish. To functionally assess the Goldfish IL-10, we generated a recombinant form of the molecule (rgIL-10). The rgIL-10 substantially reduced the expression of TNFα1, TNFα2, IL-1β1, IL-10, CXCL-8, and NADPH oxidase component, p47phox in monocytes activated with heat-killed Aeromonas salmonicida and reduced the expression of IFNγ in A. salmonicida-activated splenocytes. Pre-treatment of monocytes with rgIL-10 resulted in substantial reduction of the ROI response of the A. salmonicida or rgIFNγ-primed monocytes. The rgIL-10 bound to Goldfish monocytes and induced phosphorylation and nuclear translocation of Stat3. The rgIL-10 also induced rapid and robust increase in the mRNA levels of the Goldfish monocyte SOCS-3. Our results indicate that the function of IL-10 is highly conserved through evolution.
-
comparison of macrophage antimicrobial responses induced by type ii interferons of the Goldfish carassius auratus l
Journal of Biological Chemistry, 2010Co-Authors: Leon Grayfer, Erick Garcia Garcia, Miodrag BelosevicAbstract:Unlike mammals, bony fish have two type II interferons, IFNγ and IFNγrel, whose pro-inflammatory functions have not been fully characterized. To elucidate the distinct roles of these type II interferons of bony fish, we examined the effects of recombinant Goldfish (rg) IFNγ and IFNγrel on the macrophage antimicrobial responses, immune gene expression, and their signaling pathways. Our findings indicate that rgIFNγ and rgIFNγrel possess unique capacities to mediate each of the above processes. Q-PCR analysis revealed similar expression of both cytokines in tissues and immune cell populations of the Goldfish, although IFNγ mRNA levels were generally higher in most tissues and cell types. Whereas rgIFNγ had long-lasting effects on the priming of Goldfish monocyte ROI production, the rgIFNγrel had relatively short-lived ROI priming potential and eventually down-regulated the priming of ROI production induced by rgIFNγ or rgTNFα2. Whereas rgIFNγ induced relatively modest phagocytic and nitric oxide responses of Goldfish macrophages, rgIFNγrel induced significantly higher phagocytosis, iNOSA and iNOSB gene expression and nitric oxide production compared with rgIFNγ. The rgIFNγ and rgIFNγrel induced different gene expression profiles in Goldfish monocytes. These differences included significantly higher induction of TNFα2, CXCL8, ceruloplasmin, and interferon regulatory factor (IRFs) expression after activation of monocytes with rgIFNγrel. The rgIFNγrel was more abundant in whole cell lysates compared with rgIFNγ. Both cytokines induced the phosphorylation of Stat1, while the nuclear localization of Stat1 was only observed following treatment of monocytes with rgIFNγ. Our findings suggest the presence of functional segregation of the induction of macrophage antimicrobial functions by type II interferons of bony fish.
-
Molecular characterization of novel interferon gamma receptor 1 isoforms in zebrafish (Danio rerio) and Goldfish (Carassius auratus L.)
Molecular immunology, 2009Co-Authors: Leon Grayfer, Miodrag BelosevicAbstract:Interferon gamma (IFNγ) is a highly pleotropic pro-inflammatory and anti-viral cytokine that mediates its effects by binding to a receptor complex composed of interferon gamma receptors 1 and 2 (IFNGR1 and IFNGR2). Using gene synteny analysis, we identified a distinct isoform of the zebrafish IFNGR1. The two zebrafish IFNGR1 called here IFNGR1-1 and IFNGR1-2 were used to identify the respective cDNA sequences of the Goldfish IFNGR1-1 and IFNGR1-2. Analysis of protein sequences revealed that all fish IFNGR1 species have potential JAK1 and STAT1 docking sites. Phylogenetically, teleost IFNGR1 proteins grouped separately from those of higher vertebrates. Q-PCR analysis revealed that while the constitutive mRNA levels of the two zebrafish IFNGR1 isoforms were comparable in different tissues examined, the Goldfish IFNGR1-1 tissue expression was substantially higher than that of IFNGR1-2. Q-PCR analysis of Goldfish immune cell populations revealed highest expression of both receptor isoforms in monocytes. Incubation of Goldfish macrophages with recombinant Goldfish IFNγ2 (rgIFNγ2) up-regulated expression of both IFNGR1-1 and IFNGR1-2, while treatment of cells with rgTNFα2 only increased the expression of IFNGR1-1. Treatment with rgTGFβ resulted in more modest increases in expression of both receptor isoforms only after prolonged treatment. In vitro binding studies indicated that rgIFNGR1-1 bound to rgIFNγ1 but not rgIFNγ2, while the rgIFNGR1-2 bound to rgIFNγ2. Thus, unlike mammals that have a single IFNGR1, cyprinid fish have two distinct IFNGR1 isoforms that preferentially bind corresponding ligands, IFNγ1 and IFNγ2, respectively, suggesting that the type II interferon system of these fish species is distinct from that of higher vertebrates.
-
molecular characterization expression and functional analysis of Goldfish carassius aurutus l interferon gamma
Developmental and Comparative Immunology, 2009Co-Authors: Leon Grayfer, Miodrag BelosevicAbstract:Abstract We report on the characterization and functional analysis of IFNγ of the Goldfish. Quantitative analysis indicated the highest expression of Goldfish IFNγ in the spleen, with lower expressions in brain, gill, kidney, heart, intestine and muscle. An increase in IFNγ expression was observed in kidney leukocytes following stimulation with PHA, PolyI:C and during mixed leukocyte reaction. IFNγ was expressed in different Goldfish immune cell populations and this expression was upregulated in several of these populations following treatment of cells with recombinant Goldfish TNFα-2. A recombinant form of Goldfish IFNγ (rgIFNγ) was produced and functionally analyzed. The rgIFNγ primed Goldfish macrophages and neutrophils for enhanced respiratory burst responses and increased the phagocytic and nitric oxide responses of macrophages. Furthermore, treatment of Goldfish macrophages with rgIFNγ induced increased expression of several proinflammatory genes including TNFα-1, TNFα-2, IL-β-1, IL-β-2, IL-12-p35, IL-12-p40, RSAD-2 (=viparin), CXCL-8 (=IL-8), CCL-1, iNOS A and B and IFNγ, and decreased the expression of TLR-3, while the expression of TGFβ remained unchanged. Our results indicate that IFNγ is a central proinflammatory cytokine of teleosts.
-
characterization and functional analysis of Goldfish carassius auratus l tumor necrosis factor alpha
Developmental and Comparative Immunology, 2008Co-Authors: Leon Grayfer, John G Walsh, Miodrag BelosevicAbstract:We identified and characterized two isoforms of tumor necrosis factor-alpha (TNFα) from the Goldfish, TNFα-1 and TNFα-2. At the protein level, Goldfish TNFα-1 and TNFα-2 were most homologous to carp TNFα-1 and TNFα-2, respectively. Phylogenetically, the two Goldfish isoforms grouped most closely with the carp TNFα isoforms and TNF species of other cyprinids. Real-time PCR analysis revealed constitutive expression of Goldfish TNFα-1 and TNFα-2 in all tissues with TNFα-2 mRNA levels higher than TNFα-1 in all tissues examined. A modest up-regulation in expressions of Goldfish TNFα-1 and TNFα-2 in kidney-derived monocytes and significant increase in expression of both isoforms in mature macrophages were observed in response to activation with macrophage-activating factors. TNFα-2 was subsequently expressed using a prokaryotic expression system and the recombinant molecule (rTNFα-2) was functionally characterized. The rTNFα-2 induced a dose-dependent chemotactic response and enhanced phagocytosis of primary Goldfish macrophages. Furthermore, rTNFα-2 primed the respiratory burst in monocytes and induced nitric oxide production of primary Goldfish macrophages. Our results indicate that Goldfish TNFα is a central regulatory and effector cytokine of inflammatory and antimicrobial responses of the Goldfish.