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
Objective To investigate changes in the expression of sarcoplamic reticular Ca 2+ ATPase (SERCA) and IP 3 I receptors (IP 3R 1) mRNA in patients with atrial fibrillation Methods Thirty eight patients wi...Objective To investigate changes in the expression of sarcoplamic reticular Ca 2+ ATPase (SERCA) and IP 3 I receptors (IP 3R 1) mRNA in patients with atrial fibrillation Methods Thirty eight patients with mitral stenosis undergoing open heart surgery were studied 100 mg of atrial tissue was obtained during surgery from the right appendage and the right atrium The amount of messenger ribonucleic acid (mRNA) amount of SERCA and IP 3R 1 was measured by reverse transcription polymerase chain reaction (RT PCR) and normalized to the mRNA levels of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) Results Levels of mRNA expression of SERCA in patients with AF, as compared with subjects in sinus rhythm, was lower and that of IP 3R 1 was higher The longer AF was sustained, the higher the levels of mRNA There was no significant difference between right atrial free wall and right appendage Conclusions The expression changes of SERCA and IP3R mRNA may correlate with the initiation or maintenance of AF展开更多
The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the...The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells(NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3 r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor(IP3 R) type-1 and ryanodine receptor(RyR) type-2 are differentially expressed in cells with neuron-or radial glial-like properties. Furthermore, ATP but not caffeine(IP3 R and RyR agonists, respectively), induced the generation of Ca^(2+) transients in cells exhibiting neuron-or glial-like morphology. These results indicate the differential expression of components of the Ca^(2+) -signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca^(2+) signaling as a read-out.展开更多
The Atri intracellular calcium oscillations model was extended, and two new models were established. Furthermore, a unified model of the protein phosphorylation driven by cytosolic calcium oscillations was constructed...The Atri intracellular calcium oscillations model was extended, and two new models were established. Furthermore, a unified model of the protein phosphorylation driven by cytosolic calcium oscillations was constructed. The numerical results obtained verified related experimental conclusions. And the analytical expressions of intracellular calcium spiral and target waves in the Xenopus laevis oocyte were obtained, resulting in velocity and waveform of calcium solitary pulse wave were found.展开更多
Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate(IP3)-induced Ca2+release(IICR)via IP3R1,but the mechanism remains largely unexplored.Using extracellular ATP to induce intracellular cal...Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate(IP3)-induced Ca2+release(IICR)via IP3R1,but the mechanism remains largely unexplored.Using extracellular ATP to induce intracellular calcium transient as an IICR model,Ca2+image,pull down assay,and Western blotting experiments were carried out in the present study.We found that extracellular ATP induced calcium transient via IP3Rs(IICR)and the IICR were markedly decreased in ERp44 overexpressed Hela cells.The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331–377)mutants of ERp44 on IICR were significantly decreased compared with ERp44.However,the binding capacity of ERp44 to L3V domain of IP3R1(1L3V)was enhanced by ERp44 C160S/C212S mutation.Taken together,these results suggest that the mutants of ERp44,C160/C212,can more tightly bind to IP3R1 but exhibit a weak inhibition of IP3R1 channel activity in Hela cells.展开更多
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.
文摘Objective To investigate changes in the expression of sarcoplamic reticular Ca 2+ ATPase (SERCA) and IP 3 I receptors (IP 3R 1) mRNA in patients with atrial fibrillation Methods Thirty eight patients with mitral stenosis undergoing open heart surgery were studied 100 mg of atrial tissue was obtained during surgery from the right appendage and the right atrium The amount of messenger ribonucleic acid (mRNA) amount of SERCA and IP 3R 1 was measured by reverse transcription polymerase chain reaction (RT PCR) and normalized to the mRNA levels of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) Results Levels of mRNA expression of SERCA in patients with AF, as compared with subjects in sinus rhythm, was lower and that of IP 3R 1 was higher The longer AF was sustained, the higher the levels of mRNA There was no significant difference between right atrial free wall and right appendage Conclusions The expression changes of SERCA and IP3R mRNA may correlate with the initiation or maintenance of AF
基金supported by the ANR/RGC Joint Research Scheme Award (A-HKUST601/ 13)the HK RGC General Research Fund awards (662113, 16101714, 16100115)Funding from the HKITC (ITCPD/17-9)
文摘The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells(NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3 r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor(IP3 R) type-1 and ryanodine receptor(RyR) type-2 are differentially expressed in cells with neuron-or radial glial-like properties. Furthermore, ATP but not caffeine(IP3 R and RyR agonists, respectively), induced the generation of Ca^(2+) transients in cells exhibiting neuron-or glial-like morphology. These results indicate the differential expression of components of the Ca^(2+) -signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca^(2+) signaling as a read-out.
文摘The Atri intracellular calcium oscillations model was extended, and two new models were established. Furthermore, a unified model of the protein phosphorylation driven by cytosolic calcium oscillations was constructed. The numerical results obtained verified related experimental conclusions. And the analytical expressions of intracellular calcium spiral and target waves in the Xenopus laevis oocyte were obtained, resulting in velocity and waveform of calcium solitary pulse wave were found.
基金by grants from the National Basic Research Program of China(Grant Nos.2011CB8091004 and 2009CB918701)the National Natural Science Foundation of China(Grant No.81100539).
文摘Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate(IP3)-induced Ca2+release(IICR)via IP3R1,but the mechanism remains largely unexplored.Using extracellular ATP to induce intracellular calcium transient as an IICR model,Ca2+image,pull down assay,and Western blotting experiments were carried out in the present study.We found that extracellular ATP induced calcium transient via IP3Rs(IICR)and the IICR were markedly decreased in ERp44 overexpressed Hela cells.The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331–377)mutants of ERp44 on IICR were significantly decreased compared with ERp44.However,the binding capacity of ERp44 to L3V domain of IP3R1(1L3V)was enhanced by ERp44 C160S/C212S mutation.Taken together,these results suggest that the mutants of ERp44,C160/C212,can more tightly bind to IP3R1 but exhibit a weak inhibition of IP3R1 channel activity in Hela cells.
