A protein kinase C inhibitor is found in bovine sper-matozoa. This inhibitor was purified by Sephadex G-200and isoelectrofocusion electrophoresis. The molecularweight of this protein kinase C inhibitor was about 63 00...A protein kinase C inhibitor is found in bovine sper-matozoa. This inhibitor was purified by Sephadex G-200and isoelectrofocusion electrophoresis. The molecularweight of this protein kinase C inhibitor was about 63 000,the isoelectric point of the inhibitor was pH 4.5.展开更多
Aim Evidence has shown that stimulation of alA-adrenorecetors receptor （alA-AR） or angiotensin II type 1 receptor （AT1R） acutely down-regulates the rapid component of the delayed rectifier K ＋ current （IKr） via...Aim Evidence has shown that stimulation of alA-adrenorecetors receptor （alA-AR） or angiotensin II type 1 receptor （AT1R） acutely down-regulates the rapid component of the delayed rectifier K ＋ current （IKr） via protein kinase C （PKC）. This study was designed to investigate which PKC isozymes mediate down-regulations of IKr by alA-AR and AT1R. Method The whole-cell patch-clamp technique was used to record IKr in native cardio- myocytes and in human embryonic kidney （HEK） 293 cells co-transfected with human ether-a-go-go related gene （hERG） encoding α-subunit of IKr and human alA-AR or AT1R gene. Result In isolated guinea-pig ventricular cardiomyocytes the inhibitory action of Ang II on IKr was little affected by Go6976 （selectively inhibiting PKCα, β and γ） and Go6983 （selectively inhibiting PKCα, β, γ , δ, and ζ）, but was significantly antagonized by an inter- nal dialysis with PKCe-selective inhibitory peptide εV1 -2. In contrast, the inhibitory action of alA-AR agonist A61603 on IKr was remarkably attenuated by Go6976 or Go6983, but not affected by peptide εV1 -2. Moreover, specific PKC-selective inhibitory peptide antagonized the effect of A61603. The results suggested that PKCe and PKCα isoform respectively mediated the inhibitory effect of AT1R and a1A-AR. In heterologous expression system, both PKCα and e-selective activator peptides down regulated hERG current with different manner. PKCα activator peptide shifted the activation curve of the channel to the right, but PKCe-selective activator peptide did not. Simi- larly, A61603 shifted the activation curve to the right, whereas Ang Ⅱ had no effect. In addition, both A61603 and PKCα activator peptide showed inhibitory action on bERG A PKC current （an bERG mutant in which 17 of the 18 ROSITE-predicted PKC acceptor serines/threonines were changed to alanine） with a similar potency to wild type bERG current. But, both Ang Ⅱ and PKCe-selective activator peptide exhibited no effects on bERG △ PKC cur- rent. The results indicated that PKCα and PKCe isoforms down-regulated bERG current through different mecha- nism. Conclusion PKCα and PKCe isoform respectively mediates the inhibition on IKr by stimulation of AT1R and alA-AR via different molecular mechanism.展开更多
OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) us...OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.展开更多
Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-reg...Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.展开更多
文摘A protein kinase C inhibitor is found in bovine sper-matozoa. This inhibitor was purified by Sephadex G-200and isoelectrofocusion electrophoresis. The molecularweight of this protein kinase C inhibitor was about 63 000,the isoelectric point of the inhibitor was pH 4.5.
文摘Aim Evidence has shown that stimulation of alA-adrenorecetors receptor （alA-AR） or angiotensin II type 1 receptor （AT1R） acutely down-regulates the rapid component of the delayed rectifier K ＋ current （IKr） via protein kinase C （PKC）. This study was designed to investigate which PKC isozymes mediate down-regulations of IKr by alA-AR and AT1R. Method The whole-cell patch-clamp technique was used to record IKr in native cardio- myocytes and in human embryonic kidney （HEK） 293 cells co-transfected with human ether-a-go-go related gene （hERG） encoding α-subunit of IKr and human alA-AR or AT1R gene. Result In isolated guinea-pig ventricular cardiomyocytes the inhibitory action of Ang II on IKr was little affected by Go6976 （selectively inhibiting PKCα, β and γ） and Go6983 （selectively inhibiting PKCα, β, γ , δ, and ζ）, but was significantly antagonized by an inter- nal dialysis with PKCe-selective inhibitory peptide εV1 -2. In contrast, the inhibitory action of alA-AR agonist A61603 on IKr was remarkably attenuated by Go6976 or Go6983, but not affected by peptide εV1 -2. Moreover, specific PKC-selective inhibitory peptide antagonized the effect of A61603. The results suggested that PKCe and PKCα isoform respectively mediated the inhibitory effect of AT1R and a1A-AR. In heterologous expression system, both PKCα and e-selective activator peptides down regulated hERG current with different manner. PKCα activator peptide shifted the activation curve of the channel to the right, but PKCe-selective activator peptide did not. Simi- larly, A61603 shifted the activation curve to the right, whereas Ang Ⅱ had no effect. In addition, both A61603 and PKCα activator peptide showed inhibitory action on bERG A PKC current （an bERG mutant in which 17 of the 18 ROSITE-predicted PKC acceptor serines/threonines were changed to alanine） with a similar potency to wild type bERG current. But, both Ang Ⅱ and PKCe-selective activator peptide exhibited no effects on bERG △ PKC cur- rent. The results indicated that PKCα and PKCe isoforms down-regulated bERG current through different mecha- nism. Conclusion PKCα and PKCe isoform respectively mediates the inhibition on IKr by stimulation of AT1R and alA-AR via different molecular mechanism.
文摘OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.
基金Shanghai Medical Key Discipline Construction Foundation(05-Ⅲ-005-017).
文摘Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.
