When calcium ions enter the cytosol,it is a stimulatory signal for cellular events.The calcium sensor calmodulin picks up the change in calcium concentration and relays this information to its more than 300 downstream...When calcium ions enter the cytosol,it is a stimulatory signal for cellular events.The calcium sensor calmodulin picks up the change in calcium concentration and relays this information to its more than 300 downstream interaction partners.In this way,calmodulin affects cellular processes such as fertilization,muscle contraction,neuronal firing,and apoptosis.That is,calmodulin is involved in(nearly)everything!The significance of calmodulin is emphasized by the fact that we all carry three different genes(CALM1,2,3)on different chromosomes that encode the exact same calmodulin protein,and these are all expressed in all cell types.Moreover,throughout vertebrate evolution,the protein sequence has remained completely unchanged.展开更多
INTRODUCTIONCalmodulin（CaM）,widely distributed in almost alleukaryotic cells,is a major intracellular calcium receptorresponsible for mediating the Ca<sup>2+</sup> signal to a multitude ofdifferent enzym...INTRODUCTIONCalmodulin（CaM）,widely distributed in almost alleukaryotic cells,is a major intracellular calcium receptorresponsible for mediating the Ca<sup>2+</sup> signal to a multitude ofdifferent enzyme systems and is thought to play a vital rolein the regulation of cell proliferative cycle.Recently,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
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
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
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
The interaction between dansyl-labeled pollen calmodulin (D-pCaM) and synthesizedpeptides was studied in the presence of Ca2+ by fluorescence spectra. It is found that Gly/L- Ala→D-Ala substitution in peptide chains ...The interaction between dansyl-labeled pollen calmodulin (D-pCaM) and synthesizedpeptides was studied in the presence of Ca2+ by fluorescence spectra. It is found that Gly/L- Ala→D-Ala substitution in peptide chains caused great changes in their affinity for pCaM. Besides.our data provided evidence on the dissimilarity of different CaMs although they haVe highlyconserved structures. A preliminary study was carried out on the effects of CaM-binding peptideson cellular signal transduction, cell proliferation, showing the participation of CaM in cellfunctions mentioned above.展开更多
Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine ...Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes.Run-down was induced by the inside-out patch formation.Calpastatin(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion proteins.Results(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS's effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm.Conclusions The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor).展开更多
Calmodulin is a calcium binding protein that modulates the activity of diverse groups of protein including some protein kinase, adenylate cyclases and ATPase. Here we use the total DNA of Sonneratia paracaseolaris as ...Calmodulin is a calcium binding protein that modulates the activity of diverse groups of protein including some protein kinase, adenylate cyclases and ATPase. Here we use the total DNA of Sonneratia paracaseolaris as the template of the polymerase chain reaction (PCR). The PCR primers have been designed and synthesized according to the 5-and 3-terminal oligonucleotide sequences of Calmodulin gene of plants in Genbank and ligated with cloning vector pBsk(+).The recombinant clones have been obtained from the selected medium. The results of DNA sequences analysis show that the nucleotide sequences of ORF share more than 85% homologies as compared with those of calmodulin genes of several other plants.Similar to rice and apple, the ORF is interrupted by an intron behind the 75th nucleotide.展开更多
The effects of chronic hypoxia on calmodulin levels of lung tissues and small pulmonary ar-terial walls were studied in young pigs, The tissue specimens of hypoxic animals were obtained underhypoxic conditions. The fo...The effects of chronic hypoxia on calmodulin levels of lung tissues and small pulmonary ar-terial walls were studied in young pigs, The tissue specimens of hypoxic animals were obtained underhypoxic conditions. The following results were collected:(1) The swine exposed to chronic intermittent hypoxia showed a significant pulmonary pressor re-sponse at a simulated high altitude of 4000 m.(2) A higher level of calmodulin was found in the lung tissues of chronic hypoxic animals. It maybe related to the increased release of some vaosactive substances from pulmonary non-muscularcalls.(3) No significant difference of calmodulin level of small arterial walls was demonstrated between theexperimental animals and the control.The findings suggest that pulmonary vasoconstriction due to hypoxia is not likely to be associatedwith obvious change in calmodulin level in the smooth muscle of blood vessels.展开更多
We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 c...We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 cells. After treatment with 1 mM db-cAMP for 1 hour and 2 hours, there was an early and repldly reduced in gene expression of Calmodulin and c-fos respectively. After db-cAMP treatment for 4 -5 days, the number of capping cells of ConA binding decreased significantly and the cell surface microvllll decreased as well. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed In G1 phase, we have found that the db- cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, mlcrofilaments and fibronectln were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the Inhibition of proliferation, alteration, of phenotype and reco- very of cytoskeleton is展开更多
Aquaporin-0 (AQP0) contributes to the nurturing and cleaning of the eye lens of waste products. It is a tetrameric protein composed of four identical monomers, each of which has its own water pore. AQP0 water conducti...Aquaporin-0 (AQP0) contributes to the nurturing and cleaning of the eye lens of waste products. It is a tetrameric protein composed of four identical monomers, each of which has its own water pore. AQP0 water conduction is regulated by pH, Ca<sup>2+</sup> concentration, and the phosphorylation of serine residues at the C-terminal. High cellular Ca<sup>2+</sup> concentration enhances the binding of Calmodulin (CaM), a Ca<sup>2+</sup> dependent protein, to AQP0 from cytoplasm. This study focuses on determining the differences between the AQP0-CaM and the open AQP0 systems, by using Molecular Dynamics (MD) methods. The water conduction energy profiles are measured with two separate MD simulation techniques revealed two distinct channel profiles for the AQP0-CaM combined model. While the CaM bound channels’ energy barriers exceed the 6 kcal/mol, the no CaM bound AQP0 energy profile stays below 3 kcal/mol. The structural analysis of these different pores during the free equilibrations also supported this conclusion with distinct pore diameters. Unlike the previous report, this study observed Phe75 and His66 taking role in stabilizing the CSII restriction site in CaM bound AQP0.展开更多
OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) us...OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.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 rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.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
基金supported by Lundbeckfonden(R250-2017-134,to HHJ).
文摘When calcium ions enter the cytosol,it is a stimulatory signal for cellular events.The calcium sensor calmodulin picks up the change in calcium concentration and relays this information to its more than 300 downstream interaction partners.In this way,calmodulin affects cellular processes such as fertilization,muscle contraction,neuronal firing,and apoptosis.That is,calmodulin is involved in(nearly)everything!The significance of calmodulin is emphasized by the fact that we all carry three different genes(CALM1,2,3)on different chromosomes that encode the exact same calmodulin protein,and these are all expressed in all cell types.Moreover,throughout vertebrate evolution,the protein sequence has remained completely unchanged.
基金the Natural Science Fundation of Jiangsu Province,№BK95141307
文摘INTRODUCTIONCalmodulin（CaM）,widely distributed in almost alleukaryotic cells,is a major intracellular calcium receptorresponsible for mediating the Ca<sup>2+</sup> signal to a multitude ofdifferent enzyme systems and is thought to play a vital rolein the regulation of cell proliferative cycle.Recently,
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 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.
文摘The interaction between dansyl-labeled pollen calmodulin (D-pCaM) and synthesizedpeptides was studied in the presence of Ca2+ by fluorescence spectra. It is found that Gly/L- Ala→D-Ala substitution in peptide chains caused great changes in their affinity for pCaM. Besides.our data provided evidence on the dissimilarity of different CaMs although they haVe highlyconserved structures. A preliminary study was carried out on the effects of CaM-binding peptideson cellular signal transduction, cell proliferation, showing the participation of CaM in cellfunctions mentioned above.
基金supported by the grants from the Japan Society for the Promotion of Science and the National Natural Science Foundation of China(No.30670761,No.30671726)
文摘Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes.Run-down was induced by the inside-out patch formation.Calpastatin(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion proteins.Results(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS's effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm.Conclusions The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor).
基金Supported by the national science and technology ministry plant transgenic and industrialization foundation of China (J00-B-14) the foundation of Key Laboratory of halobios engineering SOA
文摘Calmodulin is a calcium binding protein that modulates the activity of diverse groups of protein including some protein kinase, adenylate cyclases and ATPase. Here we use the total DNA of Sonneratia paracaseolaris as the template of the polymerase chain reaction (PCR). The PCR primers have been designed and synthesized according to the 5-and 3-terminal oligonucleotide sequences of Calmodulin gene of plants in Genbank and ligated with cloning vector pBsk(+).The recombinant clones have been obtained from the selected medium. The results of DNA sequences analysis show that the nucleotide sequences of ORF share more than 85% homologies as compared with those of calmodulin genes of several other plants.Similar to rice and apple, the ORF is interrupted by an intron behind the 75th nucleotide.
文摘The effects of chronic hypoxia on calmodulin levels of lung tissues and small pulmonary ar-terial walls were studied in young pigs, The tissue specimens of hypoxic animals were obtained underhypoxic conditions. The following results were collected:(1) The swine exposed to chronic intermittent hypoxia showed a significant pulmonary pressor re-sponse at a simulated high altitude of 4000 m.(2) A higher level of calmodulin was found in the lung tissues of chronic hypoxic animals. It maybe related to the increased release of some vaosactive substances from pulmonary non-muscularcalls.(3) No significant difference of calmodulin level of small arterial walls was demonstrated between theexperimental animals and the control.The findings suggest that pulmonary vasoconstriction due to hypoxia is not likely to be associatedwith obvious change in calmodulin level in the smooth muscle of blood vessels.
文摘We have demonstrated that the distribution of microtubules (MT), mlcrofilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c- fos enhanced in the transformed C3H10T1/2 cells. After treatment with 1 mM db-cAMP for 1 hour and 2 hours, there was an early and repldly reduced in gene expression of Calmodulin and c-fos respectively. After db-cAMP treatment for 4 -5 days, the number of capping cells of ConA binding decreased significantly and the cell surface microvllll decreased as well. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed In G1 phase, we have found that the db- cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, mlcrofilaments and fibronectln were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the Inhibition of proliferation, alteration, of phenotype and reco- very of cytoskeleton is
文摘Aquaporin-0 (AQP0) contributes to the nurturing and cleaning of the eye lens of waste products. It is a tetrameric protein composed of four identical monomers, each of which has its own water pore. AQP0 water conduction is regulated by pH, Ca<sup>2+</sup> concentration, and the phosphorylation of serine residues at the C-terminal. High cellular Ca<sup>2+</sup> concentration enhances the binding of Calmodulin (CaM), a Ca<sup>2+</sup> dependent protein, to AQP0 from cytoplasm. This study focuses on determining the differences between the AQP0-CaM and the open AQP0 systems, by using Molecular Dynamics (MD) methods. The water conduction energy profiles are measured with two separate MD simulation techniques revealed two distinct channel profiles for the AQP0-CaM combined model. While the CaM bound channels’ energy barriers exceed the 6 kcal/mol, the no CaM bound AQP0 energy profile stays below 3 kcal/mol. The structural analysis of these different pores during the free equilibrations also supported this conclusion with distinct pore diameters. Unlike the previous report, this study observed Phe75 and His66 taking role in stabilizing the CSII restriction site in CaM bound AQP0.
文摘OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.
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 rcticulum and polyribosomes. Characteristically, calmodulin was present in the amyloplasts of root-cap cells. The widespread distribution of calmodulin may reflect its plciotropic functions in plant cellular activities.
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）.
文摘用药理学和生物化学的途径，在过氧化氢( H ( 2 ) O ( 2 ))之间的发信号的小径，钙( Ca (2+)) -calmodulin (凸轮)，并且氮的氧化物(没有)在 abscisic 酸(骆驼毛的织物)，导致的抗氧化剂防卫在玉米( Zea mays L.)的叶子被调查植物。有骆驼毛的织物， H (2 ) O (2 ) ，和 CaCl (2 ) 的处理在产生导致了增加不，在玉米叶肉房间和氮的氧化物的活动，在 cytosolic 和玉米的 microsomal 部分的 synthase (NOS ) 离开。然而，如此的增加被预告的处理与 Ca (2+) 禁止者和凸轮对手堵住。同时， Ca (2+)导致了的有也压制的二个 NOS 禁止者的预告的处理在另一方面在号码的生产增加，有骆驼毛的织物和 NO 施主钠 nitroprusside ( SNP )的处理也在叶肉房间的原物并且在 calmodulin 的表示在 cytosolic Ca (2+)的集中导致了增加 1 ( CaM1 )基因和在玉米的叶子的凸轮的内容种，并且骆驼毛的织物导致的增加被预告的处理减少与一没有 scavenger 和一个 NOS 禁止者。而且，抗氧化剂基因的表示的 导致SNP 的增加超级氧化物 dismutase 4 ( SOD4 )， cytosolic ascorbate 过氧化物酶(帽子 X )，并且谷胱甘肽 reductase 1 ( GR1 )并且 chloroplastic 和 cytosolic 抗氧化剂酶的活动被预告的处理与 Ca (2+)禁止者和凸轮对手逮捕。我们的结果建议那个 Ca (2+) 凸轮没有生产在上游、下游的功能，主要来自 NOS，导致骆驼毛的织物并且 H (2 ) O (2 ) 在玉米植物的叶子导致了抗氧化剂防卫。
