Two new aequorin genes,aeqxm and aeqxxm,were isolated from jellyfish Aequorea macrodactyla and Aequorea parva respectively,which are commonly found in the warmer waters on the coastal region of the East China Sea.The ...Two new aequorin genes,aeqxm and aeqxxm,were isolated from jellyfish Aequorea macrodactyla and Aequorea parva respectively,which are commonly found in the warmer waters on the coastal region of the East China Sea.The DNA sequences of the two genes have no introns and each one contains an ORF of 585 bp in full-length encoding a 195-aa protein.The two genes of aeqxm and aeqxxm share nucleotide homologies of 80.7%and 85.1%with AEVAQ440X respectively,and the corresponding proteins share amino acid homologies of 84.7%and 84.2%with AEVAQ440X.High amino acid homology was found between apoaeqxm and apoaeqxxm.The two genes were cloned into expression vector pTO-T7 respectively,and the expression yields amounted to 40%of the total protein in E.coli BL21.The activities of the two photoproteins were reconstituted by incubating the expressed apoproteins with coelenterazine f.In the presence of Ca ion,both of the regenerated aeqxm and aeqxxm exhibited an emission peak at the wave length of 470 nm.展开更多
Calcium ion is a versatile second messenger for diverse cell signaling in response to developmental and environmental cues. The specificity of Ca^2＋-mediated signaling is defined by stimulus-elicited Ca^2＋ signature...Calcium ion is a versatile second messenger for diverse cell signaling in response to developmental and environmental cues. The specificity of Ca^2＋-mediated signaling is defined by stimulus-elicited Ca^2＋ signature and down-stream decoding processes. Here, an Aequorin-based luminescence recording system was developed for monitoring Ca^2＋ in response to various stimuli in Arabidopsis. With the simple, highly sensitive, and robust Ca^2＋ recording, this system revealed stimulus-and tissue-specific Ca^2＋ signatures in seedlings. Cellular Ca^2＋ dynamics and relationship to Aequorin-based Ca^2＋ recording were explored using a GFP-based Ca^2＋ indicator, which suggested that a synchronous cellular Ca^2＋ signal is responsible for cold-induced Ca^2＋ response in seedlings, whereas asynchronous Ca^2＋ oscillation contributes to osmotic stress-induced Ca^2＋ increase in seedlings. The optimized recording system would be a powerful tool for the iden-tification and characterization of novel components in Ca^2＋ -mediated stress-signaling pathways,展开更多
In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent repo...In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the biolumines- cent Ca2＋ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation withf-coelenterazine. The temporal nature of the Ca2＋ signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca2＋ transients （generated by re- lease from intracellular stores） were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KC1 or CaC12, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor （IP3R） agonist, small Ca2＋transients were generated from day 1 onward. That ATP was inducing Ca2＋ release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor （RyR） agonist, a minima/Ca2＋ response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.展开更多
The present study was designed to establish a suitable assay to explore CCR2 b receptor antagonists from the natural products of Artemisia rupetris and Leontopodium leontopodioides. An aequorin assay was developed as ...The present study was designed to establish a suitable assay to explore CCR2 b receptor antagonists from the natural products of Artemisia rupetris and Leontopodium leontopodioides. An aequorin assay was developed as a cell-based assay suitable for 384-well microplate and used for screening CCR2 b receptor antagonists from natural products. Through establishing suitable conditions, the assay was shown to be suitable for screening of CCR2 b receptor antagonists. Seven compounds were identified in preliminary screening. Five of them showed evident dose-response relationship in secondary screening. The structure–activity relationship study suggested that 7-position hydroxyl group of flavonoids was necessary, a polar group should be introduced on the 3-position, and the substituents on 2-position benzene ring of flavonoids have little influence on the potentency of the inhibition activity on CCR2 b receptor. The ortho-position dihydroxyl structure in quinic acid compounds may be important. In conclusion, Compounds HR-1, 5, 7, and AR-20, 35 showed activity as antagonist of CCR2 b receptor, which shed lights on the development of novel drugs as CCR2 b receptor antagonists for preventing inflammation related diseases.展开更多
Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases an...Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death （PCD） in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species （ROS） has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium （DPI）, indicating the involvement of NADPH oxidase（s） in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum （La^3＋）. Therefore, ROS production occurs downstream of DHS-induced Ca^2＋ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La^3＋ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.展开更多
Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2＋ levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism o...Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2＋ levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants＇ responses to Ca2＋ depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca2＋ （[Ca2＋]ext） depletion-responsive genes in Arabidopsis thaliana L., many of which are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca2＋ （[Ca2＋]cyt） sensors were significantly upregulated, implying that [Ca2＋]cyt has a role in sensing [Ca2＋]ext depletion. Consistent with this observation, [Ca2＋]ext depletion stimulated a transient rise in [Ca2＋]cyt that was negatively influenced by [K＋]ext, suggesting the involvement of a membrane potential-sensitive component. The [Ca2＋]cyt response to [Ca2＋]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca2＋ sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd3＋, an inhibitor of Ca2＋ channels, suppressed the [Ca2＋]ext-triggered rise in [Ca2＋]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca2＋]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca2＋]ext depletion of plant cells.展开更多
基金This work was supported in parts by Hi-tech Research and Development Programme of China("863"Programme)under contract No.819-Q-06the National Natural Science Foundation of China under contract No.C01040101 and the Natural Science Foundation of Fujian of China under cotract No.C0010001
文摘Two new aequorin genes,aeqxm and aeqxxm,were isolated from jellyfish Aequorea macrodactyla and Aequorea parva respectively,which are commonly found in the warmer waters on the coastal region of the East China Sea.The DNA sequences of the two genes have no introns and each one contains an ORF of 585 bp in full-length encoding a 195-aa protein.The two genes of aeqxm and aeqxxm share nucleotide homologies of 80.7%and 85.1%with AEVAQ440X respectively,and the corresponding proteins share amino acid homologies of 84.7%and 84.2%with AEVAQ440X.High amino acid homology was found between apoaeqxm and apoaeqxxm.The two genes were cloned into expression vector pTO-T7 respectively,and the expression yields amounted to 40%of the total protein in E.coli BL21.The activities of the two photoproteins were reconstituted by incubating the expressed apoproteins with coelenterazine f.In the presence of Ca ion,both of the regenerated aeqxm and aeqxxm exhibited an emission peak at the wave length of 470 nm.
文摘Calcium ion is a versatile second messenger for diverse cell signaling in response to developmental and environmental cues. The specificity of Ca^2＋-mediated signaling is defined by stimulus-elicited Ca^2＋ signature and down-stream decoding processes. Here, an Aequorin-based luminescence recording system was developed for monitoring Ca^2＋ in response to various stimuli in Arabidopsis. With the simple, highly sensitive, and robust Ca^2＋ recording, this system revealed stimulus-and tissue-specific Ca^2＋ signatures in seedlings. Cellular Ca^2＋ dynamics and relationship to Aequorin-based Ca^2＋ recording were explored using a GFP-based Ca^2＋ indicator, which suggested that a synchronous cellular Ca^2＋ signal is responsible for cold-induced Ca^2＋ response in seedlings, whereas asynchronous Ca^2＋ oscillation contributes to osmotic stress-induced Ca^2＋ increase in seedlings. The optimized recording system would be a powerful tool for the iden-tification and characterization of novel components in Ca^2＋ -mediated stress-signaling pathways,
基金supported by the Hong Kong Theme-based Research Scheme award(T13-706/11-1)the Hong Kong Research Grants Council(RGC)General Research Fund awards(662113,16101714,16100115)+2 种基金the ANR/RGC joint research scheme award(A-HKUST601/13)the Innovation and Technology Commission(ITCPD/17-9)supported by a Hong Kong University Grants Council post-graduate studentship(T13-706/11-11PG)
文摘In order to develop a novel method of visualizing possible Ca2＋ signaling during the early differentiation of hESCs into cardi- omyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the biolumines- cent Ca2＋ reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation withf-coelenterazine. The temporal nature of the Ca2＋ signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca2＋ transients （generated by re- lease from intracellular stores） were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KC1 or CaC12, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor （IP3R） agonist, small Ca2＋transients were generated from day 1 onward. That ATP was inducing Ca2＋ release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor （RyR） agonist, a minima/Ca2＋ response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.
基金supported by the Major Scientific and Technological Special Project for Significant New Drugs Creation(No.2012ZX09504001-001)National Natural Science Foundation of China(No.81102876+2 种基金81430082)the Fundamental Research Funds for the Central Universities(No.2015ZD005)333 high level project of Jiangsu Province(NO.BRA2014245)
文摘The present study was designed to establish a suitable assay to explore CCR2 b receptor antagonists from the natural products of Artemisia rupetris and Leontopodium leontopodioides. An aequorin assay was developed as a cell-based assay suitable for 384-well microplate and used for screening CCR2 b receptor antagonists from natural products. Through establishing suitable conditions, the assay was shown to be suitable for screening of CCR2 b receptor antagonists. Seven compounds were identified in preliminary screening. Five of them showed evident dose-response relationship in secondary screening. The structure–activity relationship study suggested that 7-position hydroxyl group of flavonoids was necessary, a polar group should be introduced on the 3-position, and the substituents on 2-position benzene ring of flavonoids have little influence on the potentency of the inhibition activity on CCR2 b receptor. The ortho-position dihydroxyl structure in quinic acid compounds may be important. In conclusion, Compounds HR-1, 5, 7, and AR-20, 35 showed activity as antagonist of CCR2 b receptor, which shed lights on the development of novel drugs as CCR2 b receptor antagonists for preventing inflammation related diseases.
文摘Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death （PCD） in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species （ROS） has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium （DPI）, indicating the involvement of NADPH oxidase（s） in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum （La^3＋）. Therefore, ROS production occurs downstream of DHS-induced Ca^2＋ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La^3＋ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.
基金supported by the Program for New Century Excellent Talents in University from the Ministry of Education (NCET-10-0906)the Major Basic Science Research Open Program from the Inner Mongolia Science and Technology DepartmentProgram for Innovative Research Team in Universities of Inner Mongolia Autonomous Region for Z. Qi
文摘Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2＋ levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants＇ responses to Ca2＋ depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca2＋ （[Ca2＋]ext） depletion-responsive genes in Arabidopsis thaliana L., many of which are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca2＋ （[Ca2＋]cyt） sensors were significantly upregulated, implying that [Ca2＋]cyt has a role in sensing [Ca2＋]ext depletion. Consistent with this observation, [Ca2＋]ext depletion stimulated a transient rise in [Ca2＋]cyt that was negatively influenced by [K＋]ext, suggesting the involvement of a membrane potential-sensitive component. The [Ca2＋]cyt response to [Ca2＋]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca2＋ sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd3＋, an inhibitor of Ca2＋ channels, suppressed the [Ca2＋]ext-triggered rise in [Ca2＋]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca2＋]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca2＋]ext depletion of plant cells.
