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1.
Frontiers of Medicine ; (4): 263-274, 2023.
Article in English | WPRIM | ID: wpr-982570

ABSTRACT

Melanoma is the most aggressive cutaneous tumor. Neuropilin and tolloid-like 2 (NETO2) is closely related to tumorigenesis. However, the functional significance of NETO2 in melanoma progression remains unclear. Herein, we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients. Disrupting NETO2 expression markedly inhibited melanoma proliferation, malignant growth, migration, and invasion by downregulating the levels of calcium ions (Ca2+) and the expression of key genes involved in the calcium signaling pathway. By contrast, NETO2 overexpression had the opposite effects. Importantly, pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis. Overall, this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression, indicating that targeting NETO2 may effectively improve melanoma treatment.


Subject(s)
Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Line, Tumor , Cell Proliferation , Melanoma/genetics , Membrane Proteins/genetics , Phosphorylation , Signal Transduction
2.
Zhongguo yi xue ke xue yuan xue bao ; Zhongguo yi xue ke xue yuan xue bao;(6): 685-695, 2021.
Article in Chinese | WPRIM | ID: wpr-921527

ABSTRACT

Objective To study the stemness characteristics of uterine corpus endometrial carcinoma(UCEC)and its potential regulatory mechanism.Methods Transcriptome sequencing data of UCEC was obtained from The Cancer Genome Atlas.Gene expression profile was normalized by edgeR package in R3.5.1.A one-class logistic regression machine learning algorithm was employed to calculated the mRNA stemness index(mRNAsi)of each UCEC sample.Then,the prognostic significance of mRNAsi and candidate genes was evaluated by survminer and survival packages.The high-frequency sub-pathways mining approach(HiFreSP)was used to identify the prognosis-related sub-pathways enriched with differentially expressed genes(DEGs).Subsequently,a gene co-expression network was constructed using WGCNA package,and the key gene modules were analyzed.The clusterProfiler package was adopted to the function annotation of the modules highly correlated with mRNAsi.Finally,the Human Protein Atlas(HPA)was retrieved for immunohistochemical validation.Results The mRNAsi of UCEC samples was significantly higher than that of normal tissues(


Subject(s)
Female , Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Endometrial Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Mad2 Proteins , Multigene Family , Neoplastic Stem Cells , Prognosis , Securin
3.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;52(7): e8732, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011598

ABSTRACT

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.


Subject(s)
Animals , Rats , Myocytes, Cardiac/pathology , Toll-Like Receptors/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Signal Transduction/physiology , Gene Expression , Lipopolysaccharides/pharmacology , NF-kappa B/metabolism , Rats, Wistar , Chaperonin 60/pharmacology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , RNA, Small Interfering , Inflammation/metabolism
4.
Article in English | WPRIM | ID: wpr-713586

ABSTRACT

Liquiritigenin (LQ) is a flavonoid that can be isolated from Glycyrrhiza radix. It is frequently used as a tranditional oriental medicine herbal treatment for swelling and injury and for detoxification. However, the effects of LQ on cognitive function have not been fully explored. In this study, we evaluated the memory-enhancing effects of LQ and the underlying mechanisms with a focus on the N-methyl-D-aspartic acid receptor (NMDAR) in mice. Learning and memory ability were evaluated with the Y-maze and passive avoidance tests following administration of LQ. In addition, the expression of NMDAR subunits 1, 2A, and 2B; postsynaptic density-95 (PSD-95); phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII); phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2); and phosphorylation of cAMP response element binding (CREB) proteins were examined by Western blot. In vivo, we found that treatment with LQ significantly improved memory performance in both behavioral tests. In vitro, LQ significantly increased NMDARs in the hippocampus. Furthermore, LQ significantly increased PSD-95 expression as well as CaMKII, ERK, and CREB phosphorylation in the hippocampus. Taken together, our results suggest that LQ has cognition enhancing activities and that these effects are mediated, in part, by activation of the NMDAR and CREB signaling pathways.


Subject(s)
Animals , Mice , Behavior Rating Scale , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cognition , Glycyrrhiza , Hippocampus , In Vitro Techniques , Learning , Medicine, East Asian Traditional , Memory , N-Methylaspartate , Phosphorylation , Phosphotransferases , Protein Kinases , Receptors, N-Methyl-D-Aspartate , Response Elements
5.
Article in English | WPRIM | ID: wpr-728546

ABSTRACT

Reducing [Mg2+]o to 0.1 mM can evoke repetitive [Ca2+]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg2+]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca2+ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg2+]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca2+ channel antagonist nimodipine, which blocked 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca2+]i spikes. The intracellular Ca2+ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca2+]i spikes. While Go6976, a specific inhibitor of PKCalpha had no effect on the tolerance, both the PKCepsilon translocation inhibitor and the PKCzeta pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca2+]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg2+]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca2+]i spike-induced activation of PKCepsilon and PKCxi, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.


Subject(s)
Animals , Rats , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cell Death , Cycloheximide , N-Methylaspartate , Neurons , Nimodipine , Phosphatidylinositol 3-Kinases , Phosphotransferases , Receptors, AMPA , Seizures
6.
Zhongguo Zhong Yao Za Zhi ; (24): 4075-4079, 2015.
Article in Chinese | WPRIM | ID: wpr-279282

ABSTRACT

<p><b>OBJECTIVE</b>To observe the dynamic time-phase expressions of key genes of brain-gut CaM signal pathway of spleen Qi deficiency rats and the intervention effect of Sijunzi decoction.</p><p><b>METHOD</b>Male Wistar rats were randomly divided into the normal control group, model 14 d, 21 d, 28 d groups, and Sijunzi decoction 14 d, 21 d, 28 d groups. Except for the normal control group, the remaining groups were included into the spleen Qi deficiency model with the bitter cold breaking Qi method (ig 7.5 g · kg⁻¹ · d⁻¹ of Rheum officinale, Fructus aurantii immaturus, Magnolia officinalis preparation) and the exhaustive swimming method. On the 7th day after the modeling, the Sijunzi decoction groups were orally administered with Sijunzi decoction 20 g · kg⁻¹ · d⁻¹. The expressions of key genes CaM/CaMK II of CaM signaling pathway in hippocampus and intestine at different time points by immunohistochemical method and Western blot. At the same time, the intervention effect of Sijunzi decoction on spleen Qi deficiency rats and its mechanism were analyzed.</p><p><b>RESULT</b>Spleen Qi deficiency rats showed higher intestinal CaM/CaMK II expression and lower hippocampus CaM/CaMK II expression than normal rats (P < 0.05, P < 0.01). After the treatment of Sijunzi decoction, spleen Qi deficiency rats showed reduction in intestinal CaM/CaMK II expression and increase in hippocampus CaM/CaMK II expression (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>The formation of spleen Qi deficiency syndrome may be related to the high expression of CaM/CaMK II in small intestine tissues and its low expression in hippocampus tissues. Sijunzi decoction may achieve the therapeutic effect in spleen Qi deficiency syndrome by reducing the CaM/CaMK II expression in intestinal tissues and increasing it in hippocampus tissues.</p>


Subject(s)
Animals , Humans , Male , Rats , Brain , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Genetics , Metabolism , Calmodulin , Metabolism , Drugs, Chinese Herbal , Intestines , Metabolism , Qi , Rats, Wistar , Spleen , Splenic Diseases , Drug Therapy , Genetics , Metabolism
7.
Sheng Li Xue Bao ; (6): 201-206, 2015.
Article in Chinese | WPRIM | ID: wpr-255956

ABSTRACT

This study was aimed to establish an experimental mouse model of combined transgenic inhibition of both multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and inward rectifier potassium current (Ik1), and to observe whether the specific inhibition of both CaMKII and Ik1 can bring about any effects on cardiac remodeling. Mice were divided into 4 groups: wild type (WT), CaMKII inhibited (AC3-I), Ik1 inhibited (Kir2.1-AAA) and combined inhibition of both CaMKII and Ik1 (AC3-I+Kir2.1-AAA). Mice in each group received electrocardiogram (ECG) and echocardiography examination. ECG in the condition of isoproterenol (ISO) injection was also checked. The whole cell patch clamp technique was used to measure Ik1 and the transient outward potassium current (Ito) from enzymatically isolated myocytes of left ventricle. In the condition of basal status, no significant changes of heart rate, PR interval and QRS interval were observed. No mouse showed ventricular arrhythmias in all of the 4 groups. After ISO injection, each group presented no significant ventricular arrhythmias either. The indexes measured by M-mode (motion-mode) and two-dimensional echocardiography had no significant differences among the four groups. Ik1 in AC3-I group was significantly higher than those in other three groups (P < 0.01) because of the results brought about by CaMKII inhibition. Among the latter three groups, both Kir2.1-AAA group and AC3-I+Kir2.1-AAA group had a significant reduced Ik1 compared with that of WT group, which was due to the Ik1 inhibition (P < 0.01). Ito in AC3-I group was higher than that of the other three groups (P < 0.01), but there were no significant differences in Ito among WT, Kir2.1-AAA and AC3-I+Kir2.1-AAA groups. Thus, combined transgenic myocardial CaMKII and Ik1 inhibition eliminated the up-regulation of Ik1 in CaMKII inhibited mice, and had no effects on cardiac remodeling including heart structure and function as well as arrhythmias at the basic and ISO conditions. The results of this study may provide a basis for the further investigation of combined inhibition of CaMKII and Ik1 in pathogenic cardiac remodeling.


Subject(s)
Animals , Mice , Arrhythmias, Cardiac , Brugada Syndrome , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Physiology , Cardiac Conduction System Disease , Disease Models, Animal , Electrocardiography , Heart , Physiology , Heart Conduction System , Congenital Abnormalities , Heart Ventricles , Isoproterenol , Mice, Transgenic , Patch-Clamp Techniques , Potassium Channels, Inwardly Rectifying , Physiology , Up-Regulation , Ventricular Remodeling
8.
Anatomy & Cell Biology ; : 235-243, 2015.
Article in English | WPRIM | ID: wpr-208411

ABSTRACT

Kaempferol exerts cardioprotective actions through incompletely understood mechanisms. This study investigated the molecular mechanisms underlying the cardioprotective effects of kaempferol in sinus node dysfunction (SND) heart. Here, we demonstrate that angiotensin II (Ang II) infusion causes SND through oxidized calmodulin kinase II (CaMKII). In contrast to this, kaempferol protects sinus node against Ang II-induced SND. Ang II evoked apoptosis with caspase-3 activation in sinus nodal cells. However, kaempferol lowered the CaMKII oxidization and the sinus nodal cell death. To block the CaMKII oxidization, gene of p47phox, a cytosolic subunit of NADPH oxidase, was deleted using Cas9 KO plasmid. In the absence of p47phox, sinus nodal cells were highly resistance to Ang II-induced apoptosis, suggesting that oxidized-CaMKII contributed to sinus nodal cell death. In Langendorff heart from Ang II infused mice, kaempferol preserved normal impulse formation at right atrium. These data suggested that kaempferol protects sinus node via inhibition of CaMKII oxidization and may be useful for preventing SND in high risk patients.


Subject(s)
Animals , Humans , Mice , Angiotensin II , Apoptosis , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases , Caspase 3 , Cell Death , Cytosol , Heart , Heart Atria , NADPH Oxidases , Plasmids , Sick Sinus Syndrome , Sinoatrial Node
9.
Article in Chinese | WPRIM | ID: wpr-333669

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of KN93, a calmodulin-dependent protein kinase II (CaMK II) inhibitor, on SH-SY5Y cell injury induced by bupivacaine hydrochloride.</p><p><b>METHODS</b>SH-SY5Y cells exposed for 24 h to 1 mmol/L KN93, 1 mmol/L bupivacaine hydrochloride, or both were examined for morphological changes and Cav3.1 protein expressions using Western blotting. The vitality and apoptosis rate of the cells at different time points during the exposures were assessed with MTT assay and flow cytometry, respectively.</p><p><b>RESULTS</b>Bupivacaine hydrochloride exposure caused obvious cell morphologial changes, reduced cell viability, increased cell apoptosis, and enhanced Cav3.1 protein expression. All these changes were partly reversed by treatment of the cells with 1 mmol/L KN93.</p><p><b>CONCLUSIONS</b>CaMKII may play a role in bupivacaine hydrochloride-induced SH-SY5Y cells injury, which is related with upregulated Cav3.1 protein expression.</p>


Subject(s)
Humans , Apoptosis , Bupivacaine , Calcium Channels, T-Type , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Cell Line , Cell Survival , Up-Regulation
10.
Article in English | WPRIM | ID: wpr-812475

ABSTRACT

The present study was aimed at determining the effects of Tongqiao Huoxue Decoction (TQHXD) on the Ca(2+)-CaMKII-CREB pathway and the memory and learning capacities of rats with vascular dementia (VD). The rat VD model was established by using an improved bilateral carotid artery ligation method. The Morris water maze experiment was used to evaluate the ethology of the VD rats following treatments with TQHXD at 3.01, 6.02, and 12.04 g·kg(-1) per day for 31 days. At the end of experiment, the hippocampus were harvested and analyzed. Western blotting and RT-PCR were used to measure the expression levels of calmodulin-binding protein kinase II(CaMKII), protein kinase A(PKA), cAMP-response element binding protein(CREB), and three N-methyl-D-aspartic acid receptor subunits (NR1, NR2A, and NR2B). Our results revealed that TQHXD could alleviate the loss of learning abilities and increase the memory capacity (P < 0.05 and P < 0.01 vs the model group, respectively). The treatment with 6.02 and 12.04 g·kg(-1) of TQHXD significantly up-regulated the Ca(2+)-CaMKII-CREB pathway in the hippocampus. In conclusion, TQHXD showed therapeutic effects on a bilateral carotid artery ligation-induced vascular dementia model, through the up-regulation of calcium signalling pathways.


Subject(s)
Animals , Female , Male , Calcium , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Cyclic AMP Response Element-Binding Protein , Metabolism , Dementia, Vascular , Drug Therapy , Metabolism , Psychology , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Hippocampus , Metabolism , Learning Disabilities , Drug Therapy , Metabolism , Maze Learning , Memory , Memory Disorders , Drug Therapy , Metabolism , Phytotherapy , Rats, Sprague-Dawley , Signal Transduction , Up-Regulation
11.
Zhongguo Zhong Yao Za Zhi ; (24): 2065-2070, 2014.
Article in Chinese | WPRIM | ID: wpr-299829

ABSTRACT

<p><b>OBJECTIVE</b>Effects of ginsenoside Rb1, Rg1 and Re on neurotrophic factor signal transduction pathway using liposome-mediated transfection of eukaryotic cells approach.</p><p><b>METHOD</b>The injury model was established by treating SH-SY5Y cells with 0.6 mmol x L(-1) of corticosterone (CORT) by 24 h. SH-SY5Y cell were pretreated with CORT for 30 min followed by co-treated with 120,60 and 20 micromol x L(-1) of Rb1, 120, 80 and 40 micromol x L(-1) of Rg1 and 120, 80 and 40 micromol x L(-1) of Re for 24 h. Cells viability was determined by Cell Counting Kit (CCK) assay. CREB expressing Luciferase reporter gene was constructed and transfected with plasmid containing hRaf, hcAMP, hAkt, hCaMK gene into human embryonic kidney (HEK293) cells using liposornal transfection reagent lipofection 2000. The expression of CREB before and after it addion of Rb1, Rg1 and Re was examined by Luc assay system and Western blotting.</p><p><b>RESULT</b>Compared with normal control group, CORT significantly decreased the viability of SH-SY5Y cells to 67.21% (P < 0.01). CCK results show that Rb1 (60 micromol x L(-1)), Rg1 (80 micromol x L(-1)) and Re (80 micromol x L(-1)) on SH-SY5Y cells have significant protective effect (P < 0.01). Lucassay and Western blotting results show that the gene and protein levels of CREB increased significantly through the pathway of Raf and Akt with Rb1 and Rg1 (P < 0.01), Re can increase significantly the gene and protein levels of CREB through the pathway of Raf and CaMK II.</p><p><b>CONCLUSION</b>Rb1, Rg1 and Re protects SH-SY5Y cells from CORT-induced damage and the neuroprotective mechanism may be associated with the Raf-CREB, Akt-CREB and CaMK II -CREB pathways.</p>


Subject(s)
Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Genetics , Metabolism , Cell Line , Cell Survival , Cyclic AMP Response Element-Binding Protein , Genetics , Metabolism , Drugs, Chinese Herbal , Pharmacology , Genes, Reporter , Ginsenosides , Pharmacology , Panax , Chemistry , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , Signal Transduction , raf Kinases , Genetics , Metabolism
12.
Sheng Li Xue Bao ; (6): 365-372, 2014.
Article in English | WPRIM | ID: wpr-297480

ABSTRACT

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and phosphorylates a large number of synaptic proteins, including major ionotropic glutamate receptors (iGluRs) and group I metabotropic glutamate receptors (mGluRs), to constitutively and/or activity-dependently regulate trafficking, subsynaptic localization, and function of the receptors. Among iGluRs, the N-methyl-D-aspartate receptor (NMDAR) is a direct target of CaMKII. By directly binding to an intracellular C-terminal (CT) region of NMDAR GluN2B subunits, CaMKII phosphorylates a serine residue (S1303) in the GluN2B CT. CaMKII also phosphorylates a serine site (S831) in the CT of α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors. This phosphorylation enhances channel conductance and is critical for synaptic plasticity. In addition to iGluRs, CaMKII binds to the proximal CT region of mGluR1a, which enables the kinase to phosphorylate threonine 871. Agonist stimulation of mGluR1a triggers a CaMKII-mediated negative feedback to facilitate endocytosis and desensitization of the receptor. CaMKII also binds to the mGluR5 CT. This binding seems to anchor and accumulate inactive CaMKII at synaptic sites. Active CaMKII dissociates from mGluR5 and may then bind to adjacent GluN2B to mediate the mGluR5-NMDAR coupling. Together, glutamate receptors serve as direct substrates of CaMKII. By phosphorylating these receptors, CaMKII plays a central role in controlling the number and activity of the modified receptors and determining the strength of excitatory synaptic transmission.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Neuronal Plasticity , Phosphorylation , Receptor, Metabotropic Glutamate 5 , Metabolism , Receptors, Metabotropic Glutamate , Metabolism , Receptors, N-Methyl-D-Aspartate , Metabolism , Serine , Metabolism , Synapses , Synaptic Transmission
13.
Zhonghua xinxueguanbing zazhi ; (12): 327-333, 2014.
Article in Chinese | WPRIM | ID: wpr-316464

ABSTRACT

<p><b>OBJECTIVE</b>Endothelial oxidative stress plays an important role in the pathogenesis of cardiovascular disease. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, could exert potent antioxidant properties. In this study, we investigated the protective effects, and related mechanism of salidroside against high glucose (33 mmol/L)-induced cell damage in human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>HUVECs were cultured in normal glucose (5.5 mmol/L), high glucose (33 mmol/L), high salidroside (10 µg/ml+33 mmol/L glucose), moderate salidroside (4 µg/ml+33 mmol/L glucose), low salidroside (1 µg/ml+33 mmol/L glucose) and very low salidroside (0.1 µg/ml+33 mmol/L glucose) for 48 h. Cell viability, the level of malondialdehyde (MDA) , reactive oxygen species (ROS) , nitric oxide (NO) , [Ca(2)+]i, calmodulin (CaM) , calmodulin-dependent kinase (CaMK) IIδ, endothelial nitric oxide synthase (eNOS) , active caspase-3 protein expression and eNOS ser 1177 phosphorylation of HUVECs post various treatments were measured. The cell viability was assessed with MTT assay, and the level of ROS, and [Ca(2)+]i was analyzed using flow cytometry. Nitric oxide and MDA was detected by Nitric Oxide Assay Kit and MDA Assay Kit. Western blot was performed to detect the protein expressions of eNOS, active caspase-3 and eNOS ser 1177 phosphorylation.</p><p><b>RESULTS</b>Comparing to the normal glucose group, high glucose treatment increased the cell damage, the level of NO and [Ca(2)+]i (P < 0.05) , downregulated CAMKIIδ, eNOS expression and eNOS ser 1177 phosphorylation (P < 0.05), elevated the concentration of MDA and ROS (P < 0.05) in HUVECs. Salidroside treatment significantly attenuated high glucose-induce cell damage on cultured HUVECs in a dose-dependent manner. Comparing to the high glucose group, 10 µg/ml Salidroside significantly increased cell viability (P < 0.05) , inhibited high glucose-induced release of MDA , generation of ROS, active caspase 3 protein expression (P < 0.05) , upregulated the release of nitric oxide and [Ca(2)+]i by HUVECs (P < 0.05) , enhanced CaM, CAMKIIδ, eNOS expression and eNOS ser 1177 phosphorylation in HUVECs (P < 0.05) .</p><p><b>CONCLUSIONS</b>These findings suggeste that salidroside could attenuate high glucose induced apoptosis in HUVEC, partly through activating the Ca(2)+/CaM/CAMKIIδ/eNOS pathway.</p>


Subject(s)
Humans , Apoptosis , Calcium , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Calmodulin , Metabolism , Cell Survival , Cells, Cultured , Glucose , Toxicity , Glucosides , Pharmacology , Human Umbilical Vein Endothelial Cells , Metabolism , Pathology , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type III , Metabolism , Phenols , Pharmacology , Phosphatidylinositol 3-Kinases , Metabolism , Signal Transduction
14.
Exp. mol. med ; Exp. mol. med;: e93-2014.
Article in English | WPRIM | ID: wpr-163226

ABSTRACT

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIalpha) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIalpha-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice generated by crossing CaMKIIalpha-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIalpha-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Delta)) without inheriting the Cre transgene. The Gnao(Delta/Delta) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIalpha-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIalpha-Cre mice as breeding pairs.


Subject(s)
Animals , Female , Male , Mice , Brain/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Gene Deletion , Gene Knockout Techniques/methods , RNA, Untranslated/genetics , Recombination, Genetic , Spermatozoa/metabolism
15.
Article in English | WPRIM | ID: wpr-727691

ABSTRACT

Phasic and tonic gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated inhibition critically regulate neuronal information processing. As these two inhibitory modalities have distinctive features in their receptor composition, subcellular localization of receptors, and the timing of receptor activation, it has been thought that they might exert distinct roles, if not completely separable, in the regulation of neuronal function. Inhibition should be maintained and regulated depending on changes in network activity, since maintenance of excitation-inhibition balance is essential for proper functioning of the nervous system. In the present study, we investigated how phasic and tonic inhibition are maintained and regulated by different signaling cascades. Inhibitory postsynaptic currents were measured as either electrically evoked events or spontaneous events to investigate regulation of phasic inhibition in layer 2/3 pyramidal neurons of the rat visual cortex. Tonic inhibition was assessed as changes in holding currents by the application of the GABA(A) receptor blocker bicuculline. Basal tone of phasic inhibition was maintained by intracellular Ca2+ and Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, maintenance of tonic inhibition relied on protein kinase A activity. Depolarization of membrane potential (5 min of 0 mV holding) potentiated phasic inhibition via Ca2+ and CaMKII but tonic inhibition was not affected. Thus, phasic and tonic inhibition seem to be independently maintained and regulated by different signaling cascades in the same cell. These results suggest that neuromodulatory signals might differentially regulate phasic and tonic inhibition in response to changes in brain states.


Subject(s)
Animals , Rats , Electronic Data Processing , Bicuculline , Brain , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cyclic AMP-Dependent Protein Kinases , Inhibitory Postsynaptic Potentials , Membrane Potentials , Nervous System , Neurons , Protein Kinases , Receptors, GABA-A , Visual Cortex
16.
J. forensic med ; Fa yi xue za zhi;(6): 169-177, 2014.
Article in Chinese | WPRIM | ID: wpr-983899

ABSTRACT

OBJECTIVE@#To observe the time-course expression of calcium-calmodulin dependent protein kinase II delta (CaMK II delta) in cerebral cortex after traumatic brain injury (TBI).@*METHODS@#The TBI rat model was established. The expression of CaMK II delta in cerebral cortex around injured area was tested by Western blotting and immunohistochemical staining.@*RESULTS@#Western blotting revealed expression of CaMK II delta in normal rat brain cortex. It gradually increased after TBI, peaked after 3 days, and then returned to normal level. The result of immunohistochemical staining was consistent with that of Western blotting.@*CONCLUSION@#The expression of CaMK II delta around injured area after TBI increased initially and then decreased. It could be used as a new indicator for wound age determination following TBI.


Subject(s)
Animals , Rats , Blotting, Western , Brain Injuries/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cerebral Cortex/metabolism , Forensic Medicine , Immunohistochemistry , Time Factors
17.
Chinese Journal of Stomatology ; (12): 694-698, 2013.
Article in Chinese | WPRIM | ID: wpr-274156

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of zoledronate acid on osteoclast differentiation and gene expression of calmodulin (CAM) and calmodulin-dependent protein kinase (CAMK)II.</p><p><b>METHODS</b>Receptor activation of nuclear factor κB ligand (RANKL) was used to induce differentiation of RAW264.7 cells into osteoclasts in vitro. The cells were divided into two groups, group A and group B. Both groups were treated with RANKL for 5 days, whereas group B was also treated with zoledronate for the last 2 days.Osteoclastogenesis and gene expression of CAM and CAMK II were examined.</p><p><b>RESULTS</b>In group B, the number of new-generated osteoclasts (≥3 nuclei), number and size of dentin resorption lacunaes were (23 ± 3) , (19 ± 2) and (4951 ± 223) µm(2) respectively, which were significantly lower than those [(44 ± 3) , (46 ± 1) and (13 331 ± 248) µm(2)] in group A (P < 0.01).mRNA and protein level of CAM and CAMK II were also significantly down-regulated in group B when compared with group A (P < 0.01) and the decrease was 26.7% and 37.2% respectively for CAM, 57.0% and 76.1% respectively for CAMK II.</p><p><b>CONCLUSIONS</b>Zoledronate acid could significantly inhibit formation and resorption function of osteoclasts. CAM and CAMKII may be involved in the inhibition process.</p>


Subject(s)
Animals , Mice , Bone Density Conservation Agents , Pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Genetics , Metabolism , Calmodulin , Genetics , Metabolism , Cell Differentiation , Cell Line , Diphosphonates , Pharmacology , Imidazoles , Pharmacology , Macrophages , Cell Biology , Osteoclasts , Cell Biology , Metabolism , RNA, Messenger , Metabolism , Receptor Activator of Nuclear Factor-kappa B , Pharmacology
18.
Article in English | WPRIM | ID: wpr-251418

ABSTRACT

Abnormal enhanced transmural dispersion of repolarization (TDR) plays an important role in the maintaining of the severe ventricular arrhythmias such as torsades de pointes (TDP) which can be induced in long-QT (LQT) syndrome. Taking advantage of an in vitro rabbit model of LQT2, we detected the effects of KN-93, a CaM-dependent kinase (CaMK) II inhibitor on repolarization heterogeneity of ventricular myocardium. Using the monophasic action potential recording technique, the action potentials of epicardium and endocardium were recorded in rabbit cardiac wedge infused with hypokalemic, hypomagnesaemic Tyrode's solution. At a basic length (BCL) of 2000 ms, LQT2 model was successfully mimicked with the perfusion of 0.5 μmol/L E-4031, QT intervals and the interval from the peak of T wave to the end of T wave (Tp-e) were prolonged, and Tp-e/QT increased. Besides, TDR was increased and the occurrence rate of arrhythmias like EAD, R-on-T extrasystole, and TDP increased under the above condition. Pretreatment with KN-93 (0.5 μmol/L) could inhibit EAD, R-on-T extrasystole, and TDP induced by E-4031 without affecting QT interval, Tp-e, and Tp-e/QT. This study demonstrated KN-93, a CaMKII inhibitor, can inhibit EADs which are the triggers of TDP, resulting in the suppression of TDP induced by LQT2 without affecting TDR.


Subject(s)
Animals , Rabbits , Action Potentials , Anti-Arrhythmia Agents , Pharmacology , Arrhythmias, Cardiac , Benzylamines , Pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Electrocardiography , Electrophysiologic Techniques, Cardiac , Endocardium , Heart , In Vitro Techniques , Long QT Syndrome , Pericardium , Piperidines , Pharmacology , Protein Kinase Inhibitors , Pharmacology , Pyridines , Pharmacology , Sulfonamides , Pharmacology , Torsades de Pointes
19.
Sheng Li Xue Bao ; (6): 1-7, 2013.
Article in Chinese | WPRIM | ID: wpr-333142

ABSTRACT

Sustained activation of β adrenergic receptor (βAR) leads to pathologic cardiac hypertrophy. However, the related mechanisms still remain unclear. In this study, we observe how N-acetylcysteine (NAC) affects the oxidative stress and calcium/calmodulin-dependent protein kinase II (CaMKII) expression in heart of isoproterenol (ISO)-stimulated rats, and investigate whether oxidative stress and CaMKII contribute to the development of sustained βAR-stimulated cardiac hypertrophy. Healthy male Wistar rats were randomly separated into 4 groups: control (CTRL), ISO-treated (ISO), control with NAC supplement (CTRL+NAC) and ISO-treated with NAC supplement (ISO+NAC) groups (6 rats in each group). Systolic blood pressure (SBP) was measured in awake rats with the tail-cuff method every week for two weeks. Heart weight/body weight ratio (HW/BW) and HE staining were used for the detection of myocardial hypertrophy. Myocardial mitochondrial reactive oxygen species (ROS) levels were measured by DCF fluorometry. The expressions of activated-CaMKII (p-CaMKII/CaMKII) and NADPH oxidase 4 (NOX(4)) were determined by Western blot analysis. The results showed that ISO-treated (i.p., daily 3 mg/kg, 2 weeks) rats developed an obvious cardiac hypertrophy as expressed by increases of HW/BW and myocyte cross-section area. Cardiac mitochondrial ROS level was significantly enhanced in ISO group as compared to CTRL group (P < 0.05). The expressions of NOX(4) and p-CaMKII in ISO group were also up-regulated as compared to CTRL group (1.4 and 1.6 times of CTRL, respectively, P < 0.05). NAC supplement significantly suppressed the hypertrophic development of heart in ISO-stimulated rats. The cardiac mitochondrial ROS level showed a significant decrease in rats of ISO+NAC group (P < 0.05 vs ISO). In accordance with this, ISO+NAC group rats also showed marked reductions in the expressions of NOX(4) and p-CaMKII/CaMKII compared to ISO group rats (P < 0.05). There were no significant differences of the detected indices between the rats from CTRL+NAC and CTRL groups. SBP showed no differences among four groups. These results suggest that both oxidative stress and CaMKII play important roles in sustained βAR-stimulated cardiac hypertrophy. NAC may suppress ISO-induced cardiac hypertrophy by down-regulating the expression of activated-CaMKII, and by reducing the level of oxidative stress originated from mitochondria and NADPH oxidase pathways.


Subject(s)
Animals , Male , Rats , Acetylcysteine , Pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Metabolism , Cardiomegaly , Isoproterenol , Pharmacology , Mitochondria, Heart , Metabolism , Myocardium , Pathology , NADPH Oxidase 4 , NADPH Oxidases , Metabolism , Oxidative Stress , Rats, Wistar , Reactive Oxygen Species , Metabolism , Receptors, Adrenergic, beta , Metabolism
20.
Article in Chinese | WPRIM | ID: wpr-306493

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of the γ isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIγ) on colorectal cancer (CRC) cell growth in vitro and in vivo and explore the mechanisms.</p><p><b>METHODS</b>The mRNA levels of CaMKIIγ in 5 CRC cell lines, tumor tissues and matched adjacent tissues from 20 CRC patients were examined by semi-quantitative RT-PCR. The lentiviral vector pLenti6.3-MCS-IRES2-eGFP was used to generate the lentivirus particle Lenti-CaMKIIγ for transfecting SW620 cells. The proliferation ability of the transfected SW620-CaMKIIγ cells was assessed by growth curve and colony formation assay. The expression of IKKα, IKKβ, IKKγ, p-IKKα/β, p-IκB andIκB of the transfected cells were determined by Western blotting, and the expression and localization of nuclear factor-κB (NF-κB) p65 were detected by immunofluorescence. In nude mouse models bearing the transfected SW620-CaMKIIγ cell xenograft, the tumor volume was measured twice a week.</p><p><b>RESULTS</b>CaMKIIγ mRNA showed high expressions in the 5 colorectal cancer cell lines. Eighteen of the 20 tumor tissues showed higher expressions of CaMKIIγ than the adjacent non-tumor tissues. The proliferation of transfected SW620-CaMKIIγ cells was enhanced significantly. CaMKIIγ activated NF-κB signaling pathway and led to NF-κB p65 nuclear translocation. In the tumor-bearing mouse model, the volume of the tumors generated by the transfected SW620-CaMKIIγ cells was 1.46- and 1.68-fold higher than that of the tumors with the control cells at the 8th and 12th day, respectively.</p><p><b>CONCLUSION</b>CaMKIIγ can effectively promote the growth of colorectal cancer cells in vitro and in vivo by activating NF-κB signaling pathway.</p>


Subject(s)
Humans , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms , Metabolism , Pathology , NF-kappa B , Metabolism , RNA, Messenger , Genetics , Signal Transduction , Up-Regulation
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