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
Plant calmodulin(CaM) has been extracted from cauliflower, and the purified CaM has been identified with the activation of NAD kinase(NADK) and the inhibition effect of CaM antagonist W 7. CaM′s intrinsic fluorescen...Plant calmodulin(CaM) has been extracted from cauliflower, and the purified CaM has been identified with the activation of NAD kinase(NADK) and the inhibition effect of CaM antagonist W 7. CaM′s intrinsic fluorescence and Tb 3+ fluorescence showed that there was one tyrosine residue and four metal binding sites in cauliflower CaM. Based on Frster type nonradiative energy theory, the distances of Tyr→site Ⅲ, Ⅳ have been determined , and these are 1 23 nm(Tyr→site Ⅲ) and 1 18 nm(Tyr→site Ⅳ). The Eu 3+ and Tb 3+ fluorescence probes showed that the combination of CaM with W 7 resulted in significant change on CaM′s conformation, but did not affect coordination environment of metal binding sites.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
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
Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successf...Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.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
The interaction between dansyl-labeled pollen calmodulin (D-pCaM) and synthesized peptides was studied in the presence of Ca2+ by fluorescence spectra. It is Found that Gly/L-Ala --> D-Ala substitution in peptide c...The interaction between dansyl-labeled pollen calmodulin (D-pCaM) and synthesized peptides 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 highly-conserved structures. A preliminary study was carried out on the effects of CaM-binding peptides on cellular signal transduction, cell proliferation, showing the participation of CaM in cell functions mentioned above.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
In this paper, the adsorption process of calmodulin (CaM) on Au substrate was first investigated with electrochemical impedance spectroscopy (EIS) method. The result reveals that the adsorption of the protein-calmodu...In this paper, the adsorption process of calmodulin (CaM) on Au substrate was first investigated with electrochemical impedance spectroscopy (EIS) method. The result reveals that the adsorption of the protein-calmodulin contains two steps, i.e., one short quick step followed by a slow one. The complexation of calmodulin with Ca2+ was also first probed using EIS technique, in which the complexation of CaM with Ca2+ could be reflected by the change of apparent membrane capacitance(Capp) clearly. In all above measurements, a redox couple Fe(CN)63-/ Fe(CN)64- was used as probing-pin to reflect all the changes occurring in the above process. Our work suggests that some biological processes of CaM could be studied using EIS method conveniently.展开更多
To clarify the possible mechanisms of α anordin and probimane on calmodulin Ca ++ Mg ++ APTase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentra...To clarify the possible mechanisms of α anordin and probimane on calmodulin Ca ++ Mg ++ APTase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentration(ATP) response curve, dose(inhibitors) response curve and time response curve]. Our data have shown that the inhibitory rates of α anordrin and probimane are unrelated to substrate(APT) concentrations, but related to calmodulin concentrations. The inhibition of α anordrin and probimane is very quick that is completed within 1 to 5 min and can maintain more than 1 hr in the same inhibitory rates. So it is possible that α anordrin and probimane are calmodulin competitors with calmodulin like binding whose actions can occur by affecting the reaction balance of substrate and product on target enzymes of calmodulin( Ca ++ Mg ++ ATPase).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
Injection of TFP(a specific antagonist of CaM)into the lateral ventricles of the ratbrain on thefourth day of pregnancy causes marked antifertility effect in 83%of the rats(0%in the control group).Examination with the...Injection of TFP(a specific antagonist of CaM)into the lateral ventricles of the ratbrain on thefourth day of pregnancy causes marked antifertility effect in 83%of the rats(0%in the control group).Examination with the pontamone blue reaction for theimplantation elucidated that the antifertility effect was due to the blockage ofimplantation of the follicle.Injection of TFP into the lateral ventricles of the brainmarkedly reduced the concentration of CaM in hypothalamus,ovary and uterus.Serumprogesterone was also reduced.However,injection given on day 7 was ineffective to terminate the pregnancy.Injection given on certain time of pregnancy was able to reduce thecellular CaM content and to cause antifertility.This finding demonstrated that CaMplayed an important role during the course of pregnancy.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
Calmodulin (CAM) is involved in the regulation of a variety of cellular signaling pathways. To accomplish its physiological functions, CaM binds with Ca2+ at its EF-hand Ca2+ binding sites which induce the conform...Calmodulin (CAM) is involved in the regulation of a variety of cellular signaling pathways. To accomplish its physiological functions, CaM binds with Ca2+ at its EF-hand Ca2+ binding sites which induce the conformational switching of CaM. However, the molecular mechanism by which Ca2+ binds with CaM and induces conformational switching is still obscure. Here we combine molecular dynamics with targeted molecular dynamics simulation and achieve the state-transition pathway of CaM. Our data show that Ca2+ binding speeds up the conformational transition of CaM by weakening the interactions which stabilize the closed state. It spends about 6.5 ns and 5.25 ns for transition from closed state to open state for apo and holo CaM, respectively. Regarding the contribution of two EF-hands, our data indicate that the first EF-hand triggers the conformational transition and is followed by the second one. We determine that there are two interaction networks which contribute to stabilize the closed and open states, respectively.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.
文摘Plant calmodulin(CaM) has been extracted from cauliflower, and the purified CaM has been identified with the activation of NAD kinase(NADK) and the inhibition effect of CaM antagonist W 7. CaM′s intrinsic fluorescence and Tb 3+ fluorescence showed that there was one tyrosine residue and four metal binding sites in cauliflower CaM. Based on Frster type nonradiative energy theory, the distances of Tyr→site Ⅲ, Ⅳ have been determined , and these are 1 23 nm(Tyr→site Ⅲ) and 1 18 nm(Tyr→site Ⅳ). The Eu 3+ and Tb 3+ fluorescence probes showed that the combination of CaM with W 7 resulted in significant change on CaM′s conformation, but did not affect coordination environment of metal binding sites.
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 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This work is financially supported by the NNSF (No.29890280).
文摘Monoclonal antibody technique was employed to detect the conformational difference of CaM induced by metal ions. A trivalent europium ion induced conformation-specific anti-calmodulin monoclonal antibody was successfully prepared with europium-saturated calmodulin as antigen.
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The interaction between dansyl-labeled pollen calmodulin (D-pCaM) and synthesized peptides 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 highly-conserved structures. A preliminary study was carried out on the effects of CaM-binding peptides on cellular signal transduction, cell proliferation, showing the participation of CaM in cell functions mentioned above.
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In this paper, the adsorption process of calmodulin (CaM) on Au substrate was first investigated with electrochemical impedance spectroscopy (EIS) method. The result reveals that the adsorption of the protein-calmodulin contains two steps, i.e., one short quick step followed by a slow one. The complexation of calmodulin with Ca2+ was also first probed using EIS technique, in which the complexation of CaM with Ca2+ could be reflected by the change of apparent membrane capacitance(Capp) clearly. In all above measurements, a redox couple Fe(CN)63-/ Fe(CN)64- was used as probing-pin to reflect all the changes occurring in the above process. Our work suggests that some biological processes of CaM could be studied using EIS method conveniently.
文摘To clarify the possible mechanisms of α anordin and probimane on calmodulin Ca ++ Mg ++ APTase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentration(ATP) response curve, dose(inhibitors) response curve and time response curve]. Our data have shown that the inhibitory rates of α anordrin and probimane are unrelated to substrate(APT) concentrations, but related to calmodulin concentrations. The inhibition of α anordrin and probimane is very quick that is completed within 1 to 5 min and can maintain more than 1 hr in the same inhibitory rates. So it is possible that α anordrin and probimane are calmodulin competitors with calmodulin like binding whose actions can occur by affecting the reaction balance of substrate and product on target enzymes of calmodulin( Ca ++ Mg ++ ATPase).
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Injection of TFP(a specific antagonist of CaM)into the lateral ventricles of the ratbrain on thefourth day of pregnancy causes marked antifertility effect in 83%of the rats(0%in the control group).Examination with the pontamone blue reaction for theimplantation elucidated that the antifertility effect was due to the blockage ofimplantation of the follicle.Injection of TFP into the lateral ventricles of the brainmarkedly reduced the concentration of CaM in hypothalamus,ovary and uterus.Serumprogesterone was also reduced.However,injection given on day 7 was ineffective to terminate the pregnancy.Injection given on certain time of pregnancy was able to reduce thecellular CaM content and to cause antifertility.This finding demonstrated that CaMplayed an important role during the course of pregnancy.
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.
基金Supported by the Natural Science Fund for Distinguished Young Scholars of Hebei Province under Grant Nos C2015202340 and C2013202244the Fund for Outstanding Talents of Hebei Province under Grant No C201400305+3 种基金the National Natural Science Fund of China under Grant Nos 11247010,11175055,11475053,11347017,31600594,31400711 and 11647121the Fund for the Science and Technology Program of Higher Education Institutions of Hebei Province under Grant No QN2016113the Scientific Innovation Grant for Excellent Young Scientists of Hebei University of Technology under Grant No 2015010the Natural Science Foundation of Hebei Province under Grant No C2017202208
文摘Calmodulin (CAM) is involved in the regulation of a variety of cellular signaling pathways. To accomplish its physiological functions, CaM binds with Ca2+ at its EF-hand Ca2+ binding sites which induce the conformational switching of CaM. However, the molecular mechanism by which Ca2+ binds with CaM and induces conformational switching is still obscure. Here we combine molecular dynamics with targeted molecular dynamics simulation and achieve the state-transition pathway of CaM. Our data show that Ca2+ binding speeds up the conformational transition of CaM by weakening the interactions which stabilize the closed state. It spends about 6.5 ns and 5.25 ns for transition from closed state to open state for apo and holo CaM, respectively. Regarding the contribution of two EF-hands, our data indicate that the first EF-hand triggers the conformational transition and is followed by the second one. We determine that there are two interaction networks which contribute to stabilize the closed and open states, respectively.