AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cell...AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.展开更多
Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct.The absorption,distribution and elimination of drugs are impaired during this pa...Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct.The absorption,distribution and elimination of drugs are impaired during this pathology.Prolonged cholestasis may alter both liver and kidney function.Lactam antibiotics,diuretics,non-steroidal anti-inflammatory drugs,several antiviral drugs as well as endogenous compounds are classified as organic anions.The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds.It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions.The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis,such as multidrug resistanceassociated protein 2,organic anion transporting polypeptide 1,organic anion transporter 3,bilitranslocase,bromosulfophthalein/bilirubin binding protein,organic anion transporter 1 and sodium dependent bile salt transporter.The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.展开更多
Odorant binding proteins （OBPs） are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. Here, we injected...Odorant binding proteins （OBPs） are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. Here, we injected AlinOBP4-siRNA into the conjunctivum between prothorax and mesothorax of the lucerne plant bug, Adelphocoris lineolatus and evaluated the silencing of AlinOBP4 by reverse transcription polymerase chain reaction （RT-PCR） analysis, quantitative real-time PCR （qPCR） test and electroantennogram （EAG） assay. The combination of RT-PCR and qPCR analyses revealed that the levels of mes- senger RNA transcript were significantly reduced ~95% in AlinOBP4-siRNA-treated A. lineolatus males and ~75% in RNAi-treated females within 48 hours. It was found that there are different EAG responses between male and female bugs when the AlinOBP4 gene was silenced by RNAi. The EAGs of A. lineolatus to two plant volatiles, tride- canal and hexyl alcohol, were reduced 9.09% and 79.45% in RNAi-treated males, 62.08% and 62.08% in RNAi-treated females compared to the controls, separately. Antennae of RNAi-treated bugs showed significantly lower electrophysiological responses to four sex pheromone analogs, butyl butanoate, 1-hexyl butyrate, （E）-2-hexenyl butyrate and hexyl hexanoate. The EAG recordings were reduced 35.43%, 35.24%, 39.96% and 78.47% in RNAi-treated males and 64.52%, 18.13%, 36.88% and 49.52% in RNAi-treated females, respectively. The results suggested that AlinOBP4 might play dual-roles in the identifi- cation of plant volatiles and sex pheromones. It was suspected that AlinOBP4 may have different functions in odor perception between male and female A. lineolatus.展开更多
Recent studies on enzymes regulating dynamic N6-methyl-adenosine （m6A） in RNA together with the findings from m6A-methylated RNA immunoprecipitation followed by high-throughput sequencing （MeRIP-seq/m6A- seq） have...Recent studies on enzymes regulating dynamic N6-methyl-adenosine （m6A） in RNA together with the findings from m6A-methylated RNA immunoprecipitation followed by high-throughput sequencing （MeRIP-seq/m6A- seq） have revealed a broad biological role of m6A in RNA processing, development, differentiation, metabolism and fertility. RNA m6A methylation is catalyzed by a multi- component methyltransferase complex composed of at least three subunits： METTL3, METTL14 and Wilms tumor 1-associated protein （WTAP）, in which METTL3 and METTL14 serve as catalytic subunits, while WTAP as reg- ulatory subunit. Dioxygenases FTO and ALKBH5, as the first two known m6A demethylases, catalyze m6A removal. Five m6A-binding proteins are classified into cytoplasmic YT521-B homology （YTH） domain-containing family YT- HDF1-3 and nuclear YTHDC1-2. Perturbation of enzy- matic activities catalyzing dynamic m6A results in altered expression of thousands of genes and affects mRNA stability and splicing at the cellular level. Here, we summarize recent discoveries about m6A methyltransferases （writers）,demethylases （erasers） and binding proteins （readers）, and further discuss the potential impacts of m6A on RNA pro- cessing, especially on mRNA splicing.展开更多
基金Supported by Medical Specialty Development Projects of Beijing Municipal Administration of Hospitals,No.ZYLX201402Ministry of Education of The People’s Republic of China,No.20121107110012+1 种基金Beijing Municipal Commission of Education,No.11320016Collaborative Innovation Center of Infectious Diseases and Beijing Key Laboratory of Emerging Infectious Diseases,Beijing,China
文摘AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.
基金Supported by Grants from FONCYT(PICT 2007,No.00966, PICT 2010,No.2127)CONICET(PIP 2009-2011,No.1665, PIP2012-2015,No.00014)UNR PID(2008-2011/2012-2015)
文摘Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct.The absorption,distribution and elimination of drugs are impaired during this pathology.Prolonged cholestasis may alter both liver and kidney function.Lactam antibiotics,diuretics,non-steroidal anti-inflammatory drugs,several antiviral drugs as well as endogenous compounds are classified as organic anions.The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds.It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions.The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis,such as multidrug resistanceassociated protein 2,organic anion transporting polypeptide 1,organic anion transporter 3,bilitranslocase,bromosulfophthalein/bilirubin binding protein,organic anion transporter 1 and sodium dependent bile salt transporter.The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.
文摘Odorant binding proteins （OBPs） are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. Here, we injected AlinOBP4-siRNA into the conjunctivum between prothorax and mesothorax of the lucerne plant bug, Adelphocoris lineolatus and evaluated the silencing of AlinOBP4 by reverse transcription polymerase chain reaction （RT-PCR） analysis, quantitative real-time PCR （qPCR） test and electroantennogram （EAG） assay. The combination of RT-PCR and qPCR analyses revealed that the levels of mes- senger RNA transcript were significantly reduced ~95% in AlinOBP4-siRNA-treated A. lineolatus males and ~75% in RNAi-treated females within 48 hours. It was found that there are different EAG responses between male and female bugs when the AlinOBP4 gene was silenced by RNAi. The EAGs of A. lineolatus to two plant volatiles, tride- canal and hexyl alcohol, were reduced 9.09% and 79.45% in RNAi-treated males, 62.08% and 62.08% in RNAi-treated females compared to the controls, separately. Antennae of RNAi-treated bugs showed significantly lower electrophysiological responses to four sex pheromone analogs, butyl butanoate, 1-hexyl butyrate, （E）-2-hexenyl butyrate and hexyl hexanoate. The EAG recordings were reduced 35.43%, 35.24%, 39.96% and 78.47% in RNAi-treated males and 64.52%, 18.13%, 36.88% and 49.52% in RNAi-treated females, respectively. The results suggested that AlinOBP4 might play dual-roles in the identifi- cation of plant volatiles and sex pheromones. It was suspected that AlinOBP4 may have different functions in odor perception between male and female A. lineolatus.
基金supported by the National BasicResearch Program of China(2011CB510103,2014CB964902)the National Science Foundation of China(91319308,31430022 and31400672)Strategic Priority Research Program of Chinese Academy ofSciences(XDB14030300)
文摘Recent studies on enzymes regulating dynamic N6-methyl-adenosine （m6A） in RNA together with the findings from m6A-methylated RNA immunoprecipitation followed by high-throughput sequencing （MeRIP-seq/m6A- seq） have revealed a broad biological role of m6A in RNA processing, development, differentiation, metabolism and fertility. RNA m6A methylation is catalyzed by a multi- component methyltransferase complex composed of at least three subunits： METTL3, METTL14 and Wilms tumor 1-associated protein （WTAP）, in which METTL3 and METTL14 serve as catalytic subunits, while WTAP as reg- ulatory subunit. Dioxygenases FTO and ALKBH5, as the first two known m6A demethylases, catalyze m6A removal. Five m6A-binding proteins are classified into cytoplasmic YT521-B homology （YTH） domain-containing family YT- HDF1-3 and nuclear YTHDC1-2. Perturbation of enzy- matic activities catalyzing dynamic m6A results in altered expression of thousands of genes and affects mRNA stability and splicing at the cellular level. Here, we summarize recent discoveries about m6A methyltransferases （writers）,demethylases （erasers） and binding proteins （readers）, and further discuss the potential impacts of m6A on RNA pro- cessing, especially on mRNA splicing.
