Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

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.

The calmodulin-dependent protein kinase II inhibitor KN-93 protects rat cerebral cortical neurons from N-methyl-D-aspartic acid-induced injury

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.

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABA_A receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and IMUS), respectively in the Mg2+-free external solution containing 1 μmol/L glycine at a holding potential (VH) of -40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 μmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 nmol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 μmol/L) or La3+(30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the

Tale of two kinases:Protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ in pre-diabetic cardiomyopathy

Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations,including insulin resistance,visceral fat accumulation,and dyslipidemias,which increase the risk for developing cardiovascular disease.Metabolic syndrome is associated with augmented sympathetic tone,which could account for the etiology of pre-diabetic cardiomyopathy.This review summarizes the current knowledge of the pathophysiological consequences of enhanced and sustainedβ-adrenergic response in pre-diabetes,focusing on cardiac dysfunction reported in diet-induced experimental models of pre-diabetic cardiomyopathy.The research reviewed indicates that both protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ play important roles in functional responses mediated byβ1-adrenoceptors;therefore,alterations in the expression or function of these kinases can be deleterious.This review also outlines recent information on the role of protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ in abnormal Ca^(2+)handling by cardiomyocytes from diet-induced models of pre-diabetic cardiomyopathy.

Effects of Calmodulin-dependent Protein Kinase Ⅱ Inhibitor,KN-93,on Electrophysiological Features of Rabbit Hypertrophic Cardiac Myocytes

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.

MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

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.

Amelioration of mitochondrial dysfunction in heart failure through S-sulfhydration of Ca^2＋/calmodulin-dependent protein kinase Ⅱ

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.

Role of inositol polyphosphate-4-phosphatase type II in oncogenesis of digestive system tumors

Inositol polyphosphate-4-phosphatase type II(INPP4B)is a newly discovered PI(3,4,5)P3 phosphatase.Many studies have revealed that INPP4B is upregulated or downregulated in tumors of the digestive system,and the abnormal expression of INPP4B may be attributed to the occurrence,development,and prognosis of tumors of the digestive system.This paper reviews studies on the correlations between INPP4B and digestive system tumors and the roles of INPP4B in the development of different tumors to provide a theoretical basis for further research on its molecular mechanism and clinical application."INPP4B"and"tumor"were searched as key words in PubMed and in the CNKI series full text database retrieval system from January 2000 to August 2023.A total of 153 Englishlanguage studies and 30 Chinese-language studies were retrieved.The following enrollment criteria were applied:(1)Studies contained information on the biological structure and functions of INPP4B;(2)studies covered the influence of abnormal expression of INPP4B in digestive system tumors;and(3)studies covered the role of INPP4B in the diagnosis,treatment,and prognosis of digestive system tumors.After excluding the literature irrelevant to this study,61 papers were finally included in the analysis.INPP4B expression is low in gastric cancer,colon cancer,pancreatic cancer,and liver cancer but it has high expression in esophageal cancer,colon cancer,pancreatic cancer,and gallbladder cancer.INPP4B is involved in the occurrence and development of digestive system tumors through the regulation of gene expression and signal transduction.The abnormal expression of INPP4B plays an important role in the development of digestive system tumors.Studies on INPP4B provide new molecular insights for the diagnosis,treatment,and prognosis evaluation of digestive system tumors.

黄连总生物碱通过调控CaMKII减轻局灶性脑缺血(MCAO)大鼠神经功能损伤的研究

目的:研究黄连总生物碱调节钙调素依赖性蛋白激酶Ⅱ(CaMKII)对局灶性脑缺血(MCAO)大鼠神经功能损伤后的影响。方法:随机将50只雄性SD大鼠分为假手术组、对照组、黄连总生物碱(高浓度、中浓度和低浓度)治疗组,采用线栓法建立大鼠局灶性脑缺血(MCAO)模型,假手术组不给予线栓处理,术后对大鼠进行神经功能评分。各治疗组给予黄连总生物碱灌胃治疗,按照人与大鼠等效剂量关系折算,假手术组用等量生理盐水灌胃;用酶联免疫吸附法(ELISA)检测大鼠脑组织中CaMKII的含量;采用BCA法和WST-1法分别测超氧化物歧化酶(SOD)的活力和丙二醛(MDA)的含量;制备脑组织病理切片,HE染色观察脑组织形态学变化。结果:与假手组相比,模型组评分和脑梗死体积明显升高,各个实验组经过黄连治疗后神经功能评分和脑梗死体积都有明显降低(P<0.05)。模型组与假手术组的CaMKII的含量相比明显升高(P<0.01)。与假手术组比较,模型组大鼠脑组织损伤程度加重,外周血氧化因子SOD活性降低,MDA含量升高;黄连治疗后,低、中、高剂量组SOD水平升高,MDA水平降低。HE染色观察脑组织病变程度,给药组大鼠的病变程度较模型组有明显改善。结论:黄连总生物碱通过调节脑缺血大鼠模型中CaMKII活性的影响,对海马细胞的神经损伤有一定的恢复和治疗作用。

Ginsenoside Rb1 Pretreatment Attenuates Myocardial Ischemia by Reducing Calcium/Calmodulin-Dependent Protein Kinase Ⅱ-Medicated Calcium Release

Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A myocardial ischemia(IS)rat.model and a myocardial H9 C2 cell hypoxia model were established.MI was induced by occluding the left anterior descending artery for 120 min.Ginsenoside Rb1(10 mg/kg)was administered 30 min before ischemia induction,and the treatment continued for 7 days.Results:In the rat IS injury model,ginsenoside Rb1 reduced myocardial infarct size,mean left ventricular diastolic pressure,incidence of arrhythmia,and levels of serum creatine kinase,lactate dehydrogenase,and malondialdehyde.However,the mean left ventricular systolic pressure,and maximal rising and falling rates of ventricular pressure(±dp/dtmax)increased.In the myocardial H9 C2 cell hypoxia model,ginsenoside Rb1 reduced intracellular calcium concentrations([Ca2+]i)during hypoxia,and markedly reversed the hypoxia-induced decrease in cell survival.Ginsenoside Rb1 was involved in the downregulation of CaMKⅡand the ryanodine receptor,as well as hypoxia-induced H9 C2 cell survival.Conclusion:The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats,partially through the downregulation of CaMKⅡexpression.

Mediation of flowering by a calmodulin-dependent protein kinase

A calmodulin-dependent protein kinase (MCK1) appeared important in regulating flowering in tobacco. The expression of modified MCK1 that lacks the C-terminal including calmodulin-binding domain upsets the flowering developmental program, leading to the abortion of flower primordia initiated on the main axis of the plant and, as well, caused the prolongation of the vegetative phase in axillary buds. The abortion process of flowers began first in the developing anthers and subsequently the entire flower senesces. In axillary buds the prolonged vegetative phase was characterized by atypical elongated, narrow, twisted leaves. These results suggested a role for calmodulin-dependent protein kinase homologs in mediating flowering.

Cloning and Characterization of a Homologous Ca^(2+)/Calmodulin-Dependent Protein Kinase PSKH1 from Pearl Oyster Pinctada fucata

Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the calcium metabolism in oysters will help elucidate the pearl formation mechanism. This paper describes a full-length PSKH1 cDNA isolated from pearl oyster Pinctada fucata. Oyster PSKH1 shares 65% homology with human PSKH1 and 48% similarity with rat CaM kinase I in the amino acid sequence, and contains a calmodulin-binding domain. The results of semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization revealed that oyster PSKH1 mRNA is highly expressed in the outer epithelial cells of the mantle pallial and in the gill epithelial cells. These studies provide important information describing the complex Ca^2＋ signaling mechanism in oyster calcium metabolism.

阿片类药物对NG108-15细胞Ca^(2+)/钙调蛋白依赖的蛋白激酶II信息通路的作用

目的 观察阿片类依赖时Ca2 + 钙调蛋白依赖的蛋白激酶II信息通路的变化。方法 以NG10 8 15细胞作为体外的细胞模型 ,分别用竞争性蛋白结合法及放射免疫法、PDE法、γ 32 P参入法测定cAMP水平、钙调蛋白(CaM)活性和钙调蛋白依赖的蛋白激酶II(CaMKII)活性。结果 DPDPE作用NG10 8 15细胞 48h可使细胞浆和细胞核CaM和CaMKII活性升高 ,该变化可被CaM特异性拮抗剂W 7所抑制 ;CaMKII特异性抑制剂KN 6 2可抑制CaMKII活性的增高 ,而对CaM活性无明显影响。DPDPE作用NG10 8 15细胞 48h后 ,加入纳洛酮 ,CaM活性、CaMKII活性进一步增高。结论 Ca2 + CaMKII信息通路参与了阿片依赖的机制。

CKII抑制剂——肝癌治疗新靶点

目的探讨蛋白激酶(casein kinase 2,CK2)抑制剂影响肝癌细胞株HepG-2凋亡的机制,为其临床治疗肝癌提供可能的实验依据。方法本研究通过MTT法和流式细胞仪检测和观察不同作用时间、不同浓度CK2抑制剂四溴苯三唑(4,5,6,7-tetrabromobenzotriazole,TBB)作用HepG-2肝癌细胞株后对其增殖和凋亡的影响,分别应用荧光定量PCR和Western blot实验分析可能机制。结果 TBB在48 h浓度为200μmol/L时对肝癌细胞的抑制作用最明显;150μmol/L TBB作用48 h后,早期凋亡率明显增高,至(13.2±0.67)%,磷酸化P65在100μmol/L TBB实验组表达下调,而caspase-3、caspase-8、Bcl-2和Bcl-x等在两组间表达未见明显差异。结论 CK2抑制剂TBB能够通过参与调控NF-κB信号转导通路抑制肿瘤细胞增殖,促进其凋亡,可能成为未来肝癌治疗的一个新的靶点。

PTSD大鼠杏仁核CaMKIIα及pCaMKIIα表达的变化

目的观察创伤后应激障碍(PTSD)样行为异常大鼠杏仁核神经元CaM依赖性蛋白激酶IIα(CaMKIIα)及磷酸化CaM依赖性蛋白激酶IIα(pCaMKIIα)表达变化。方法采用国际认定的SPS方法刺激建立大鼠PTSD模型,取成年健康雄性Wistar大鼠75只,随机分为SPS模型的12h、1d、4d、7d组及正常对照组,采用免疫组织化学、免疫印迹法检测大鼠杏仁核CaMKIIα及pCaMKIIα的表达变化。结果SPS刺激后大鼠杏仁核神经元细胞内CaMKIIα于12h开始减少,1d表达最少,4d开始逐渐增多,至7d时基本恢复正常;pCaMKIIα的表达则于SPS12h开始增多,1d时表达最多,4d时开始减少,至7d时基本恢复正常。CaMKIIα阳性细胞在SPS各组模型均出现,1d表达最少。结论杏仁核CaMKIIα及pCaMKIIα的表达变化,可能是PTSD大鼠情感行为异常的重要病理生理基础之一。

丝裂素活化蛋白激酶参与AngII诱导心肌细胞血小板衍生生长因子受体的表达

目的 探讨丝裂素活化蛋白激酶在血管紧张素II(AngII)诱导心肌细胞血小板衍生生长因子受体 - β(PDGF - β)表达中的作用。 方法 分离纯化培养的乳鼠心肌细胞 ,以10 -7mol·L-1AngII刺激为AngII组 ;以 10 -5mol·L-1PD980 5 9(一种丝裂素活化蛋白激酶抑制剂 )预孵育 30min10 -7后再用AngII刺激为PD980 5 9组 ,以正常的乳鼠心肌细胞为对照组 ;免疫印迹法测定培养 2 4h时心肌细胞PDGF - β受体的含量。 结果 AngII刺激培养 2 4h的乳鼠心肌细胞PDGF - β受体表达增强 (P <0 .0 5 ) ,PD980 5 9可部分抑制AngII对PDGF - β受体表达的诱导作用。结论 丝裂素活化蛋白激酶参与AngII上调心肌细胞PDGF -

PP^(60c-Src)在血管紧张素II诱导的大鼠血管平滑肌细胞信息转导中的作用(英文)

目的:本研究通过观察c -Src在AngII对大鼠血管平滑肌细胞(VSMC)丝裂原激活的蛋白激酶(MAPK)的激活和c -Fos蛋白表达中的影响,以进一步阐明AngII促VSMC增殖的细胞内信息转导机制。方法:原代和传代培养SD大鼠主动脉VSMC ,以脂质体包裹反义c -Src寡脱氧核苷酸(oligodeoxynucleotides,ODNs)转染培养的VSMC以抑制c -Src蛋白表达和激酶活性。以未转染的VSMC为对照,观察10 -7mol/LAngII刺激对转染的VSMC的MAPK活性和c -Fos蛋白表达的影响。蛋白免疫沉淀和酶自身磷酸化率法测定c -Src激酶活性;髓鞘碱性蛋白(MBP)底物磷酸化率测定MAPK激酶活性;Westernblotting免疫印迹法测定c-Src和c -Fos蛋白表达情况。结果:转染不同浓度反义c-SrcODNs的VSMC ,c -Src蛋白含量呈浓度依赖性降低,c-Src激酶活性也显著抑制,以AngII刺激经转染反义c -SrcODNs的VSMC ,c -Src激酶活性增幅仅为对照组的8 .7% ;MAPK活性仅为对照的1 .6 % ;c -Fos蛋白表达的增幅为对照组的30 .0 %。结论:AngII可诱导VSMCc -Src激活和细胞内信息转导,且AngII引起的MAPK和c -fos的激活依赖于c-Src的激活,提示c -Src是AngII促血管平滑肌细胞增殖的重要信息分子。

Phosphorylation of OsRbohB by the protein kinase OsDMI3 promotes H_(2)O_(2) production to potentiate ABA responses in rice

In rice, the Ca^(2+)/calmodulin-dependent protein kinase OsDMI3 is an important positive regulator of abscisic acid (ABA) signaling. In ABA signaling, H_(2)O_(2) is required for ABA-induced activation of OsDMI3, which in turn increase H_(2)O_(2) production. However, how OsDMI3 regulates H_(2)O_(2) production in ABA signaling remains unknown. Here we show that OsRbohB is the main NADPH oxidase involved in ABA-induced H_(2)O_(2) production and ABA-mediated physiological responses. OsDMI3 directly interacts with and phosphorylates OsRbohB at Ser-191, which is OsDMI3-mediated site-specific phosphorylation in ABA signaling. Further analyses revealed that OsDMI3-mediated OsRbohB Ser-191 phosphorylation positively regulates the activity of NADPH oxidase and the production of H_(2)O_(2) in ABA signaling, thereby enhancing the sensitivity of seed germination and root growth to ABA and plant tolerance to water stress and oxidative stress. Moreover, we discovered that the OsDMI3-mediated OsRbohB phosphorylation and H_(2)O_(2) production is dependent on the sucrose non-fermenting 1-related protein kinases SAPK8/9/10, which phosphorylate OsRbohB at Ser-140 in ABA signaling. Taken together, these results not only reveal an important regulatory mechanism that directly activates Rboh for ABA-induced H_(2)O_(2) production but also uncover the importance of this regulatory mechanism in ABA signaling.

人pEGFP-PKARIIβ重组质粒的构建及其在BGC-823胃癌细胞中的表达

目的:研究人的蛋白激酶A调节亚基RIIβ(PKARIIβ)基因的绿色荧光蛋白(GFP)真核表达载体融合蛋白在胃癌细胞BGC-823内的表达和定位,初步探讨其在肿瘤细胞中的分子作用机制.方法:以pRSETB-PKARIIβ质粒为模板,PCR扩增出全长PKARIIβ编码序列,用XhoⅠ和EcoRⅠ双酶切后,亚克隆至含有GFP标签的融合表达载体pEGFP-C1中.测序正确后,将重组质粒转染到胃癌细胞BGC-823中,提取细胞蛋白进行Westernbolt检测.利用共聚焦激光扫描显微镜观察pEGFP-PKARIIβ在人胃癌细胞BGC-823内的定位.结果:将人全长PKARIIβ编码序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段为1.2kb,在预期的位置产生条带.Western blot检测到了融合蛋白正确表达,分子量约为72000Da.并观察到GFP-PKARIIβ在HEK293和BGC-823细胞内都有表达,且定位以细胞质为主,在细胞核内少量表达.结论:成功构建了人全长PKARIIβ编码序列的pEGFP-PKARIIβ真核表达载体,证实GFP-PKARIIβ蛋白在BGC-823细胞质内特异表达,为进一步研究PKARIIβ的在胃癌中的功能提供了重要的实验材料.

抑制CaMKII减轻线粒体氧化应激可改善离体心脏缺血再灌注损伤

目的探讨钙/钙调蛋白依赖的蛋白激酶Ⅱ(CaMKII)在离体心脏缺血再灌注(IR)损伤中的作用和可能机制。方法采用大鼠离体心脏缺血再灌注(IR 45 min/120 min)模型,将40只大鼠随机分为对照组(Control)、KN-93药物对照组(2.5μmol/L KN-93)、IR组和CaMKII特异性抑制剂KN-93干预缺血再灌注组(KN-93+IR),以左室心脏功能、冠脉流出液中乳酸脱氢酶(LDH)活性和心肌肌钙蛋白(cTnI)含量、心肌梗死面积评价心脏损伤程度,Western-blot检测CaMKII磷酸化(p-CaMKII)、CaMKII氧化(ox-CaMKII)和PLN磷酸化(p-PLN)蛋白的表达,试剂盒检测线粒体超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量。结果与Control组相比,KN-93组各项指标均无明显变化;IR组心脏功能、线粒体SOD的活性降低(P<0.01),而心肌梗死面积、冠脉流出液中LDH活性和cTnI含量、p-CaMKII、ox-CaMKII和p-PLN的表达、线粒体MDA的含量均明显升高(P<0.01);KN-93干预IR组可明显改善心脏功能(P<0.01),增加线粒体SOD活性,减小心肌梗死面积、LDH活性、cTnI含量、p-CaMKII、ox-CaMKII和p-PLN的表达以及线粒体MDA含量(P<0.01)。结论 CaMKII参与离体心脏缺血再灌注损伤,抑制CaMKII可通过降低线粒体氧化应激进而减轻离体心脏缺血再灌注损伤。