Involvement of calmodulin-dependent protein kinase I in the regulation of the expression of connexin 43 in MA-10 tumor Leydig cells.

Connexin 43 (Cx43, also known as Gja1) is the most abundant testicular gap junction protein. It has a crucial role in the support of spermatogenesis by Sertoli cells in the seminiferous tubules as well as in androgen synthesis by Leydig cells. The multifunctional family of Ca2+/calmodulin-dependent protein kinases (CaMK) is composed of CaMK I, II, and IV and each can serve as a mediator of nuclear Ca2+ signals. These kinases can control gene expression by phosphorylation of key regulatory sites on transcription factors. Among these, AP-1 members cFos and cJun are interesting candidates that seem to cooperate with CaMKs to regulate Cx43 expression in Leydig cells. In this study, the Cx43 promoter region important for CaMK-dependent activation is characterized using co-transfection of plasmid reporter-constructs with different plasmids coding for CaMKs and/or AP-1 members in MA-10 Leydig cells. Here we report that the activation of Cx43 expression by cFos and cJun is increased by CaMKI. Furthermore, results from chromatin immunoprecipitation suggest that the recruitment of AP-1 family members to the proximal region of the Cx43 promoter may involve another uncharacterized AP-1 DNA regulatory element and/or protein-protein interactions with other partners. Thus, our data provide new insights into the molecular regulatory mechanisms that control mouse Cx43 transcription in testicular Leydig cells.

Calcium Signalling in Heart and Vessels: Role of Calmodulin and Downstream Calmodulin-Dependent Protein Kinases.

Cardiovascular disease is the major cause of death worldwide. The success of medication and other preventive measures introduced in the last century have not yet halted the epidemic of cardiovascular disease. Although the molecular mechanisms of the pathophysiology of the heart and vessels have been extensively studied, the burden of ischemic cardiovascular conditions has risen to become a top cause of morbidity and mortality. Calcium has important functions in the cardiovascular system. Calcium is involved in the mechanism of excitation-contraction coupling that regulates numerous events, ranging from the production of action potentials to the contraction of cardiomyocytes and vascular smooth muscle cells. Both in the heart and vessels, the rise of intracellular calcium is sensed by calmodulin, a protein that regulates and activates downstream kinases involved in regulating calcium signalling. Among them is the calcium calmodulin kinase family, which is involved in the regulation of cardiac functions. In this review, we present the current literature regarding the role of calcium/calmodulin pathways in the heart and vessels with the aim to summarize our mechanistic understanding of this process and to open novel avenues for research.

Discovery of a New CaMKII-Targeted Synthetic Lethal Therapy against Glioblastoma Stem-like Cells.

Glioblastoma stem-like cells (GSCs) drive tumor initiation, cancer invasion, immune evasion, and therapeutic resistance and are thus a key therapeutic target for improving treatment for glioblastoma multiforme (GBM). We previously identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an emerging molecular target for eliminating GSCs. In this study, we aim to explore a new CaMKII-targeted synthetic lethal therapy for GSCs. Through high-throughput drug combination screening using CaMKII inhibitors and a bioactive compound library in GSCs, neurokinin 1 receptor (NK1R) inhibitors such as SR 140333 and aprepitant are found to be potential anticancer agents that exhibit chemical synthetic lethal interactions with CaMKII inhibitors, including hydrazinobenzoylcurcumin (HBC), berbamine, and KN93. Combined treatment with NK1R and CaMKII inhibitors markedly suppresses the viability and neurosphere formation of U87MG- and U373MG-derived GSCs. In addition, the combination of HBC and NK1R inhibitors significantly inhibits U87MG GSC tumor growth in a chick embryo chorioallantoic membrane (CAM) model. Furthermore, the synthetic lethal interaction is validated using RNA interference of CaMKIIγ and NK1R. Notably, the synthetic lethal effects in GSCs are associated with the activation of caspase-mediated apoptosis by inducing p53 expression and reactive oxygen species generation, as well as the suppression of stemness marker expression by reducing nuclear factor-kappa B (NF-κB) activity. This follows the downregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling and a decrease in intracellular calcium concentration. Moreover, NK1R affects CaMKIIγ activation. These findings demonstrate that NK1R is a potential synthetic lethal partner of CaMKII that is involved in eradicating GSCs, and they suggest a new CaMKII-targeted combination therapy for treating GBM.

Anti-echinococcal effect of verapamil involving the regulation of the calcium/calmodulin-dependent protein kinase II response in vitro and in a murine infection model.

BACKGROUND: Echinococcosis, which is caused by the larvae of cestodes of the genus Echinococcus, is a parasitic zoonosis that poses a serious threat to the health of humans and animals globally. Albendazole is the drug of choice for the treatment of echinococcosis, but it is difficult to meet clinical goals with this chemotherapy due to its low cure rate and associated side effects after its long-term use. Hence, novel anti-parasitic targets and effective treatment alternatives are urgently needed. A previous study showed that verapamil (Vepm) can suppress the growth of Echinococcus granulosus larvae; however, the mechanism of this effect remains unclear. The aim of the present study was to gain insight into the anti-echinococcal effect of Vepm on Echinococcus with a particular focus on the regulatory effect of Vepm on calcium/calmodulin-dependent protein kinase II (Ca2+/CaM-CaMKII) in infected mice. METHODS: The anti-echinococcal effects of Vepm on Echinococcus granulosus protoscoleces (PSC) in vitro and Echinococcus multilocularis metacestodes in infected mice were assessed. The morphological alterations in Echinococcus spp. induced by Vepm were observed by scanning electron microscopy (SEM), and the changes in calcium content in both the parasite and mouse serum and liver were measured by SEM-energy dispersive spectrometry, inductively coupled plasma mass spectrometry and alizarin red staining. Additionally, the changes in the protein and mRNA levels of CaM and CaMKII in infected mice, and in the mRNA levels of CaMKII in E. granulosus PSC, were evaluated after treatment with Vepm by immunohistochemistry and/or real-time quantitative polymerase chain reaction. RESULTS: In vitro, E. granulosus PSC could be killed by Vepm at a concentration of 0.5 µg/ml or higher within 8 days. Under these conditions, the ultrastructure of PSC was damaged, and this damage was accompanied by obvious calcium loss and downregulation of CaMKII mRNA expression. In vivo, the weight and the calcium content of E. multilocularis metacestodes from mice were reduced after treatment with 40 mg/kg Vepm, and an elevation of the calcium content in the sera and livers of infected mice was observed. In addition, downregulation of CaM and CaMKII protein and mRNA expression in the livers of mice infected with E. multilocularis metacestodes was found after treatment with Vepm. CONCLUSIONS: Vepm exerted a parasiticidal effect against Echinococcus both in vitro and in vivo through downregulating the expression of Ca2+/CaM-CaMKII, which was over-activated by parasitic infection. The results suggest that Ca2+/CaM-CaMKII may be a novel drug target, and that Vepm is a potential anti-echinococcal drug for the future control of echinococcosis.

Increased calcium-mediated cerebral processes after peripheral injury: possible role of the brain in complex regional pain syndrome.

BACKGROUND: Among various diseases that accompany pain, complex regional pain syndrome (CRPS) is one of the most frustrating for patients and physicians. Recently, many studies have shown functional and anatomical abnormalities in the brains of patients with CRPS. The calcium-related signaling pathway is important in various physiologic processes via calmodulin (CaM) and calcium-calmodulin kinase 2 (CaMK2). To investigate the cerebral mechanism of CRPS, we measured changes in CaM and CaMK2 expression in the cerebrum in CRPS animal models. METHODS: The chronic post-ischemia pain model was employed for CRPS model generation. After generation of the animal models, the animals were categorized into three groups based on changes in the withdrawal threshold for the affected limb: CRPS-positive (P), CRPS-negative (N), and control (C) groups. Western blot analysis was performed to measure CaM and CaMK2 expression in the rat cerebrum. RESULTS: Animals with a decreased withdrawal threshold (group P) showed a significant increment in cerebral CaM and CaMK2 expression (P = 0.013 and P = 0.021, respectively). However, groups N and C showed no difference in CaM and CaMK2 expression. CONCLUSIONS: The calcium-mediated cerebral process occurs after peripheral injury in CRPS, and there can be a relationship between the cerebrum and the pathogenesis of CRPS.

CaMKII(δ) regulates osteoclastogenesis through ERK, JNK, and p38 MAPKs and CREB signalling pathway.

Calcium/calmodulin-dependent protein kinases (CaMKs) are a group of important molecules mediating calcium signal transmission and have been proved to participate in osteoclastogenesis regulation. CaMKII, a subtype of CaMKs is expressed during osteoclast differentiation, but its role in osteoclastogenesis regulation remains controversial. In the present study, we identified that both mRNA and protein levels of CaMKII (δ) were upregulated in a time-dependent manner during osteoclast differentiation. CaMKII (δ) gene silencing significantly inhibited osteoclast formation, bone resorption, and expression of osteoclast-related genes, including nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), and c-Src. Furthermore, CaMKII (δ) gene silencing downregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38, which were transiently activated by RANKL. Specific inhibitors of ERK, JNK, and p38 also markedly inhibited expression of osteoclast-related genes, osteoclast formation, and bone resorption like CaMKII (δ) gene silencing. Additionally, CaMKII (δ) gene silencing also suppressed RANKL-triggered CREB phosphorylation. Collectively, these data demonstrate the important role of CaMKII (δ) in osteoclastogenesis regulation through JNK, ERK, and p38 MAPKs and CREB pathway.

Chronic Calmodulin-Kinase II Activation Drives Disease Progression in Mutation-Specific Hypertrophic Cardiomyopathy.

BACKGROUND: Although the genetic causes of hypertrophic cardiomyopathy (HCM) are widely recognized, considerable lag in the development of targeted therapeutics has limited interventions to symptom palliation. This is in part attributable to an incomplete understanding of how point mutations trigger pathogenic remodeling. As a further complication, similar mutations within sarcomeric genes can result in differential disease severity, highlighting the need to understand the mechanism of progression at the molecular level. One pathway commonly linked to HCM progression is calcium homeostasis dysregulation, though how specific mutations disrupt calcium homeostasis remains unclear. METHODS: To evaluate the effects of early intervention in calcium homeostasis, we used 2 mouse models of sarcomeric HCM (cardiac troponin T R92L and R92W) with differential myocellular calcium dysregulation and disease presentation. Two modes of intervention were tested: inhibition of the autoactivated calcium-dependent kinase (calmodulin kinase II [CaMKII]) via the AC3I peptide and diltiazem, an L-type calcium channel antagonist. Two-dimensional echocardiography was used to determine cardiac function and left ventricular remodeling, and atrial remodeling was monitored via atrial mass. Sarcoplasmic reticulum Ca2+ATPase activity was measured as an index of myocellular calcium handling and coupled to its regulation via the phosphorylation status of phospholamban. RESULTS: We measured an increase in phosphorylation of CaMKII in R92W animals by 6 months of age, indicating increased autonomous activity of the kinase in these animals. Inhibition of CaMKII led to recovery of diastolic function and partially blunted atrial remodeling in R92W mice. This improved function was coupled to increased sarcoplasmic reticulum Ca2+ATPase activity in the R92W animals despite reduction of CaMKII activation, likely indicating improvement in myocellular calcium handling. In contrast, inhibition of CaMKII in R92L animals led to worsened myocellular calcium handling, remodeling, and function. Diltiazem-HCl arrested diastolic dysfunction progression in R92W animals only, with no improvement in cardiac remodeling in either genotype. CONCLUSIONS: We propose a highly specific, mutation-dependent role of activated CaMKII in HCM progression and a precise therapeutic target for clinical management of HCM in selected cohorts. Moreover, the mutation-specific response elicited with diltiazem highlights the necessity to understand mutation-dependent progression at a molecular level to precisely intervene in disease progression.

Changes in CaMK Ⅱ α expression in spinal cord and dorsal root ganglia during remifentanil-induced hyperalgesia in a rat model of incisional pain / 中华麻醉学杂志

Objective To investigate the changes in the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ α (CaMK Ⅱ α) in the spinal cord and dorsal root ganglia (DRG) during remifentanil-induced hyperalgesia in a rat model of incisional pain (IP).Methods Thirty-two male Sprague-Dawley rats in which caudal vein catheter was successfully placed,aged 260-280 g,were divided into 4 groups (n=8 each) using a random number table method:control group (group C),IP group,remifentanil group (group R) and remifentanil plus IP group (group RIP).Normal saline was infused via the caudal vein for 60 min at a rate of 0.1 ml · kg-1 · min-1 in group C.Normal saline was infused via the caudal vein for 60 min at a rate of 0.1 ml · kg-1 · min-1,and the model of IP was simultaneously established in group IP.Remifentanil was infused via the caudal vein for 60 min at a rate of 1.0 μg · kg-1 · min-1 in group R.Remifentanil was infused via the caudal vein for 60 min at a rate of 1.0 μg · kg-1 · min-1,and the model of IP was simultaneously established in group RIP.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion and 2,6,24 and 48 h after infusion (T0-4).The rats were sacrificed after the last behavioral test,and L4-6 segment of the spinal cord and DRGs were removed for determination of the expression of total and phosphorylated CaMK Ⅱ α (tCaMK Ⅱ α,pCaMK Ⅱ α) by Western blot.The ratio of pCaMK Ⅱ /tCaMK Ⅱ α was calculated.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the expression of tCaMK Ⅱ α and pCaMK Ⅱ α in the spinal cord and DRGs was up-regulated,and the ratio of pCaMK Ⅱ α/tCaMK Ⅱ α was increased in I,R and RIP groups (P＜0.05 or 0.01).Compared with group IP and group R,MWT was significantly decreased,TWL was shortened,the expression of tCaMK Ⅱ α and pCaMK Ⅱ α in the spinal cord and DRGs was up-regulated,and the ratio of pCaMK Ⅱ α/tCaMK Ⅱ α was increased in group RIP (P＜0.05 or 0.01).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to upregulated expression of CaMK Ⅱ α in the spinal cord and DRGs in a rat model of IP.

Relationship between NR2B and CaMK Ⅱ α in spinal cord during remifentanil-induced hyperalgesia in a rat model of incisional pain / 中华麻醉学杂志

Objective To evaluate the relationship between NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptors (NR2B receptors) and Ca2+/calmodulin-dependent protein kinase Ⅱ α (CaMK Ⅱ α) in the spinal cord during remifentanil-induced hyperalgesia in a rat model of incisional pain (IP).Methods Forty male Sprague-Dawley rats in which intrathecal and caudal catheters were successfully placed,weighing 260-280 g,aged 2-3 months,were divided into 4 groups (n=10 each) using a random number table method:control group (group C),remifentanil plus IP group (group RI),NR2B antagonist Ro 25-6981 group (group Ro) and remifentanil plus IP plus Ro 25-6981 group (group RI+Ro).In group C,normal saline 0.1 ml was intrathecally injected,and 10 min later normal saline was infused for 60 min via the tail vein at a rate of0.1 ml · kg-1 · min-1.In group RI,normal saline 0.1 ml was intrathecally injected,and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1.0 μg · kg-1 · min-1,and IP was established immediately after onset of remifentanil infusion.In group Ro,Ro 25-6981 (0.1 ml) 10 μg was intrathecally injected,and 10 min later normal saline was infused for 60 min via the tail vein at a rate of 0.1 ml · kg-1 · min-1.In group RI+Ro,Ro 25-6981 (0.1 ml) 10 μg was intrathecally injected,and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1.0 μg · kg-1 · min-1,and IP was established immediately after onset of remifentanil infusion.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before intravenously infusing normal saline or remifentanil and at 2,6,24 and 48 h after the end of infusion (T0-4).The rats were sacrificed after the last behavioral test,and the L4-6 segment of the spinal cord was removed for determination of the expression of NR2B in total and membrane protein (tNR2B and mNR2B) and expression of CaMK Ⅱ α in total protein (tCaMK Ⅱ α) and phosphorylated CaMK Ⅱ α (pCaMKⅡα).The ratios of mNR2B/tNR2B and pCaMKⅡα/tCaMK Ⅱα were calculated.Results Compared with group C,the MWT was significantly decreased,TWL was shortened,the expression of tNR2B,mNR2B,tCaMKⅡα and pCaMKⅡα was up-regulated,and the ratios of mNR2B/tNR2B and pCaMK Ⅱ α/tCaMK Ⅱ α were increased in group RI (P＜0.05 or 0.01).Compared with group RI,the MWT was significantly increased,TWL was prolonged,the expression of tNR2B,mNR2B,tCaMKⅡα and pCaMKⅡα was down-regulated,and the ratios of mNR2B/tNR2B and pCaMK Ⅱ α/tCaMK Ⅱ α were decreased in group RI+ Ro (P＜0.05 or 0.01).Conclusion Enhanced function of NR2B can activate CaMKⅡα during remifentanil-induced hyperalgesia,which may be involved in the mechanism of remifentanil-induced hyperalgesia in a rat model of IP.

The calmodulin gene in Neurospora crassa is required for normal vegetative growth, ultraviolet survival, and sexual development.

We isolated a Neurospora crassa mutant of the calmodulin (cmd) gene using repeat-induced point mutation and studied its phenotypes. The cmd RIP mutant showed a defect in growth, reduced aerial hyphae, decreased carotenoid accumulation, a severe reduction in viability upon ultraviolet (UV) irradiation, and a fertility defect. Moreover, meiotic silencing of the cmd gene resulted in a barren phenotype. In addition, we also performed site-directed mutational analysis of the calcium/calmodulin-dependent kinase-2 (Ca2+/CaMK-2), a target of the CaM protein encoded by the cmd gene. The camk-2 S247A and the camk-2 T267A mutants in a homozygous cross, or in a cross with a Δcamk-2 mutant, displayed an intermediate phenotype, suggesting that serine 247 and threonine 267 phosphorylation sites of the Ca2+/CaMK-2 are essential for full fertility in N. crassa. Therefore, CaM in N. crassa is required for normal vegetative growth, UV survival, and sexual development. Additionally, serine 247 and threonine 267 phosphorylation sites are important for the Ca2+/CaMK-2 function.

Effects of luteolin on CaM-CaMPK signaling pathway in hippocampus in epileptic rats / 国际中医中药杂志

Objective To explore the effects of luteolin on cognition function in pentylenetetrazol (PTZ)-induced epileptic rats and related mechanism.Methods Fifty male SD rats were randomly divided into a normal control group(n=8), a model group(n=12), and groups of 25, 50 mg/kg luteolin(both ofn=11), as well as 100 mg/kg luteolin group(n=8). Those rats were given different doses of luteolin (25, 50 and 100 mg/kg, daily, intragastric administration) for 36 consecutive days. Similarly, rats of the normal control group and the model group were given 0.5% sodium carboxymethyl cellulose suspension liquid via intragastric administration. Thirty minutes later, a model of epilepsy was induced using PTZ (40 mg/kg, daily) via intraperitoneal injection except the control group. Learning and memory of rats were evaluated by Morris water maze and novel objective recognition trials(including escape latency and recognition index). The levels of CaM and CaMPK were determined by ELISA methods, and expression of Ras proteins in the hippocampus were detected by Western Blot.Results Compared with the model group, luteolin treatment groups significantly shorten the escape latency(28.51 ± 3.84 s, 19.77 ± 5.41 s, 14.86 ± 2.76 svs. 37.08 ± 5.18 s) in the Morris water maze, and increased recognition index(18.77% ± 2.02%, 25.06% ± 4.32%, 31.92% ± 2.65%vs. 13.87% ± 2.14%) in the novel objection trial(P<0.05 orP<0.01). Meanwhile, CaM(140.33 ± 13.52 ng/L, 124.26 ± 9.97 ng/L, 113.52 ± 11.57 ng/Lvs. 158.36 ± 10.68 ng/L) and CaMPK(8.25 ± 1.37 ng/ml, 7.69 ± 0.84 ng/ml, 6.74 ± 0.93 ng/mlvs. 9.87 ± 1.02 ng/ml) were significantly decreased(P<0.05 orP<0.01). What’s more, the expression of Ras proteins(0.99 ± 0.08, 0.76 ± 0.07, 0.52 ± 0.07vs. 1.58 ± 0.12) was obviously decreased compared with the model group(P<0.05 orP<0.01).Conclusion Luteolin could effectively improve the cognition dysfunction of epileptic rats, and the mechanism might be relevant to regulate the CaM-CaMPK signaling pathway via down-regulation of CaM, CaMPK, as well as Ras protein.

Role of calcium/calmodulin-dependent kinase Ⅱ alpha in central nucleus of amygdale in fentanyl-induced hyperalgesia in rats: the relationship with mEPSCs / 中华麻醉学杂志

Objective To evaluate the role of calcium/calmodulin-dependent kinase Ⅱ alpha (CaMK Ⅱα) in the central nucleus of the amygdale (CeA) in fentanyl-induced hyperalgesia in rats and the relationship with miniature excitatory postsynaptic currents (mEPSCs).Methods Thirty-two male Sprague-Dawley rats,weighing 50-80 g,in which the CeA was successfully cannulated,were randomly divided into 4 groups (n=8 each) using a random number table:control 1 group (group C1),fentanylinduced hyperalgesia 1 group (group FIH1),KN92 group,and KN93 group.Normal saline was injected subcutaneously,and dimethyl sulfoxide (DMSO) was given into the amygdale in group C1.In group FIH1,fentanyl was injected subcutaneously (60 μg/kg per time,4 times in total,15-min interval,cumulative dose of 240 μg/kg) to establish the model of hyperalgesia.In KN92 and KN93 groups,KN92 and KN93 10 nmol were given into the CeA after establishing the model.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal threshold (TWT) were measured at 6 and 7 h after fentanyl or normal saline injection.Another 12 Sprague-Dawley rats were selected and randomly divided into either control 2 group (group C2) or fentanyl-induced hyperalgesia 2 group (group FIH2) using a random number table with 6 rats in each group.The brains were removed and sliced 12 h later,and the frequency and amplitude of mEPSCs were recorded.KN93 10 nmol was then added to the artificial cerebral spinal fluid,and the frequency and amplitude of mEPSCs were recorded by whole cell patch-clamp technique.Results Compared with group C 1,the MWT and TWT were significantly decreased at 6 h after fentanyl or normal saline injection in FIH1,KN92 and KN93 groups,and at 7 h after fentanyl or normal saline injection in FIH and KN92 groups (P＜0.05).Compared with group FIH1,the MWT and TWT were significantly increased at 7 h after fentanyl or normal saline injection in group KN93 (P＜0.05),and no significant change was found in group KN92 (P＞0.05).Compared with group C2,the frequency and amplitude of mEPSCs were significantly increased before administration of KN93 (P ＜ 0.05),and no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group FIH2 (P＞0.05).Compared with the value before KN93 administration,no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group C2 (P＞0.05),and the frequency and amplitude of mEPSCs were significantly decreased after administration of KN93 in group FIH2 (P＜ 0.05).Conclusion Activation of CaMK Ⅱ α in the CeA enhances synaptic excitation in neurons,which is involved in fentanyl-induced hyperalgesia in rats.

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Potentiated macrophage activation by acid sensing under low adiponectin levels.

Adiponectin can protect against inflammation; one of the mechanisms involves direct, inhibition of macrophages (MΦ). We postulated that adiponectin anti-sense transgenic (AsTg) mice raised in our laboratory are prone to inflammation because of systemic low adiponectin levels. The writhing response to acetic acid was utilized as an in vivo inflammatory model, and using Ca(2)(+), response to the acid was exploited in vitro to evaluate the function of resident peritoneal MΦ. The in vivo response to the acid was increased and the Ca(2)(+) response of MΦ was enhanced in AsTg mice, compared with those in wild type (WT) mice. In parallel with these enhanced responses, MΦ from AsTg mice augmented TNF-α and IL-6 mRNA expression. We further analyzed the enhancement in activity of MΦ from AsTg mice by acid sensing using specific inhibitors, amiloride for acid-sensing ion channels (ASICs) and KB-R7943 for Na(+)/Ca(2)(+) exchangers (NCXs). Our results indicated that in AsTg mice, the Ca(2)(+) response to the acid was facilitated in MΦ by a low threshold of ASIC1 and NCX1 molecules and the activity of these channel was possibly regulated by adiponectin.

Structural and functional diversity in the activity and regulation of DAPK-related protein kinases.

Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field.

Promoter methylation of DAPK1, RAR-β and MGMT in exfoliated cervical cytology and its clinicalapplication / 中华妇产科杂志

Objective To assess the correlation of promoter methylation of DAPK1,RAR-β and MGMT with cervical lesions from cytology to histology,and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia (CIN).Methods A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology,Peking Union Medical Collage Hospital from March 2010 to October 2010.Informed consent was obtained from each woman before the initiation of the study.The methylation seusitive-high resolution melt (MS-HRM) assay was used to evaluate promoter methylation of three genes ( DAPKI,RAR-β and MGMT) in 103 biopsy-confirmed liquid-based cervical cytology samples.Methylation levels and high-risk HPV DNA loading ( HC Ⅱ values) were analyzed in relation to both cytological and histological diagnosis.Results The methylation level of all three genes showed significant difference among the different cytological groups ( P =0.000,0.011 and 0.002,respectively).The methylation level of DAPK1 and RAR-β showed significant difference among the different histological groups ( P =0.000 and 0.021 ),while there was no significant difference for MGMT.DAPK1 methylation levels was 1.47％ in the CIN Ⅱ/high-grade precancerous lesions group,and 20.98％ in the normal/CIN I groups ( P =0.000 ),but there was no significant difference between CIN I/high-grade precancerous lesions and normal/CIN Ⅰ groups for RAR-β and MGMT.The combination of DAPK1/HR-HPV loading showed a sensitivity of 0.825 and an area under the receiver operating characteristic curve (ROC) curve (AUC) of 0.695 as diagnostic methods for detecting CIN Ⅱ/high-grade precancerous lesions.Conclusions DNA methylation such as DAPK1 and RAR-β,in combination with HR-HPV detection,may serve as biomarkers to detect CIN Ⅱ/high-grade precancerous lesions.Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.

Effects of sevoflurane or ischemic preconditioning on the expression of ERK and CaM during lung ischemia-reperfusion in rats / 中华麻醉学杂志

Objectlve To investigate the effects of sevoflurane or ischemic preconditioning on the expression of extracellular signal-regulated kinase (ERK) and calmodulin (CaM) during lung ischemia-reperfusion (I/R)in rats. Methods Twenty-four male SD rats weighing 270-320 g were randomly divided into 4 groups ( n = 6each): sham operation group (group S), group I/R, ischemic preconditioning group (group IP), and sevoflurane preconditioning group (group SP). In group S, the hilum of the left lung was dissociated after thoracotomy but not occluded. In group I/R, lung I/R was produced by occlusion of the hilum of the left lung for 45 min followed by 120 min of reperfusion. In group IP, the hilum of the left lung was occluded for 5 min and unclamped for 5 min for 2 times before the model was established. In group SP, sevoflurane was inhaled for 30 min at the end-tidal concentration of 2.1% before lung ischemia. All rats were sacrificed at 120 min of reperfusion and the lung tissues were taken for determination of the contents of TNF-α and IL-6 by ELISA and the expression of ERK mRNA and CaM mRNA by RT-PCR. Results Compared with group S, the contents of TNF-α and IL-6 and the expression of ERK mRNA and CaM mRNA were significantly increased in group I/R, IP and SP ( P ＜ 0.05). Compared with group I/R, the contents of TNF-α and IL-6 and the CaM mRNA expression were significantly decreased, while the expression of ERK mRNA was significantly increased in group IP and SP ( P ＜ 0.05). There was no significant difference in the each index metioned above between group IP and SP ( P ＞ 0.05 ). Conclusion Sevoflurane preconditioning and ischemic preconditioning can protect the lung from I/R injury through down-regulating the expression of CaM and up-regulating the expression of ERK.