To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca2+/CaMK II expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death, concentrations of cytosolic free calcium, and the expression of CaMK II by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 10(-7), 10(-6) and 10(-5) mol/L of CORT was added to culture medium, The evident effect of 10(-6) and 10(-5) mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca2+]i increased in the neurons treated with 10(-6) and 10(-5) mol/L of CORT, but no change was observed in the neuron treated with 10(-7) mol/L of CORT. The death was either by way of apoptosis or necrosis in the cells treated with 10(-6) and 10(-5) mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca2+]i and the expression of CaMK II. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca2+]i is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMK II expression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; biosynthesis ; genetics ; Cells, Cultured ; Corticosterone ; pharmacology ; Hippocampus ; cytology ; enzymology ; Neurons ; cytology ; Rats ; Rats, Wistar

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.
Humans ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Melanoma/genetics* ; Membrane Proteins/genetics* ; Phosphorylation ; Signal Transduction

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(
Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Endometrial Neoplasms/genetics* ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mad2 Proteins ; Multigene Family ; Neoplastic Stem Cells ; Prognosis ; Securin

AIMTo investigate the resting [Ca2]i level and expression of calmodulin (CaM), calmodulin-dependent protein kinase II (CaMPKII) mRNA in hippocampal neurons of the mice with vascular dementia (VD) and their roles in the pathogenesis of VD.

METHODSThe mice were subjected for ischemia/reperfusion repeatedly on bilateral common carotid arteries by knots to establish the VD models. Animals with the sham-operation were taken as control group. The changes of behavior were observed through the step-down avoidance test and water maze test on the day 29, 30 after the operations. The hippocampal neurons were obtained immediately after mice were sacrificed and the resting [Ca2+]i was measured using laser scanning confocal microscopy with Fluo-3/AM as fluorescence indicator. RT-PCR technique was used to measure the mRNA expression of CaM, CaMPKII in hippocampal neurons.

RESULTS(1) The abilities of learning and memorizing in model group were inferior to those of sham-operation group( P < 0.05). (2) The resting [Ca2]i level in model group was significantly higher than sham-operation group (P < 0.05), while the expression of CaMmRNA, CaMPKIImRNA in VD group was significantly reduced than sham-operation group (P < 0.01).

CONCLUSIONOur study indicates that excessive resting[Ca2+ ]i level and lower CaM, CaMPKII expression in hippocampal neurons might participate in the pathogenesis of VD.

Animals ; Calcium ; analysis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Calmodulin ; genetics ; metabolism ; Dementia, Vascular ; metabolism ; Hippocampus ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Neurons ; metabolism ; RNA, Messenger ; genetics

BACKGROUNDThe effect of chronic stress on cognitive functions has been one of the hot topic in neuroscience. But there has been much controversy over its mechanism. Such single stressor applied in the past could not simulate complicated living circumstances that people confronted with. The aim of this study was to investigate the effects of chronic multiple-stress on learning and memory as well as on the levels of calcium/calmodulin-dependent protein kinase II (CaMKII), calmodulin (CaM) mRNA, and cAMP-response element binding protein (CREB) mRNA in the hippocampus of rats.

METHODSThe rats were divided randomly into stressed and control groups. The stressed group was given chronic multiple-stress for 6 weeks to set up a chronic multiple-stressed model. The rats' performance of spatial learning and memory was tested using Morris Water Maze (MWM) and Y-maze. Meanwhile, the expressions of CaMKII, CaM mRNA and CREB mRNA of rats' hippocampus were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the width of synaptic cleft and the thickness of post-synaptic densities (PSD) were observed in the hippocampal CA3 region of rats by electron microscopy.

RESULTSAfter exposure to chronic multiple-stress for 6 weeks, the ability of learning and memory of the stressed group was higher than that of the control group (P < 0.05, P < 0.01). The width of synaptic cleft was smaller and the thickness of PSD was larger in the hippocampal CA3 region of the stressed group than in that of the control group (P < 0.01). The CaMK II immunostaining of the stressed group was stronger than that of the control group in the stratum radiatum and oriens of the hippocampal CA1 and CA3, especially in the stratum oriens. Quantitative analysis indicated that the expression of CaMK II, CaM mRNA, and CREB mRNA in the hippocampus of the stressed group was higher than that of the control group (P < 0.05, P < 0.01).

CONCLUSIONSThe capacity of learning and memory can be enhanced after chronic multiple-stress. The increased levels of CaMK II, CaM mRNA, and CREB mRNA may contribute to the enhancing effect of chronic multiple-stress on learning and memory.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; Calmodulin ; genetics ; Chronic Disease ; Cyclic AMP Response Element-Binding Protein ; genetics ; Hippocampus ; metabolism ; ultrastructure ; Learning ; Male ; Memory ; Microscopy, Electron ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Stress, Physiological ; metabolism ; psychology ; Synapses ; ultrastructure

OBJECTIVETo explore effect of curcumin in different concentrations on learning and memory of senescence-accelerated mice (SAM) and their possible mechanisms.

METHODSMice were randomly divided into six groups: the SAMR1 normal control group, the SAMP8 model control group, the SAMP8 + solvent (the peanut oil) control group, SAMP8 + low, middle and high dose curcumin groups. Mice were gastrogavage for 25 successive days. On the next day of ending the experiment, changes of learning and memory in mice of each group were observed by Morris water maze. The hippocampal [Ca2+] was determined. Expressions of hippocampal calmodulin-dependent protein kinase II (CaMK II) and Calmodulin (CaM) mRNA were detected using Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively.

RESULTSThe latency to find the hidden platform was remarkably prolonged, the hippocampal [Ca2+]i was markedly increased, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA were significantly reduced in the SAMP8-model control group (P < 0.05, P < 0.01). The latency to find the hidden platform was remarkably shortened in the SAMP8 + middle dose curcumin and the SAMP8 + high dose curcumin groups (P < 0.01). The hippocampal [Ca2+]i was markedly lowered, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA obviously increased in the SAMP8 + low, middle and high dose curcumin groups (P < 0.05, P < 0.01).

CONCLUSIONCurcumin could improve learning and memory Ca2+/capacities of SAM by lowering hippocampal [Ca2+] overload, increase the hippocampal CaM mRNA level and CaMK II expression in the hippocampal dose-dependently.

Aging ; drug effects ; metabolism ; Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Calmodulin ; metabolism ; Curcumin ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Maze Learning ; drug effects ; Memory ; drug effects ; Mice ; RNA, Messenger ; genetics

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.
Animals ; Brain/*metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics ; Female ; GTP-Binding Protein alpha Subunits, Gi-Go/*genetics ; *Gene Deletion ; Gene Knockout Techniques/*methods ; Male ; Mice ; RNA, Untranslated/genetics ; Recombination, Genetic ; Spermatozoa/*metabolism

OBJECTIVETo observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo.

METHODSGene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: naïve group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively.

RESULTSThe siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated.

CONCLUSIONPre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cells, Cultured ; Disks Large Homolog 4 Protein ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; genetics ; Neuralgia ; therapy ; Neurons ; physiology ; Phosphorylation ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

All three nitric oxide synthase (NOS) isoforms are found in the lungs. It has been demonstrated that eNOS-derived NO plays an important role in modulating pulmonary vascular tone and inhibiting pulmonary vascular remodeling. Histamine induces pulmonary vasoconstriction by activating H(1)-receptor on the smooth muscle cells and vasodilation by stimulating H(2)-receptor. It remains unclear whether histamine also modulates the pulmonary vascular tone by regulating eNOS gene expression and NO production in pulmonary artery endothelial cells. Therefore, the present study was performed on cultured primary porcine pulmonary artery endothelial cells (PAECs) to investigate the effects of histamine on eNOS gene expression, and to explore the role of CaMK II in eNOS gene expression. After treatment with different concentrations histamine for different times, the levels of eNOS mRNA and protein were measured by RT-PCR and Western blot, respectively. The results showed that histamine upregulated eNOS mRNA and protein levels in a concentration- and time-dependent manner. Incubation with 10 micromol/L histamine for 24 h could increase eNOS mRNA and protein level to 160.8+/-12.2% (P<0.05) and 136.2+/-11.2% (P<0.05), respectively, of the control values. These up-regulation effects were prevented by selective CaMK II inhibitor, KN-93 (10 micromol/L). To investigate whether or not histamine increases eNOS expression by upregulating eNOS gene transcription, PAECs were transiently transfected with 1.6-kb fragment of the human eNOS promoter driving a luciferase reporter gene. The results suggested that eNOS gene promoter activity was enhanced to 148.2+/-33.7% (P<0.05) of the control after PAECs were incubated with 10 micromol/L histamine for 24 h. The nitrite and nitrate content in culture media measured by colorimetric method after incubation with 10 micromol/L histamine for 24 h indicated that the NO production in PAECs was increased. These results suggest that histamine up-regulates eNOS gene transcription and enhances NO production in PAECs by a signaling pathway involving CaMK II, which might be one of the mechanisms of histamine modulating pulmonary vascular tone.
Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; enzymology ; Histamine ; pharmacology ; Nitric Oxide Synthase Type III ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Pulmonary Artery ; cytology ; enzymology ; RNA, Messenger ; biosynthesis ; genetics ; Swine ; Up-Regulation

OBJECTIVETo study the effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in hippocampus of rats exposed to lead.

METHODSMale SD rats were divided into five groups. (1) control group, (2) group exposed to lead+2 by drinking water with 0.40 g/L lead acetate, (3) group exposed to methionine and choline (1:1, 400 mg/kg), (4) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 100 mg/kg), (5) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 400 mg/kg). In 8 weeks after exposure, all rats were killed. Then CREB mRNA and CaMK II mRNA expression levels in hippocampus were detected by real-time PCR, CREB and CaMK II protein expression levels in hippocampus were measured by western blot assay.

RESULTSThe expression levels (0.743 ± 0.185 and 0.729 ± 0.199) of CaMKII mRNA and CREB mRNA in the hippocampus of lead group were significantly lower than those (0.950 ± 0.238 and 0.901 ± 0.232) of control group (P < 0.05), also the expression levels (0.271 ± 0.045 and 0.212 ± 0.058) of CREB protein and pCREB protein in the hippocampus of lead group were significantly lower than those (0.319 ± 0.058 and 0.506 ± 0.125) of control group (P < 0.05). The expression levels (1.014 ± 0.210 and 1.126 ± 0.379) of CaMKII mRNA and the expression levels (1.029 ± 0.335 and 0.932 ± 0.251) of CREB mRNA in the hippocampus of 2 groups exposed to lead acetate plus methionine and choline were significantly higher than those of lead group (P < 0.05). The expression levels (0.407 ± 0.951 and 0.563 ± 0.178) of CREB protein and pCREB protein in the hippocampus of group exposed to lead acetate plus 400 mg/kg methionine and choline were significantly higher than those of lead group (P < 0.05).

CONCLUSIONMethionine and choline could decrease the inhibition effects of lead on the expression of CaMKII and CREB mRNA or CREB and pCREB proteins in the hippocampus of rats.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Choline ; pharmacology ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Male ; Methionine ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley