Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor ...Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.展开更多
Aim Angiotensin II （AngII） induces vascular smooth muscle cell （VSMC） migration and growth, which is responsible for vascular remodeling during some cardiovascular diseases. It has been demonstrated to activate a ...Aim Angiotensin II （AngII） induces vascular smooth muscle cell （VSMC） migration and growth, which is responsible for vascular remodeling during some cardiovascular diseases. It has been demonstrated to activate a C1 current, but the underlying mechanism is not clear. Methods Whole-cell patch clamp, co-immunoprecipitation （co-IP）, site-specific mutagenesis, angiotensinII-infusion hypertensive mice model were used. Results In VSMCs, AngII could induce a C1C-3-dependent C1- current that was abolished in C1C-3 null mice. The activation mechanism of this AngII-induced C1- current was ascribed to the interaction between C1C-3 and Rho-kinase 2 （ROCIL2）, as re- vealed by N-terminal or C-terminal truncation of C1C-3, ROCIC2 siRNA and Co-IP experiments. Then we searched for and identified the phosphorylation site of C1C-3 at threonine 532 is critical for AngII-induced C1- current and VSMC migration through ROCK. The C1C-3 T532D mutant （mutation of threonine 532 to aspartate）, mimicking the phos- phorylation state of C1C-3, significantly potentiated AngII-induced C1- current and VSMC migration; while C1C-3 T532A （mutation of threonine 532 to alanine） had the opposite effects. Furthermore, we found a remarkably de- creased AngII-induced VSMC migration in C1C-3 null mice that is insensitive to Y27632, an inhibitor of ROCIL2. In addition, AngII-induced cerebrovascular remodeling was ameliorated in C1C-3 null mice, possibly by ROCIL2 path- way. Conclusions C1C-3 protein phosphorylation at threonine 532 by ROCIL2 is required for AngII-induced C1- cur- rent and VSMC migration that are involved in AngII-induced hypertensive vascular remodeling.展开更多
This paper reported the identification of the primary structure of two peptides using nanoelectrospray tandem mass spectrometry(Nano-ESI MS/MS).Firstly,the relative molecular mass of two peptides were determined in ES...This paper reported the identification of the primary structure of two peptides using nanoelectrospray tandem mass spectrometry(Nano-ESI MS/MS).Firstly,the relative molecular mass of two peptides were determined in ESI-TOF MS mode.Then fragmentation ions were obtained by selecting [M+2H]2+ ion using tandem mass spectrometry(MS/MS).Finally,the primary structure of triptorelin is determined to be E’HWSYWLRPG’,of which the N-terminal is pyroglutamic acid (E’) and the C-termianl is glycinamide (G’).The primary structure of unknown peptide was identified to be T’VSP VWLPPSVY by sequence docking method,of which the N-terminal occurs threonine phosphorylation (T’) and the foruth proline is added with sodium ion (P).The data suggested that electrospray Tandem mass spectrometry technique have obvious advantage in analyzing the full sequence of modified or unknown peptides.展开更多
OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 depende...OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.展开更多
Of the eleven families of cyclic nucleotide phosphodiesterases(PDEs) present in the human body,PDE4s represent the most widely expressed family of PDEs. A large body of work has been published on the expression and fu...Of the eleven families of cyclic nucleotide phosphodiesterases(PDEs) present in the human body,PDE4s represent the most widely expressed family of PDEs. A large body of work has been published on the expression and function of these PDEs,which preferentially hydrolyze cAMP in all cells studied,including neurons and supporting cells of the CNS. Four distinct genes termed PDE4 A,PDE4B,PDE4C and PDE4D encode PDE4 proteins. However,the number of PDE4s identified in different tissues and cells is estimated to be more than 30. Differences in regulation and localization explain this extreme heterogeneity. PDE4 hydrolytic activity is regulated by phosphorylation,and protein kinase A(PKA) was the first kinase identified. This PKA-dependent regulation establishes a feedback loop where cAMP regulates its own degradation to control the intensity and localization of the hormone and neurotransmitter signal. In addition,numerous additional kinases phosphorylate PDE4s to modulate the PKA-dependent activation and fine tune cAMP levels by growth factors and other extracellular cues. Thus,PDE4 can be considered a coincidence detector that integrates multiple signaling pathways. Finally,different PDE4s are involved in numerous macromolecular complexes targeting the cAMP hydrolytic activity to different subcellular domains. Thus,PDE4s function in different subcellular compartments,and inhibition of different isoforms affects cAMP levels in different subdomains with consequently different functions. The dyad space and the control of excitation/contraction will be used as examples of these localized regulations.展开更多
Prolactin （PRL） is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be n...Prolactin （PRL） is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be necessary for high levels of milk protein expression, the mechanism by which the synthesis of milk proteins is stimulated at the transcript level is less known. A major modification in the transcript level is protein phosphorylation. To gain additional insights into the molecular mechanisms at the transcript level underlying PRL action on the dairy cow mammary epithelial cells （DCMECs）, nuclear phosphoproteins whose expression distinguishes proliferating regulated by PRL in DCMECs were identified. A phosphoprotein-enriched fraction from nuclear proteins was obtained by affinity chromatography, and a two-dimensional gel electrophoresis （2-DE） and matrix assisted laser desorption/ionization time of matrix-assisted laser desorption/ionization/time of flight mass spectrometry （MALDI-TOF MS） were used to identify the changes of nuclear phosphoproteins in DCMECs treated with prolactin. Seven proteins displaying~〉2-fold difference in abundance upon PRL treatment in DCMECs were identified by MALDI-TOF MS. The protein-GARS （GlyRS）, which belonged to the class-II aminoacyl-tRNA synthetase family, played a global role in the milk protein synthesis. SERPINH1 （Heat shock protein 47）, which was the first heat shock protein found to be a member of the serpin superfamily, regulated physiologic functions, such as complement activation, programmed cell death, and inflammatory processes. PRDX3, which belonged to a family of antioxidant enzymes, played an important role in scavenging intracellular reactive oxygen species （ROS）. ACTR1A, belonged to the actin family, which was associated with transport of p53 to the nucleus. Annexin A2, a Ca2＋-dependent phospholipid-binding protein, maintained the viability and cell cycle regulation of DCMECs. PSMB2 and PSMD10, which belonged to ubiquitin-proteasome system, were involved in several cellular processes, including cell cycle control, cellular stress response, intracellular signaling. This screening revealed that prolactin influenced the level of nuclear phosphoproteins in DCMECs. This result opens new avenues for the study of the molecular mechanism linked to the synthesis of milk proteins.展开更多
文摘Dendritic cells (DCs) are the most potent antigen-presen ting cells that play crucial roles in the regulation of immune response. Triptol ide, an active component purified from the medicinal plant Tripterygium wilfor dii Hook F., has been demonstrated to act as a potent immunosuppressive drug c apab le of inhibiting T cell activation and proliferation. However, little is known a bout the effects of triptolide on DCs. The present study shows that triptolide d oes not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ml, as demonstrated by phosphatidylserin e exposure, mitochondria potential decrease, and nuclear DNA condensation. Tript olide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that t he anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs.
文摘Aim Angiotensin II （AngII） induces vascular smooth muscle cell （VSMC） migration and growth, which is responsible for vascular remodeling during some cardiovascular diseases. It has been demonstrated to activate a C1 current, but the underlying mechanism is not clear. Methods Whole-cell patch clamp, co-immunoprecipitation （co-IP）, site-specific mutagenesis, angiotensinII-infusion hypertensive mice model were used. Results In VSMCs, AngII could induce a C1C-3-dependent C1- current that was abolished in C1C-3 null mice. The activation mechanism of this AngII-induced C1- current was ascribed to the interaction between C1C-3 and Rho-kinase 2 （ROCIL2）, as re- vealed by N-terminal or C-terminal truncation of C1C-3, ROCIC2 siRNA and Co-IP experiments. Then we searched for and identified the phosphorylation site of C1C-3 at threonine 532 is critical for AngII-induced C1- current and VSMC migration through ROCK. The C1C-3 T532D mutant （mutation of threonine 532 to aspartate）, mimicking the phos- phorylation state of C1C-3, significantly potentiated AngII-induced C1- current and VSMC migration; while C1C-3 T532A （mutation of threonine 532 to alanine） had the opposite effects. Furthermore, we found a remarkably de- creased AngII-induced VSMC migration in C1C-3 null mice that is insensitive to Y27632, an inhibitor of ROCIL2. In addition, AngII-induced cerebrovascular remodeling was ameliorated in C1C-3 null mice, possibly by ROCIL2 path- way. Conclusions C1C-3 protein phosphorylation at threonine 532 by ROCIL2 is required for AngII-induced C1- cur- rent and VSMC migration that are involved in AngII-induced hypertensive vascular remodeling.
文摘This paper reported the identification of the primary structure of two peptides using nanoelectrospray tandem mass spectrometry(Nano-ESI MS/MS).Firstly,the relative molecular mass of two peptides were determined in ESI-TOF MS mode.Then fragmentation ions were obtained by selecting [M+2H]2+ ion using tandem mass spectrometry(MS/MS).Finally,the primary structure of triptorelin is determined to be E’HWSYWLRPG’,of which the N-terminal is pyroglutamic acid (E’) and the C-termianl is glycinamide (G’).The primary structure of unknown peptide was identified to be T’VSP VWLPPSVY by sequence docking method,of which the N-terminal occurs threonine phosphorylation (T’) and the foruth proline is added with sodium ion (P).The data suggested that electrospray Tandem mass spectrometry technique have obvious advantage in analyzing the full sequence of modified or unknown peptides.
文摘OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.
文摘Of the eleven families of cyclic nucleotide phosphodiesterases(PDEs) present in the human body,PDE4s represent the most widely expressed family of PDEs. A large body of work has been published on the expression and function of these PDEs,which preferentially hydrolyze cAMP in all cells studied,including neurons and supporting cells of the CNS. Four distinct genes termed PDE4 A,PDE4B,PDE4C and PDE4D encode PDE4 proteins. However,the number of PDE4s identified in different tissues and cells is estimated to be more than 30. Differences in regulation and localization explain this extreme heterogeneity. PDE4 hydrolytic activity is regulated by phosphorylation,and protein kinase A(PKA) was the first kinase identified. This PKA-dependent regulation establishes a feedback loop where cAMP regulates its own degradation to control the intensity and localization of the hormone and neurotransmitter signal. In addition,numerous additional kinases phosphorylate PDE4s to modulate the PKA-dependent activation and fine tune cAMP levels by growth factors and other extracellular cues. Thus,PDE4 can be considered a coincidence detector that integrates multiple signaling pathways. Finally,different PDE4s are involved in numerous macromolecular complexes targeting the cAMP hydrolytic activity to different subcellular domains. Thus,PDE4s function in different subcellular compartments,and inhibition of different isoforms affects cAMP levels in different subdomains with consequently different functions. The dyad space and the control of excitation/contraction will be used as examples of these localized regulations.
基金Supported by Major State Basic Research Development Program of China(973 Program,2011CB100804)
文摘Prolactin （PRL） is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be necessary for high levels of milk protein expression, the mechanism by which the synthesis of milk proteins is stimulated at the transcript level is less known. A major modification in the transcript level is protein phosphorylation. To gain additional insights into the molecular mechanisms at the transcript level underlying PRL action on the dairy cow mammary epithelial cells （DCMECs）, nuclear phosphoproteins whose expression distinguishes proliferating regulated by PRL in DCMECs were identified. A phosphoprotein-enriched fraction from nuclear proteins was obtained by affinity chromatography, and a two-dimensional gel electrophoresis （2-DE） and matrix assisted laser desorption/ionization time of matrix-assisted laser desorption/ionization/time of flight mass spectrometry （MALDI-TOF MS） were used to identify the changes of nuclear phosphoproteins in DCMECs treated with prolactin. Seven proteins displaying~〉2-fold difference in abundance upon PRL treatment in DCMECs were identified by MALDI-TOF MS. The protein-GARS （GlyRS）, which belonged to the class-II aminoacyl-tRNA synthetase family, played a global role in the milk protein synthesis. SERPINH1 （Heat shock protein 47）, which was the first heat shock protein found to be a member of the serpin superfamily, regulated physiologic functions, such as complement activation, programmed cell death, and inflammatory processes. PRDX3, which belonged to a family of antioxidant enzymes, played an important role in scavenging intracellular reactive oxygen species （ROS）. ACTR1A, belonged to the actin family, which was associated with transport of p53 to the nucleus. Annexin A2, a Ca2＋-dependent phospholipid-binding protein, maintained the viability and cell cycle regulation of DCMECs. PSMB2 and PSMD10, which belonged to ubiquitin-proteasome system, were involved in several cellular processes, including cell cycle control, cellular stress response, intracellular signaling. This screening revealed that prolactin influenced the level of nuclear phosphoproteins in DCMECs. This result opens new avenues for the study of the molecular mechanism linked to the synthesis of milk proteins.
