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.

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.

Involvement of Calcium dependent Protein Kinases in ABA regulation of Stomatal Movement

Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.

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.

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 suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a cal-cium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.

Molecular mechanisms and the role of the protein kinase pathway in rat spatial memory impairment following isoflurane anesthesia

BACKGROUND： Animal experiments have demonstrated that isoflurane exposure alone induces learning and memory deficits for weeks or months. However, the molecular mechanisms of learning and memory remain poorly understood. Hippocampal expression of calcium/phospholipid- dependent protein kinase （PKC） and cAMP-dependent protein kinase （PKA） in rats have been shown to be associated with memory processing. OBJECTIVE： To investigate changes in rat spatial memory and hippocampal CA1 neuronal kinase system following isoflurane anesthesia, and to explore the correlation between molecular changes in cerebral neurons and behavioral manifestations following anesthesia. DESIGN, TIME AND SE＇rFING： A randomized, controlled, animal study. All experiments were performed at the Department of Anesthesia, Beijing Chaoyang Hospital, Capital Medical University from November 2007 to December 2008. MATERIALS： A total of 72 male, 3 month-old （young group）, Sprague Dawley rats, and 36 male, 20 month-old （aged group）, Sprague-Dawley rats were used in the study. Isoflurane was purchased from Baxter, USA. METHODS： Young and aged rats were randomly assigned to control, training （no anesthesia, Morris water maze training）, and isoflurane （1.2% isoflurane, Morris water maze training） groups. The isoflurane group was further subdivided into four groups, which were exposed to anesthesia for 2 or 4 hours, and were subjected to Morris water maze training at 2 days or 2 weeks postanesthesia. Finally, each aged group comprised 6 rats, and the young group comprised 12 rats. MAIN OUTCOME MEASURES： Spatial learning and memory were observed during Morris water maze training. Hippocampal CA1 PKA and PKC expression and activity were detected by immunohistochemistry and enzyme-linked immunosorbent assay （ELISA）. RESULTS： A 4-hour isoflurane exposure induced spatial memory deficits in all rats for 2 days to 2 weeks. In particular, aged rats exhibited more severe spatial memory deficits. Immunohistochemistry and ELISA results showed a significant increase in PKC and PKA expression and activity in the hippocampus CA1 subfield following Morris water maze training （P 〈 0.05）. Moreover, isoflurane anesthesia inhibited PKC and PKA expression and activity, and this inhibition increased with increasing exposure duration and increasing age. CONCLUSION： Results suggested that increased isoflurane exposure and age could extensively inhibit the hippocampal CA1 kinase system. Inhibition of protein kinases could play an important role in the cognitive decline following anesthesia.

A Highly Sensitive Detection Method, Phos-tag^TMAffinity SDS-PAGE, Used to Analyze a Possible Substrate of CDPK-Related Protein Kinase5 in <i>Arabidopsis</i>

Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.

Stress kinase inhibition modulates acute experimental pancreatitis

AIM: To examine the role of p38 during acute experimental cerulein pancreatitis. METHODS: Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347) and/or a specific p38 inhibitor (SB203580) and pancreatic stress kinase activity was determined. Parameters to assess pancreatitis included trypsin, amylase, lipase, pancreatic weight and histology. RESULTS: JNK inhibition with CEP1347 ameliorated pancreatitis, reducing pancreatic edema. In contrast, p38 inhibition with SB203580 aggravated pancreatitis with higher trypsin levels and, with induction of acinar necrosis not normally found after cerulein hyperstimulation. Simultaneous treatment with both CEP1347 and SB203580 mutually abolished the effects of either compound on cerulein pancreatitis. CONCLUSION: Stress kinases modulate pancreatitis differentially. JNK seems to promote pancreatitis development, possibly by supporting inflammatory reactions such as edema formation while its inhibition ameliorates pancreatitis. In contrast, p38 may help reduce organ destruction while inhibition of p38 during induction of cerulein pancreatitis leads to the occurrence of acinar necrosis.

Salt Stress Triggers Phosphorylation of the Arabidopsis Vacuolar K＋ Channel TPK1 by Calcium-Dependent Protein Kinases （CDPKs）

14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K＋ channel 1 （TPK1） interacts with the 14-3-3 protein GRF6 （GF14-λ）. Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases （CDPKs） can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K＋ homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpkl mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca2＋, Ca2＋-dependent kinases, and 14-3-3 proteins.

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.

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。

山药块茎膨大期DoCDPK1基因生物信息学及低温胁迫下的表达分析

钙依赖性蛋白激酶(CDPK)在植物的生长发育及逆境胁迫方面发挥着重要作用。以大和长芋和毕克齐山药为试验材料,克隆钙依赖性蛋白激酶基因DoCDPK1,并对其进行生物信息学分析,为DoCDPK1基因的功能研究奠定基础。采用生物信息学方法分析DoCDPK1基因结构,构建瞬时表达载体,对DoCDPK1蛋白进行亚细胞定位;采用qRT-PCR分析DoCDPK1基因在不同山药品种的不同发育阶段及低温胁迫处理下的表达模式。结果表明(:1)DoCDPK1基因序列长度为2023 bp,编码521个氨基酸。(2)DoCDPK1与几内亚薯蓣CDPK蛋白序列的一致性为97%,且DoCDPK1存在STKc_CAMK结构域。(3)瞬时表达分析表明,DoCDPK1蛋白定位于细胞核与细胞膜。(4)大和长芋山药中的CDPK活性更高,DoCDPK1可能参与调控山药块茎膨大后期的生长发育。(5)经4℃低温胁迫后,CDPK活性降低,DoCDPK1表达量上调,且表达水平在不同时间处理下存在差异。综上所述,DoCDPK1可能参与调控山药块茎膨大后期的生长发育及低温胁迫响应过程。

生姜提取物对染铝毒大鼠学习记忆功能和神经细胞结构的影响及其可能作用机制

目的分析生姜提取物(GRE)对铝染毒大鼠学习记忆功能及神经细胞结构的影响,并基于钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、神经元型一氧化氮合酶(nNOS)、蛋白激酶C(PKC)探讨其可能的作用机制。方法将36只SD雄性大鼠随机分为空白对照组、铝染毒组、临床药物组、低剂量GRE(L⁃GRE)组、中剂量GRE(M⁃GRE)组、高剂量GRE(H⁃GRE)组,每组6只大鼠。空白对照组大鼠自由饮用不含氯化铝的饮用水,其余组大鼠饮用含10 mg/mL结晶氯化铝的饮用水。3个月后给予空白对照组和铝染毒组大鼠灌胃生理盐水,临床药物组大鼠灌胃盐酸多奈哌齐溶液,分别给予L⁃GRE组、M⁃GRE组、H⁃GRE组大鼠灌胃100 mg/kg、200 mg/kg、400 mg/kg GRE溶液。持续干预4周后,采用Morris水迷宫实验评估大鼠的学习记忆功能,通过HE染色观察大鼠海马组织形态变化,分别采用实时荧光定量PCR和Western blot检测大鼠海马组织CaMKⅡ、nNOS、PKC mRNA和蛋白表达水平。结果(1)与空白对照组相比,铝染毒组大鼠的逃避潜伏期延长且穿越平台次数减少(P<0.05),海马组织的神经细胞皱缩,神经细胞数量明显减少,CaMKⅡ、nNOS、PKC的mRNA表达水平及nNOS、PKC的蛋白表达水平降低(P<0.05)。(2)与铝染毒组比,L⁃GRE组、M⁃GRE组和H⁃GRE组大鼠的逃避潜伏期缩短,临床药物组、M⁃GRE组、H⁃GRE组大鼠的穿越平台次数增加(P<0.05);各给药组大鼠海马组织病理形态有不同程度的改善;临床药物组大鼠的CaMKⅡ、nNOS和PKC mRNA表达水平升高,各剂量GRE组大鼠nNOS和PKC mRNA和蛋白表达水平升高(P<0.05)。(3)与临床药物组相比,M⁃GRE组和H⁃GRE组大鼠的逃避潜伏期,以及各剂量GRE组的穿越平台次数和目标象限停留时间差异无统计学意义(P>0.05);各剂量GRE组大鼠海马组织病理形态改善程度更为明显;M⁃GRE组、H⁃GRE组大鼠的nNOS、PKC蛋白表达水平升高,L⁃GRE组大鼠的nNOS蛋白表达水平升高(P<0.05)。(4)各剂量GRE组组间大鼠逃避潜伏期、穿越平台次数及目标象限停留时间差异无统计意义(P>0.05);随GRE干预剂量增加,大鼠海马组织病理形态的改善效果越明显;H⁃GRE组大鼠的CaMKⅡmRNA表达水平高于L⁃GRE组(P<0.05)。结论GRE能够改善铝染毒大鼠的神经细胞结构和学习记忆功能,其中对神经细胞结构的改善效果优于盐酸多奈哌齐,对学习功能的改善效果与盐酸多奈哌齐相当。GRE的作用机制可能与拮抗铝所致CaMKⅡ、nNOS和PKC表达水平下降有关。

雨生红球藻钙依赖蛋白激酶(CDPK)家族鉴定与表达分析

【目的】钙依赖蛋白激酶(CDPK或CPK)广泛参与植物生长发育和环境胁迫响应。为探究CDPK基因在雨生红球藻(Haematococcus pluvialis)生长发育及虾青素积累中的功能,对HpCDPK基因家族进行鉴定和表达分析。【方法】利用生物信息学方法鉴定雨生红球藻HpCDPK基因家族成员,分析其编码蛋白的理化特性、系统进化、保守基序和功能结构域,以及缺氮等胁迫条件下HpCDPK成员基因的表达谱。【结果】结果表明,共鉴定7个HpCDPK基因家族成员,所有HpCDPK蛋白都含有典型EF-hand基序和蛋白激酶结构域。系统发育分析表明,这些HpCDPK与来自莱茵衣藻的CrCDPK聚为一类,与高等植物拟南芥的AtCDPK分开,暗示在进化过程中发生了种属特异性的基因复制事件。转录组数据分析表明,HpCDPK基因表达受到多种胁迫诱导,其中HpCDPK7基因转录水平在缺氮条件下上调最为明显。此外,HpCDPK与类胡萝卜素合成相关基因的表达相关性分析结果显示,HpCDPK与多个类胡萝卜素合成相关基因表达密切关联,特别是HpCDPK2与虾青素合成关键基因(BKT和BCH)的表达显著正相关(P<0.05)。【结论】本研究鉴定了7个HpCDPK基因,HpCDPK7基因在缺氮条件下的表达上调最为明显,HpCDPK2可能在类胡萝卜素及虾青素合成积累中发挥重要调控作用。研究结果为后续深入解析HpCDPK介导雨生红球藻胁迫响应和类胡萝卜素合成积累的功能及分子机制提供参考依据。

小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析

为探究钙依赖蛋白激酶(CDPK)在小麦生长和逆境应答中的作用,从普通小麦中克隆了TaCDPK17基因,并对其进行了序列结构、表达模式和抗逆功能初步分析。结果表明,TaCDPK17基因编码区长1701 bp,编码566个氨基酸,具有CDPK家族的典型结构特征,包括1个保守的丝氨酸/苏氨酸激酶结构域和4个EF手型结构域。对TaCDPK17与其他12种植物的CDPK17进行进化树分析表明,TaCDPK17与禾本科作物,特别是节节麦、大麦的CDPK17序列有较高同源性。TaCDPK17基因启动子区域富含与激素调控、光照响应,以及非生物应答胁迫相关的顺式调控元件,其中,脱落酸(ABA)响应元件(ABRE)和茉莉酸甲酯响应元件(CGTCA)数量较多。基于实时荧光定量PCR的表达分析结果显示,TaCDPK17受100μmol/L ABA、100μmol/L茉莉酸甲酯、20%PEG6000和250 mmol/L NaCl诱导后,表达量有不同程度升高。在2μmol/L ABA和100 mmol/L NaCl胁迫处理条件下,过表达TaCDPK17的拟南芥种子萌发率显著高于野生型对照。同时,过表达TaCDPK17缓解了ABA或渗透胁迫处理对幼苗根生长的抑制作用。在气孔关闭过程中,过表达TaCDPK17的转基因植物对ABA更敏感,与野生型植物相比,表现出更强的气孔关闭趋势。这些结果表明,TaCDPK17在小麦逆境胁迫和激素信号应答中发挥着重要作用。

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。

新型抗癫痫药Q808对颞叶癫痫大鼠的改善作用及其机制

目的:探讨新型抗癫痫药6-(4-氯苯氧基)-四唑^((5,1-a))酞嗪(Q808)对颞叶癫痫(TLE)大鼠神经元损伤的改善作用,并阐明其作用机制。方法:采用腹腔注射Q808的方法制备TLE大鼠模型。将45只造模成功大鼠随机分为模型组、低剂量Q808组和高剂量Q808组,每组15只,低剂量Q808组和高剂量Q808组大鼠分别采用20和80 mg·kg^(-1) Q808灌胃,模型组大鼠采用等量0.3%羧甲纤维素钠灌胃,另选15只健康SD大鼠作为对照组。持续灌胃治疗4周后,观察各组大鼠行为表现,采用PONEMAH 6. X实验动物遥测平台记录各组大鼠脑电图,采用高尔基染色观察各组大鼠海马CA1神经元树突形态表现和神经元树突棘密度,采用Western blotting法检测各组大鼠海马组织中突触可塑性特异性蛋白钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)蛋白表达水平。结果:对照组大鼠活动正常,无抽搐等异常表现;模型组、低剂量Q808和高剂量Q808组大鼠出现不同程度活动减少、震颤点头、失去平衡、肌肉强直和前肢的抽搐,逐渐转变为全身肌肉强直和站立,随后向后跌倒,发作间期无抽搐发作。与对照组比较,模型组、低剂量Q808和高剂量Q808组大鼠癫痫发作总持续时间明显延长(P<0.01);与模型组比较,低剂量Q808和高剂量Q808组大鼠癫痫发作总持续时间明显缩短(P<0.01)。对照组大鼠海马组织CA1区神经元树突分布较为规律,树突网密集有序;模型组大鼠海马组织CA1区神经元树突排列紊乱,大量树突缠结,形成较粗大的神经纤维束;与模型组比较,低剂量Q808和高剂量Q808组大鼠海马组织CA1区神经元树突网络有所恢复,排列相对规律。与对照组比较,模型组大鼠海马组织CA1区神经元树突棘密度明显降低(P<0.01);与模型组比较,低剂量Q808和高剂量Q808组大鼠海马组织CA1区神经元树突棘密度明显升高(P<0.01)。与对照组比较,模型组、低剂量Q808组和高剂量Q808组大鼠海马组织中CaMKⅡ蛋白表达水平明显降低(P<0.01);与模型组比较,低剂量Q808组和高剂量Q808组大鼠海马组织中CaMKⅡ蛋白表达水平明显升高(P<0.01)。结论:新型抗癫痫药Q808对TLE模型大鼠有改善作用,其机制可能与Q808能减轻海马组织CA1区神经元树突病变和增加突触可塑性相关蛋白CaMKⅡ蛋白表达水平有关。

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。