Changes of calmodulin (CaM) distribution in the embryo sac of rice (Oryza sativa subsp. Japonica) at various stages before and after fertilization have been investigated by using immunogold electron microscopy. Before...Changes of calmodulin (CaM) distribution in the embryo sac of rice (Oryza sativa subsp. Japonica) at various stages before and after fertilization have been investigated by using immunogold electron microscopy. Before pollination, both cytoplasm and vacuoles of the egg cell, synergids and central cell were labeled by gold particles. A small amount of gold particles were localized in the nucleus, endoplasmic reticulum, mitochondria and dictyosomes. From pollination to fertilization, CaM amount increased in these cells, especially rich in the starch of amyloplasts. Increase of gold particles in the central cell began about 2 h earlier than that in the egg cell. There was no distinct difference of CaM amount between the degenerated and the persistent synergids. It is interesting to observe an obvious change of CaM distribution form during pollination and fertilization from scattered single particles to clustered particles, and back again to single particles after the fertilization finished. CaM was also localized extracellularly in the embryo sac wall as well as in the wall and intercellular space of nucellus cells. The extracellular CaM also changes in its amount and form after pollination. These results suggest that CaM, either intra- or extra-cellular, may play important roles in fertilization and zygote formation.展开更多
An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to smal...An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.展开更多
Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized i...Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall. In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic reticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its pleiotropic functions in plant cellular activities.展开更多
Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide （H2O2）, calcium （Ca^2＋）-calmodulin （CAM）, and nitric oxide （NO） in abscisic acid （ABA）-induced antioxidant ...Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide （H2O2）, calcium （Ca^2＋）-calmodulin （CAM）, and nitric oxide （NO） in abscisic acid （ABA）-induced antioxidant defense were investigated in leaves of maize （Zea mays L.） plants. Treatments with ABA, H2O2, and CaCl2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. However, such increases were blocked by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca^2＋-induced increase in the production of NO. On the other hand, treatments with ABA and the NO donor sodium nitroprusside （SNP） also led to increases in the concentration of cytosolic Ca^2＋ in protoplasts of mesophyll cells and in the expression of calmodulin 1 （CaM1） gene and the contents of CaM in leaves of maize plants, and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor. Moreover, SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 （SOD4）, cytosolic ascorbate peroxidase （cAPX）, and glutathione reductase 1 （GR1） and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Our results suggest that Ca^2＋-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.展开更多
Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to pl...Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.展开更多
AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immun...AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.展开更多
In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chai...In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.展开更多
Sharp eyespot,mainly caused by the soil-borne fungus Rhizoctonia cerealis,affects wheat(Triticum aestivum L.)production worldwide.In this study,we isolated TaCML36 gene encoding a wheat calmodulin-like protein,and stu...Sharp eyespot,mainly caused by the soil-borne fungus Rhizoctonia cerealis,affects wheat(Triticum aestivum L.)production worldwide.In this study,we isolated TaCML36 gene encoding a wheat calmodulin-like protein,and studied its defense role in protection against R.cerealis.Transcription of TaCML36 was significantly elevated by both R.cerealis infection and exogenous ethylene treatment.Transcription was higher in resistant wheat lines than in susceptible ones.There were copies of TaCML36 on chromosomes 5A,5B,and 5D.The TaCML36 protein is composed of 183 amino acids and contains two calcium-binding EFhand domains.Subcellular localization assays in wheat indicated that TaCML36 localizes in both the cytoplasm and nucleus.Virus-induced gene silencing and disease assessment indicated that compared to the controls,TaCML36-silenced wheat plants displayed significantly reduced resistance to R.cerealis and had greater fungal biomass,suggesting that knockdown of TaCML36 impaired host resistance.Knockdown of TaCML36 also significantly repressed expression of pathogenesis-related genes such as Chitinase 1,PDF35,and PR17C,the ethylene response factor-encoding gene TaPIE1,and ethylene biosynthesis gene ACO2.Collectively,our results suggest that TaCML36 positively participates in the innate immune response to R.cerealis infection by modulating expression of defense-associated genes possibly in the ethylene signaling pathway.展开更多
Calmodulin is a ubiquitous calcium sensor to recognize the different developmental and/or stimulus-triggered calcium changes and regulate plant growth and development.However,the function of calmodulin remains elusive...Calmodulin is a ubiquitous calcium sensor to recognize the different developmental and/or stimulus-triggered calcium changes and regulate plant growth and development.However,the function of calmodulin remains elusive for fleshy fruit development.We performed expression studies of a family of six calmodulin genes(SlCaMs)in tomato fruit.All calmodulins showed a double peak expression pattern.The first flat peak appeared at 10–30 days after anthesis,but their expression rapidly declined at mature green and breaker.Then a sharp and even higher peak came at turning/pink stages.Among six calmodulins,SlCaM1 had the highest expression during fruit enlargement,whereas SlCaM2 was the major calmodulin during fruit ripening.However,SlCaMs showed different patterns in three ripening mutants rin,Nor and Nr.In particular,at the stages corresponding to mature green and breaker,the expression levels of SlCaMs in those mutants were significantly higher than wild-type.Furthermore,SlCaMs,especially SlCaM2 were upregulated by ethylene.Transiently overexpressing SlCaM2 in mature green fruit delayed ripening,while reducing SlCaM2 expression accelerated ripening.Our results suggest that SlCaMs play double roles to regulate fruit ripening.Prior to the ethylene burst,the ethylene-independent repression of SlCaMs might be critical for fruit to initiate the ripening process.After the ethylene burst,SlCaMs could participate in the ethylene coordinated rapid ripening.展开更多
Plant calmodulins(CaMs)and calmodulin-like proteins(CMLs)mediate Ca~(2+)signaling in response to abiotic stresses.Manipulation of this signaling in crops could increase stress tolerance.We review methods for detecting...Plant calmodulins(CaMs)and calmodulin-like proteins(CMLs)mediate Ca~(2+)signaling in response to abiotic stresses.Manipulation of this signaling in crops could increase stress tolerance.We review methods for detecting Ca~(2+)signals,regulatory roles of Ca Ms and CMLs,binding targets,and Ca~(2+)networks under abiotic stress in organelles.展开更多
Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to de...Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.展开更多
In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyI-D-asparti...In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyI-D-aspartic acid-induced injury. Results showed that, compared with N-methyi-D- aspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca2＋ concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 pM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin- dependent protein kinase II expression.展开更多
Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal ne...Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.展开更多
文摘Changes of calmodulin (CaM) distribution in the embryo sac of rice (Oryza sativa subsp. Japonica) at various stages before and after fertilization have been investigated by using immunogold electron microscopy. Before pollination, both cytoplasm and vacuoles of the egg cell, synergids and central cell were labeled by gold particles. A small amount of gold particles were localized in the nucleus, endoplasmic reticulum, mitochondria and dictyosomes. From pollination to fertilization, CaM amount increased in these cells, especially rich in the starch of amyloplasts. Increase of gold particles in the central cell began about 2 h earlier than that in the egg cell. There was no distinct difference of CaM amount between the degenerated and the persistent synergids. It is interesting to observe an obvious change of CaM distribution form during pollination and fertilization from scattered single particles to clustered particles, and back again to single particles after the fertilization finished. CaM was also localized extracellularly in the embryo sac wall as well as in the wall and intercellular space of nucellus cells. The extracellular CaM also changes in its amount and form after pollination. These results suggest that CaM, either intra- or extra-cellular, may play important roles in fertilization and zygote formation.
文摘An in situ hybridization technique for localization of calmodulin(CaM) mRNA in isolated entire embryo sacs and proembryos in Nicotiana tabacum L.cv.W38 has been developed. This technique can be applied to small amounts of materials in which a whole view of CaM mRNA distribution can be obtained. The authors revealed that CaM mRNA expression changes dramatically before and after fertilization. Especially interesting is that a prominent CaM mRNA band appears between the egg apparatus and polar nuclei temporarily during the period of pollination and fertilization. The band disappears just prior to fertilization and expands to a fan_shaped region that occupies the micropylar portion of the embryo sac. After fertilization, CaM mRNA accumulates in the elongated zygotes with higher concentration in their chalazal portion than in the micropylar portion. Such an asymmetrical pattern continues to manifest in the early proembryos. It is supposed that CaM mRNA may be involved in the early events and signaling steps associated with double fertilization and zygote polarization in higher plants.
文摘Methods for the localization of plant calmodulin by immuno-gold and immuno-peroxidase electron microscopy have been developed. In both corn root-cap cells and meristematic cells, calmodulin was found to be localized in the nucleus, cytoplasm, mitochondria as well as in the cell wall. In the meristematic cells, calmodulin was distinctly localized on the plasma membrane, cytoplasmic face of rough endoplasmic reticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its pleiotropic functions in plant cellular activities.
基金Acknowledgments This work was supported by the Major State Basic Research Program of China （grant no. 2003CB 114302 to M Jiang）, the National Natural Science Foundation of China （grant no. 30571122 to M Jiang）, and the Youth Scientific and Technological Innovation talent Project of Jiangsu Province （grant no. BK2007575 to A Zhang）.
文摘Using pharmacological and biochemical approaches, the signaling pathways between hydrogen peroxide （H2O2）, calcium （Ca^2＋）-calmodulin （CAM）, and nitric oxide （NO） in abscisic acid （ABA）-induced antioxidant defense were investigated in leaves of maize （Zea mays L.） plants. Treatments with ABA, H2O2, and CaCl2 induced increases in the generation of NO in maize mesophyll cells and the activity of nitric oxide synthase （NOS） in the cytosolic and microsomal fractions of maize leaves. However, such increases were blocked by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Meanwhile, pretreatments with two NOS inhibitors also suppressed the Ca^2＋-induced increase in the production of NO. On the other hand, treatments with ABA and the NO donor sodium nitroprusside （SNP） also led to increases in the concentration of cytosolic Ca^2＋ in protoplasts of mesophyll cells and in the expression of calmodulin 1 （CaM1） gene and the contents of CaM in leaves of maize plants, and the increases induced by ABA were reduced by the pretreatments with a NO scavenger and a NOS inhibitor. Moreover, SNP-induced increases in the expression of the antioxidant genes superoxide dismutase 4 （SOD4）, cytosolic ascorbate peroxidase （cAPX）, and glutathione reductase 1 （GR1） and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by the pretreatments with Ca^2＋ inhibitors and CaM antagonists. Our results suggest that Ca^2＋-CaM functions both upstream and downstream of NO production, which is mainly from NOS, in ABA- and H2O2-induced antioxidant defense in leaves of maize plants.
基金the Natural Science Fundation of Jiangsu Province,№BK95141307
文摘Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.
基金Supported by the National Natural Science Foundation of China,No.81302131
文摘AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.
基金supported by the 863 Project (Nos. 2002AA628120, 2004AA628090, 20060110A4013 and 2006AA10A413)
文摘In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.
基金funded by the National “Key Sci-Tech” Project (2016ZX08002-001-004)
文摘Sharp eyespot,mainly caused by the soil-borne fungus Rhizoctonia cerealis,affects wheat(Triticum aestivum L.)production worldwide.In this study,we isolated TaCML36 gene encoding a wheat calmodulin-like protein,and studied its defense role in protection against R.cerealis.Transcription of TaCML36 was significantly elevated by both R.cerealis infection and exogenous ethylene treatment.Transcription was higher in resistant wheat lines than in susceptible ones.There were copies of TaCML36 on chromosomes 5A,5B,and 5D.The TaCML36 protein is composed of 183 amino acids and contains two calcium-binding EFhand domains.Subcellular localization assays in wheat indicated that TaCML36 localizes in both the cytoplasm and nucleus.Virus-induced gene silencing and disease assessment indicated that compared to the controls,TaCML36-silenced wheat plants displayed significantly reduced resistance to R.cerealis and had greater fungal biomass,suggesting that knockdown of TaCML36 impaired host resistance.Knockdown of TaCML36 also significantly repressed expression of pathogenesis-related genes such as Chitinase 1,PDF35,and PR17C,the ethylene response factor-encoding gene TaPIE1,and ethylene biosynthesis gene ACO2.Collectively,our results suggest that TaCML36 positively participates in the innate immune response to R.cerealis infection by modulating expression of defense-associated genes possibly in the ethylene signaling pathway.
基金This research was funded by USDA-ARS NP306 project no.1245-43000-012-00D.
文摘Calmodulin is a ubiquitous calcium sensor to recognize the different developmental and/or stimulus-triggered calcium changes and regulate plant growth and development.However,the function of calmodulin remains elusive for fleshy fruit development.We performed expression studies of a family of six calmodulin genes(SlCaMs)in tomato fruit.All calmodulins showed a double peak expression pattern.The first flat peak appeared at 10–30 days after anthesis,but their expression rapidly declined at mature green and breaker.Then a sharp and even higher peak came at turning/pink stages.Among six calmodulins,SlCaM1 had the highest expression during fruit enlargement,whereas SlCaM2 was the major calmodulin during fruit ripening.However,SlCaMs showed different patterns in three ripening mutants rin,Nor and Nr.In particular,at the stages corresponding to mature green and breaker,the expression levels of SlCaMs in those mutants were significantly higher than wild-type.Furthermore,SlCaMs,especially SlCaM2 were upregulated by ethylene.Transiently overexpressing SlCaM2 in mature green fruit delayed ripening,while reducing SlCaM2 expression accelerated ripening.Our results suggest that SlCaMs play double roles to regulate fruit ripening.Prior to the ethylene burst,the ethylene-independent repression of SlCaMs might be critical for fruit to initiate the ripening process.After the ethylene burst,SlCaMs could participate in the ethylene coordinated rapid ripening.
基金supported by the National Science Foundation of China (32171941,31571583)。
文摘Plant calmodulins(CaMs)and calmodulin-like proteins(CMLs)mediate Ca~(2+)signaling in response to abiotic stresses.Manipulation of this signaling in crops could increase stress tolerance.We review methods for detecting Ca~(2+)signals,regulatory roles of Ca Ms and CMLs,binding targets,and Ca~(2+)networks under abiotic stress in organelles.
基金supported by grants from the Fujian Provincial Natural Science Foundation of China (No. 2008J0075)the Fujian Provincial Science and Technology Project of China(No. 2010Y0011)
文摘Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.
基金supported by Liaoning Social Development Key Projects of Scientific and Technological Department of Liaoning Province, No. 2012225019
文摘In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyI-D-aspartic acid-induced injury. Results showed that, compared with N-methyi-D- aspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca2＋ concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 pM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin- dependent protein kinase II expression.
基金supported by the National Natural Science Foundation of China,No.81101159the Natural Science Foundation of Jiangsu Province of China,No.BK20151268
文摘Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.
