Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in viv...Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.展开更多
In this study, a novel approach to measure the absolute cytoplasmic Caconcentration([Ca]cyt) using the Caindicator fluo-3 AM was established. The parameters associated with the probe fluo-3 AM were optimized to accura...In this study, a novel approach to measure the absolute cytoplasmic Caconcentration([Ca]cyt) using the Caindicator fluo-3 AM was established. The parameters associated with the probe fluo-3 AM were optimized to accurately determine fluorescence intensity from the Ca-bound probe. Using three optimized parameters(final concentration of 6 m M probe,incubation time of 135 min, loading probe before plasma treatment), the maximum fluorescence intensity(F?=?527.8 a.u.) and the minimum fluorescence intensity(F?=?63.8 a.u.) were obtained in a saturated Casolution or a solution of lacking Ca. Correspondingly, the maximum [Ca]cytinduced by cold plasma was 1232.5 n M. Therefore, the Caindicator fluo-3 AM was successfully applied to measure the absolute [Ca]cytin Saccharomyces cerevisiae stimulated by cold plasma at atmospheric air pressure.展开更多
Using the method of dual-wavelength measurement of platelet [Ca2+]i, and Fura-2 as the Ca2+ fluorophore probe, we measured the effect of acidic Mu-copolysaccharide from Sticopus Japonicus Selenka (SJAMP) on platelet [...Using the method of dual-wavelength measurement of platelet [Ca2+]i, and Fura-2 as the Ca2+ fluorophore probe, we measured the effect of acidic Mu-copolysaccharide from Sticopus Japonicus Selenka (SJAMP) on platelet [Ca2+]i. The results showed that the most significant increase in platelets [Ca2+]i was seen when the concentration of SJAMP was 100μg/ml and the elevation of normal platelet [Ca2+]i was 93. 96±10. 24 nmol/L (n = 10). In the presence of extracellular Ca2+(1 mmol/L), the magnitude of platelet [Ca2+]i response to SJAMP was increased and the [Ca2+]i, could reach 116. 72 + 10. 66 nmol/L (n= 10). On the other hand, the magnitude of increased platelet [Ca2+]i; induced by SJAMP was smaller and the duration of [Ca2+]i reaching the highest level was longer when compared with other platelet aggregation agents. In the mean time, if platelets were first incubated with cyclooxygenase inhibitor, the rise of [Ca2+]i evoked by SJAMP was inhibited. The results indicated that the mechanism of the rise of [Ca2+]i induced by SJAMP might be dependent upon the generation of prostaglandin endoperoxides and (or) TXA?.展开更多
BK channels are widely expressed in both excitable and non-excitable cells and known to be involved in many physiological processes,such as vascular smooth tone regulation,neuronal firing and endocrine cell secretion[...BK channels are widely expressed in both excitable and non-excitable cells and known to be involved in many physiological processes,such as vascular smooth tone regulation,neuronal firing and endocrine cell secretion[1].Recently, the BK channels have展开更多
基金supported by the Natural Science Foundation of Fujian Province,No.2020J02027the National Natural Science Foundation of China,No.31970461the Foundation of NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate,Fujian Maternity and Child Health Hospital,No.2022-NHP-05(all to WC).
文摘Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.
基金supported by National Natural Science Foundation of China (Grant Nos. 21246012, 21306015 and 21476032)
文摘In this study, a novel approach to measure the absolute cytoplasmic Caconcentration([Ca]cyt) using the Caindicator fluo-3 AM was established. The parameters associated with the probe fluo-3 AM were optimized to accurately determine fluorescence intensity from the Ca-bound probe. Using three optimized parameters(final concentration of 6 m M probe,incubation time of 135 min, loading probe before plasma treatment), the maximum fluorescence intensity(F?=?527.8 a.u.) and the minimum fluorescence intensity(F?=?63.8 a.u.) were obtained in a saturated Casolution or a solution of lacking Ca. Correspondingly, the maximum [Ca]cytinduced by cold plasma was 1232.5 n M. Therefore, the Caindicator fluo-3 AM was successfully applied to measure the absolute [Ca]cytin Saccharomyces cerevisiae stimulated by cold plasma at atmospheric air pressure.
基金This project was supported by grant from the National Nature Science Foundation of China (No.39370322)
文摘Using the method of dual-wavelength measurement of platelet [Ca2+]i, and Fura-2 as the Ca2+ fluorophore probe, we measured the effect of acidic Mu-copolysaccharide from Sticopus Japonicus Selenka (SJAMP) on platelet [Ca2+]i. The results showed that the most significant increase in platelets [Ca2+]i was seen when the concentration of SJAMP was 100μg/ml and the elevation of normal platelet [Ca2+]i was 93. 96±10. 24 nmol/L (n = 10). In the presence of extracellular Ca2+(1 mmol/L), the magnitude of platelet [Ca2+]i response to SJAMP was increased and the [Ca2+]i, could reach 116. 72 + 10. 66 nmol/L (n= 10). On the other hand, the magnitude of increased platelet [Ca2+]i; induced by SJAMP was smaller and the duration of [Ca2+]i reaching the highest level was longer when compared with other platelet aggregation agents. In the mean time, if platelets were first incubated with cyclooxygenase inhibitor, the rise of [Ca2+]i evoked by SJAMP was inhibited. The results indicated that the mechanism of the rise of [Ca2+]i induced by SJAMP might be dependent upon the generation of prostaglandin endoperoxides and (or) TXA?.
基金supported by Natural Science Foundation of China grants10732070,10602031
文摘BK channels are widely expressed in both excitable and non-excitable cells and known to be involved in many physiological processes,such as vascular smooth tone regulation,neuronal firing and endocrine cell secretion[1].Recently, the BK channels have
