Microtubule arrays in prothalli large-vacuolated and meristematic dividing cells of the fern Dryopteris crassirhizoma Nakai were studied using Steedman's wax, indirect immunofluorescence labelling and confocal las...Microtubule arrays in prothalli large-vacuolated and meristematic dividing cells of the fern Dryopteris crassirhizoma Nakai were studied using Steedman's wax, indirect immunofluorescence labelling and confocal laser scanning microscopy. Results showed that the use of high paraformaldehyde concentration (8%) allowed good fixation of prothallus cells, which are characterized by numerous (meristematic cells) and big (large-vacuolated cells) vacuoles. Results also plead for the efficiency of Steedman's wax embedding method in: (1) avoiding excessive use of enzyme for digesting cell wall in the process of the microtubule cytoskeleton labelling, (2) minimizing the autofluorescence effect in cells through utilization of alcohol in sample dehydration, and (3) permitting a clear visualization of microtubule patterns during the cell mitosis. Steedman's wax, coupled with immunofluorescence labelling and confocal laser scanning microscopy techniques, allows a good investigation of cell division process in plants by using simple multicellular organisms such as fern prothalli.展开更多
AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from...AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P < 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P < 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.展开更多
文摘Microtubule arrays in prothalli large-vacuolated and meristematic dividing cells of the fern Dryopteris crassirhizoma Nakai were studied using Steedman's wax, indirect immunofluorescence labelling and confocal laser scanning microscopy. Results showed that the use of high paraformaldehyde concentration (8%) allowed good fixation of prothallus cells, which are characterized by numerous (meristematic cells) and big (large-vacuolated cells) vacuoles. Results also plead for the efficiency of Steedman's wax embedding method in: (1) avoiding excessive use of enzyme for digesting cell wall in the process of the microtubule cytoskeleton labelling, (2) minimizing the autofluorescence effect in cells through utilization of alcohol in sample dehydration, and (3) permitting a clear visualization of microtubule patterns during the cell mitosis. Steedman's wax, coupled with immunofluorescence labelling and confocal laser scanning microscopy techniques, allows a good investigation of cell division process in plants by using simple multicellular organisms such as fern prothalli.
基金Supported by The National Natural Science Foundation of China,No. 30900666the Programs of Department of Health of Jiangsu Province,No. H201061
文摘AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P < 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P < 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.
