AIM： To delineate the mechanisms of renal vasoconstriction in hepatorenal syndrome （HRS）, we investigated the expression of type I inositol 1, 4, 5-triphosphate receptors （IP3R I） of kidney in mice with fulminant...AIM： To delineate the mechanisms of renal vasoconstriction in hepatorenal syndrome （HRS）, we investigated the expression of type I inositol 1, 4, 5-triphosphate receptors （IP3R I） of kidney in mice with fulminant hepatic failure （FHF）. METHODS： FHF was induced by lipopolysaccharide （LPS） in D-galactosamine （GAIN） sensitized BALB/c mice. There were 20 mice in normal saline （NS）-treated group, 20 mice in LPS-treated group, 20 mice in GaIN- treated group, and 60 mice in GalN/LPS-treated group （FHF group）. Liver and kidney tissues were obtained at 2, 6, and 9 h after administration. The liver and kidney specimens were stained with hematoxylin-eosin for studying morphological changes under light microscope. The expression of IP3R I in kidney tissue was tested by immunohistochemistry, Western blot and reverse transcription （RT）-PCR. RESULTS： Kidney tissues were morphologically normal at all time points in all groups. IP3R I proteins were found localized in the plasma region of glomerular mesangial cells （GMC） and vascular smooth muscle cells （VSMC） in kidney by immunohistochemical staining. In kidney of mice with FHF at 6 h and 9 h IP3R I staining was upregulated. Results from Western blot demonstrated consistent and significant increment of IP3R I expression in mice with FHF at 6 h and 9 h （t = 3.16, P 〈 0.05; t = 5.43, P 〈 0.01）. Furthermore, we evaluated IP3R I mRNA expression by RT-PCR and observed marked upregulation of IP3R I mRNA in FHF samples at 2 h, 6 h and 9 h compared to controls （t = 2.97, P 〈 0.05; t = 4.42, P 〈 0.01; t = 3.81, P 〈 0.01）. CONCLUSION： The expression of IP3R I protein increased in GMC and renal VSMC of mice with FHF, possibly caused by up-regulation of IP3R I mRNA.展开更多
We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled recepto...We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction （RT-PCR） and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase （AC） similar in amplitude to that produced by fully stimulated Gs coupled receptors. Moreover, sphingosine 1-phosphate （S1P） could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein （GFP） construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AG and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1 P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.展开更多
Sphingosine-1-phosphate（S1P） is a bioactive lipid messenger in the cells that regulate gene expression and NF-κB signal pathway through unknown mechanisms.Recently,Cheng Luo,associate professor of DDDC in Shanghai ...Sphingosine-1-phosphate（S1P） is a bioactive lipid messenger in the cells that regulate gene expression and NF-κB signal pathway through unknown mechanisms.Recently,Cheng Luo,associate professor of DDDC in Shanghai Institute of Materia Medica,whose project was funded by the National Natural Science Foundation of China,joined in a research team led by Professor Sarah Spiegel of Virginia Commonwealth University.The team continuously made significant breakthroughs in understanding the regulation mechanism of Sphingosine-1- Phosphate.In September 2009,in a paper published on SCIENCE magazine（Science 2009, 325：1254-7）,they firstly demonstrated that S1P is a physiologically important regulator of histone deacetylases（HDACs）,HDACs are direct intracellular targets of S1P.Furthermore,they identified the mechanism that S1P regulates gene expression through regulating the activity of HDACs.In June 24th,2010,in another paper to be published on NATURE magazine（Nature 2010,June 24th,advance online publication,（doi：10.1038/ nature09128）） which reports the regulation of NF-κB signaling pathway by S1P.They demonstrate that S1P is the missing cofactor for TRAF2（tumour-necrosis factor receptor-associated factor 2） and indicate a new paradigm for the regulation of lysine-63- linked poly-ubiquitination.The study also highlight the key role of SphK1 and its product S1P in TNF-αsignalling and the canonical NF-κB activation pathway, and then play crucial role in inflammatory,antiapoptotic and immune processes.The identification of new mechanisms fay which S1P regulates gene expression and TNF and NF-κB signaling pathway will light up the road to develop novel inhibitors that might be useful for treatment of cancer and in- flammatory diseases.展开更多
基金Supported by National Natural Science Foundation of China, No. 30270607
文摘AIM： To delineate the mechanisms of renal vasoconstriction in hepatorenal syndrome （HRS）, we investigated the expression of type I inositol 1, 4, 5-triphosphate receptors （IP3R I） of kidney in mice with fulminant hepatic failure （FHF）. METHODS： FHF was induced by lipopolysaccharide （LPS） in D-galactosamine （GAIN） sensitized BALB/c mice. There were 20 mice in normal saline （NS）-treated group, 20 mice in LPS-treated group, 20 mice in GaIN- treated group, and 60 mice in GalN/LPS-treated group （FHF group）. Liver and kidney tissues were obtained at 2, 6, and 9 h after administration. The liver and kidney specimens were stained with hematoxylin-eosin for studying morphological changes under light microscope. The expression of IP3R I in kidney tissue was tested by immunohistochemistry, Western blot and reverse transcription （RT）-PCR. RESULTS： Kidney tissues were morphologically normal at all time points in all groups. IP3R I proteins were found localized in the plasma region of glomerular mesangial cells （GMC） and vascular smooth muscle cells （VSMC） in kidney by immunohistochemical staining. In kidney of mice with FHF at 6 h and 9 h IP3R I staining was upregulated. Results from Western blot demonstrated consistent and significant increment of IP3R I expression in mice with FHF at 6 h and 9 h （t = 3.16, P 〈 0.05; t = 5.43, P 〈 0.01）. Furthermore, we evaluated IP3R I mRNA expression by RT-PCR and observed marked upregulation of IP3R I mRNA in FHF samples at 2 h, 6 h and 9 h compared to controls （t = 2.97, P 〈 0.05; t = 4.42, P 〈 0.01; t = 3.81, P 〈 0.01）. CONCLUSION： The expression of IP3R I protein increased in GMC and renal VSMC of mice with FHF, possibly caused by up-regulation of IP3R I mRNA.
基金Project supported by the National High-Tech R&D Program(863)of China(No.2006AA10Z136)a Grant-in-Aid for Innovative Training of Doctoral Students in Jiangsu Province of China(No.CXLX11-0701)
文摘We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction （RT-PCR） and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase （AC） similar in amplitude to that produced by fully stimulated Gs coupled receptors. Moreover, sphingosine 1-phosphate （S1P） could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein （GFP） construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AG and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1 P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.
基金supported by Natural Science Foundation of China(Grant No.20972174)
文摘Sphingosine-1-phosphate（S1P） is a bioactive lipid messenger in the cells that regulate gene expression and NF-κB signal pathway through unknown mechanisms.Recently,Cheng Luo,associate professor of DDDC in Shanghai Institute of Materia Medica,whose project was funded by the National Natural Science Foundation of China,joined in a research team led by Professor Sarah Spiegel of Virginia Commonwealth University.The team continuously made significant breakthroughs in understanding the regulation mechanism of Sphingosine-1- Phosphate.In September 2009,in a paper published on SCIENCE magazine（Science 2009, 325：1254-7）,they firstly demonstrated that S1P is a physiologically important regulator of histone deacetylases（HDACs）,HDACs are direct intracellular targets of S1P.Furthermore,they identified the mechanism that S1P regulates gene expression through regulating the activity of HDACs.In June 24th,2010,in another paper to be published on NATURE magazine（Nature 2010,June 24th,advance online publication,（doi：10.1038/ nature09128）） which reports the regulation of NF-κB signaling pathway by S1P.They demonstrate that S1P is the missing cofactor for TRAF2（tumour-necrosis factor receptor-associated factor 2） and indicate a new paradigm for the regulation of lysine-63- linked poly-ubiquitination.The study also highlight the key role of SphK1 and its product S1P in TNF-αsignalling and the canonical NF-κB activation pathway, and then play crucial role in inflammatory,antiapoptotic and immune processes.The identification of new mechanisms fay which S1P regulates gene expression and TNF and NF-κB signaling pathway will light up the road to develop novel inhibitors that might be useful for treatment of cancer and in- flammatory diseases.
