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Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer collateral synapses. Regulator of G protein signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca 2+ ) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca 2+ -activated proteins in CA2 neurons: calcium/calmodulin and CaMKII. Here, we investigated whether RGS14 regulates Ca 2+ signaling in its host CA2 neurons. We found that the nascent LTP of CA2 synapses caused by genetic knockout (KO) of RGS14 in mice requires Ca 2+ -dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report that RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca 2+ transients during structural plasticity induction compared with the Ca 2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca 2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca 2+ elevations in CA2 spines and downstream signaling pathways.

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium-dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Introduction: Brain-derived neurotrophic factor (BDNF) inhibits neuromuscular junction (NMJ) maturation. In this study we investigated the underlying molecular mechanisms of this process., Methods: We used a patch-clamp technique to measure spontaneous synaptic currents (SSCs) from innervated muscle cells in Xenopus nerve-muscle cocultures., Results: In the presence of Ca(2+)/calmodulin-dependent kinase (CaMK) inhibitor KN93, SSC amplitude (226.3 ± 26.5 pA), frequency (30.9 ± 10.1 events/min), and percentage of bell-shaped amplitude distributions (47.1%) were reversed to control levels (286.7 ± 48.2 pA, 26.2 ± 5.8 events/min, and 47.1%, respectively). Depletion of intracellular Ca(2+) by BAPTA-AM or thapsigargin had similar reversal effects to KN93. In addition, cotreatment with both 2-APB (IP3 receptor inhibitor) and TMB-8 (ryanodine receptor inhibitor) also reversed the inhibitory effects of BDNF, as shown by the physiological parameters., Conclusions: CaMK mediates the inhibitory effects of BDNF on NMJ maturation. Ca(2+) released from intracellular stores through either IP3 receptors or ryanodine receptors regulates neurotrophic actions on NMJ maturation., (© 2015 Wiley Periodicals, Inc.)

While the biochemical function of calcium and calmodulin-dependent protein kinase (CCaMK) is well studied, and plants impaired in the expression of CCaMK are known not to be infected by arbuscular mycorrhizal fungi (AMF) in glasshouse studies, the whole-plant and ecological consequences of CCaMK silencing are not well understood. Here we show that three independently transformed lines of Nicotiana attenuata plants silenced in CCaMK (irCCaMK) are neither infected by Rhizophagus irregularis in the glasshouse nor by native fungal inoculum in the field. The overall fungal community of field-grown roots did not differ significantly among empty vector (EV) and the transgenic lines, and the bacterial communities only showed minor differences, as revealed by the alpha-diversity parameters of bacterial OTUs, which were higher in EV plants compared with two of the three transformed lines, while beta-diversity parameters did not differ. Furthermore, growth and fitness parameters were similar in the glasshouse and field. Herbivory-inducible and basal levels of salicylic acid, jasmonic acid and abscisic acid did not differ among the genotypes, suggesting that activation of the classical defence pathways are not affected by CCaMK silencing. Based on these results, we conclude that silencing of CCaMK has few, if any, non-target effects., (© 2015 John Wiley & Sons Ltd.)

In rice, the Ca(2+) /calmodulin (CaM)-dependent protein kinase (CCaMK) OsDMI3 has been shown to be required for abscisic acid (ABA)-induced antioxidant defence. However, it is not clear how OsDMI3 participates in this process in rice. In this study, the cross-talk between OsDMI3 and the major ABA-activated MAPK OsMPK1 in ABA-induced antioxidant defence was investigated. ABA treatment induced the expression of OsDMI3 and OsMPK1 and the activities of OsDMI3 and OsMPK1 in rice leaves. In the mutant of OsDMI3, the ABA-induced increases in the expression and the activity of OsMPK1 were substantially reduced. But in the mutant of OsMPK1, the ABA-induced increases in the expression and the activity of OsDMI3 were not affected. Pretreatments with MAPKK inhibitors also did not affect the ABA-induced activation of OsDMI3. Further, a transient expression analysis in combination with mutant analysis in rice protoplasts showed that OsMPK1 is required for OsDMI3-induced increases in the activities of antioxidant enzymes and the production of H2 O2 . Our data indicate that there exists a cross-talk between OsDMI3 and OsMPK1 in ABA signalling, in which OsDMI3 functions upstream of OsMPK1 to regulate the activities of antioxidant enzymes and the production of H2 O2 in rice., (© 2013 John Wiley & Sons Ltd.)

Calcium and Ca(2+)/calmodulin-dependent protein kinase (CCaMK) plays a critical role in the signaling pathway that establishes root nodule symbiosis and arbuscular mycorrhizal symbiosis. Calcium-dependent autophosphorylation is central to the regulation of CCaMK, and this has been shown to promote calmodulin binding. Here, we report a regulatory mechanism of Medicago truncatula CCaMK (MtCCaMK) through autophosphorylation of S344 in the calmodulin-binding/autoinhibitory domain. The phospho-ablative mutation S344A did not have significant effect on its kinase activities, and supports root nodule symbiosis and arbuscular mycorrhizal symbiosis, indicating that phosphorylation at this position is not required for establishment of symbioses. The phospho-mimic mutation S344D show drastically reduced calmodulin-stimulated substrate phosphorylation, and this coincides with a compromised interaction with calmodulin and its interacting partner, IPD3. Functional complementation tests revealed that the S344D mutation blocked root nodule symbiosis and reduced the mycorrhizal association. Furthermore, S344D was shown to suppress the spontaneous nodulation associated with a gain-of-function mutant of MtCCaMK (T271A), revealing that phosphorylation at S344 of MtCCaMK is adequate for shutting down its activity, and is epistatic over previously identified T271 autophosphorylation. These results reveal a mechanism that enables CCaMK to 'turn off' its function through autophosphorylation., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Endothelial cells are the interface between hemodynamic fluid flow and vascular tissue contact. They actively translate physical and chemical stimuli into intracellular signaling cascades which in turn regulate cell function, and endothelial dysfunction leads to inflammation and diseased conditions. For example, atherosclerosis, a chronic vascular disease, favorably develops in regions of disturbed fluid flow and low shear stress. Apoptosis, or programmed cell death, must be properly regulated to maintain homeostasis in the vascular wall. The loss of apoptosis control, as seen in low shear stress regions, is implicated in various diseases such as atherosclerosis and cancer. Death-associated protein kinase (DAPK) is a pro-apoptotic regulator for various cell types that is localized in the cell cytoskeleton and regulates changes in cytoplasm associated with apoptosis. Yet its role in endothelial cells remains unclear. Laminar shear stress inhibits cytokine, oxidative stress, and serum starvation induced endothelial apoptosis, while extended shearing elicit structural changes and cell alignment. We hypothesize that DAPK potentially plays a role in attenuating endothelial apoptosis in response to shear stress. We found that shear stress regulates DAPK expression and apoptotic activity in endothelial cells. In fact, shear stress alone induces DAPK and apoptosis, but has the opposite effect in the presence of apoptotic triggers such as tissue necrosis factor α (TNFα). This review summarizes mechanisms of endothelial mechanotransduction and apoptosis, and explores the potential of DAPK as a novel signaling pathway involved in mediating protective effects of shear stress on the vasculature., (© 2013.)

Most deaths from breast cancer result from tumor recurrence, but mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is downregulated during tumor recurrence and that Par-4 downregulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer, and Par-4 is downregulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Membranous nephropathy is a glomerular disease typified by a nephrotic syndrome without infiltration of inflammatory cells or proliferation of resident cells. Although the cause of the disease is unknown, the primary pathology involves the generation of autoantibodies against antigen targets on the surface of podocytes. The mechanisms of nephrotic proteinuria, which reflect a profound podocyte dysfunction, remain unclear. We previously found a new gene, c-mip (c-maf-inducing protein), that was associated with the pathophysiology of idiopathic nephrotic syndrome. Here we found that c-mip was not detected in the glomeruli of rats with passive-type Heymann nephritis given a single dose of anti-megalin polyclonal antibody, yet immune complexes were readily present, but without triggering of proteinuria. Rats reinjected with anti-megalin develop heavy proteinuria a few days later, concomitant with c-mip overproduction in podocytes. This overexpression was associated with the downregulation of synaptopodin in patients with membranous nephropathy, rats with passive Heymann nephritis, and c-mip transgenic mice, while the abundance of death-associated protein kinase and integrin-linked kinase was increased. Cyclosporine treatment significantly reduced proteinuria in rats with passive Heymann nephritis, concomitant with downregulation of c-mip in podocytes. Thus, c-mip has an active role in the podocyte disorders of membranous nephropathy.

Background: PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are thought to be the central transducers of oncogenic signals in solid malignancies, and there has been a lot of enthusiasm for developing inhibitors of these pathways for cancer therapy. Some preclinical models have suggested that combining inhibitors of both parallel pathways may be more efficacious, but it remains unknown whether dual inhibition with high enough concentrations of the drugs to achieve meaningful target inhibition is tolerable in a clinical setting. Furthermore, the predictive factors for dual inhibition are unknown., Methods: Non-small cell lung cancer (NSCLC) cell lines (n=12) with the most frequent oncogenic backgrounds (K-Ras mut n=3, EGFR mut n=3, ALK translocated n=3, and triple-negative n=3) were exposed to PI3K inhibitors (ZSTK474, PI-103) or MEK inhibitor (CI-1040) alone or in combination and analysed with an MTS growth/cytotoxicity assay and statistically by combination index analysis. The activity of the intracellular signaling pathways in response to the inhibitor treatments was analysed with a western blot using phospho-specific antibodies to AKT, ERK1/2, S6, and 4E-BPI. For the differential dosing schedule experiments, additional breast and colon cancer cell lines known to be sensitive to dual inhibition were included., Results: Two of the 12 NSCLC cell lines tested, H3122 (ALK translocated) and H1437 (triple-negative), showed increased cytotoxicity upon dual MEK and PI3K inhibition. Furthermore, MDA-MB231 (breast) and HCT116 (colon), showed increased cytotoxicity upon dual inhibition, as in previous studies. Activation of parallel pathways in the dual inhibition-sensitive lines was also noted in response to single inhibitor treatment. Otherwise, no significant differences in downstream intracellular pathway activity (S6 and 4E-BPI) were noted between PI3K alone and dual inhibition other than the increased cytotoxicity of the latter. In the alternative dosing schedules two out of the four dual inhibition-sensitive cell lines showed similar cytotoxicity to continuous PI3K and short (15min) MEK inhibition treatment., Conclusions: Therapy with a dual PI3K and MEK inhibitor combination is more efficient than either inhibitor alone in some NSCLC cell lines. Responses to dual inhibition were not associated with any specific oncogenic genotype and no other predictive factors for dual inhibition were noted. The maximal effect of the dual PI3K and MEK inhibition can be achieved with alternative dosing schedules which are potentially more tolerable clinically.

The major neuronal gap junction protein connexin36 (Cx36) exhibits the remarkable property of "run-up", in which junctional conductance typically increases by 10-fold or more within 5-10min following cell break-in with patch pipettes. Such conductance "run-up" is a unique property of Cx36, as it has not been seen in cell pairs expressing other connexins. Because of the recent observation describing CaMKII binding and phosphorylation sites in Cx36 and evidence that calmodulin dependent protein kinase II (CaMKII) may potentiate electrical coupling in neurons of teleosts, we have explored whether CaMKII activates mammalian Cx36. Consistent with this hypothesis, certain Cx36 mutants lacking the CaMKII binding and phosphorylation sites or wild type Cx36 treated with certain cognate peptides corresponding to binding or phosphorylation sites blocked or strongly attenuated run-up of junctional conductance. Likewise, KN-93, an inhibitor of CaMKII, blocked run-up, as did a membrane permeable peptide corresponding to the CaMKII autoinhibitory domain. Furthermore, run-up was blocked by phosphatase delivered within the pipette and not affected by treatment with the phosphatase inhibitor okadaic acid. These results imply that phosphorylation by CaMKII strengthens junctional currents of Cx36 channels, thereby conferring functional plasticity on electrical synapses formed of this protein., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gonadotrophin-releasing hormone (GnRH) regulates reproduction via binding a G-protein coupled receptor on the surface of the gonadotroph, through which it transmits signals, mostly via the mitogen-activated protein (MAPK) cascade, to increase synthesis of the gonadotrophin hormones: luteinising hormone (LH) and follicle-stimulating hormone (FSH). Activation of the MAPK cascade requires an elevation in cytosolic Ca(2+) levels, which is a result of both calcium influx and mobilisation from intracellular stores. However, Ca(2+) also transmits signals via an MAPK-independent pathway, through binding calmodulin (CaM), which is then able to bind a number of proteins to impart diverse downstream effects. Although the ability of GnRH to activate CaM was recognised over 20 years ago, only recently have some of the downstream effects been elucidated. GnRH was shown to activate the CaM-dependent phosphatase, calcineurin, which targets gonadotrophin gene expression both directly and indirectly via transcription factors such as nuclear factor of activated T-cells and Nur77, the Transducer of Regulated CREB (TORC) co-activators and also the prolyl isomerase, Pin1. Gonadotrophin gene expression is also regulated by GnRH-induced CaM-dependent kinases (CaMKs); CaMKI is able to derepress the histone deacetylase-inhibition of β-subunit gene expression, whereas CaMKII appears to be essential for the GnRH-activation of all three subunit genes. Asides from activating gonadotrophin gene expression, GnRH also exerts additional effects on gonadotroph function, some of which clearly occur via CaM, including the proliferation of immature gonadotrophs, which is dependent on calcineurin. In this review, we summarise these pathways, and discuss the additional functions that have been proposed for CaM with respect to modifying GnRH-induced signalling pathways via the regulation of the small GTP-binding protein, Gem, and/or the regulator of G-protein signalling protein 2., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology.)

A field for cancerization (field defect), where genetic and epigenetic alterations are accumulated in normal-appearing tissues, is involved in human carcinogenesis, especially cancers associated with chronic inflammation. Although aberrant DNA methylation is involved in the field defect and induced by chronic inflammation, it is still unclear for trimethylation of histone H3 lysine 27 (H3K27me3), which is involved in gene repression independent of DNA methylation and functions as a pre-mark for aberrant DNA methylation. In this study, using a mouse colitis model induced by dextran sulfate sodium (DSS), we aimed to clarify whether aberrant H3K27me3 is induced by inflammation and involved in a field defect. ChIP-on-chip analysis of colonic epithelial cells revealed that H3K27me3 levels were increased or decreased for 266 genomic regions by aging, and more extensively (23 increased and 3574 decreased regions) by colitis. Such increase or decrease of H3K27me3 was induced as early as 2 weeks after the initiation of DSS treatment, and persisted at least for 16 weeks even after the inflammation disappeared. Some of the aberrant H3K27me3 in colonic epithelial cells was carried over into colon tumors. Furthermore, H3K27me3 acquired at Dapk1 by colitis was followed by increased DNA methylation, supporting its function as a pre-mark for aberrant DNA methylation. These results demonstrated that aberrant H3K27me3 can be induced by exposure to a specific environment, such as colitis, and suggested that aberrant histone modification, in addition to aberrant DNA methylation, is involved in the formation of a field defect.

Background: Calcium serves as a versatile messenger in many adaptation and developmental processes in plants. Ca2+ signals are represented by stimulus-specific spatially and temporally defined Ca2+ signatures. These Ca2+ signatures are detected, decoded and transmitted to downstream responses by a complex toolkit of Ca2+ binding proteins that function as Ca2+ sensors., Scope of Review: This review will reflect on advancements in monitoring Ca2+ dynamics in plants. Moreover, it will provide insights in the extensive and complex toolkit of plant Ca2+ sensor proteins that relay the information presented in the Ca2+ signatures into phosphorylation events, changes in protein-protein interaction or regulation of gene expression., Major Conclusions: Plants' response to signals is encoded by different Ca2+ signatures. The plant decoding Ca2+ toolkit encompasses different families of Ca2+ sensors like Calmodulins (CaM), Calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs) and their interacting kinases (CIPKs). These Ca2+ sensors are encoded by complex gene families and form intricate signaling networks in plants that enable specific, robust and flexible information processing., General Significance: This review provides new insights about the biochemical regulation, physiological functions and of newly identified target proteins of the major plant Ca2+ sensor families. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling., (Copyright © 2011. Published by Elsevier B.V.)

Calcium/calmodulin-stimulated protein kinases can be classified as one of two types - restricted or multifunctional. This family of kinases contains several structural similarities: all possess a calmodulin binding motif and an autoinhibitory region. In addition, all of the calcium/calmodulin-stimulated protein kinases examined in this chapter are regulated by phosphorylation, which either activates or inhibits their kinase activity. However, as the multifunctional calcium/calmodulin-stimulated protein kinases are ubiquitously expressed, yet regulate a broad range of cellular functions, additional levels of regulation that control these cell-specific functions must exist. These additional layers of control include gene expression, signaling pathways, and expression of binding proteins and molecular targeting. All of the multifunctional calcium/calmodulin-stimulated protein kinases examined in this chapter appear to be regulated by these additional layers of control, however, this does not appear to be the case for the restricted kinases.

Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca(2+)-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5-50 μM), sorafenib (1-40 μM), staurosporine (20-500 nM), or thapsigargin (20-500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca(2+) chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10-30 μg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1-10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca(2+) mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α(1)/α(2)-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca(2+)-dependent signaling, including CaMKI independent of AMPKα(1)/α(2). Our data also suggest that AMPKα(1)/α(2) might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.

Preventing Ca(2+)-leak during diastole may provide a means to improve overall cardiac function. The immunosuppressant FK506-binding protein 12.6 (FKBP12.6) regulates ryanodine receptor-2 (RyR2) gating and binds to and inhibits calcineurin (Cn). It is also involved in the pathophysiology of heart failure (HF). Here, we investigated the effects of FKBP12.6 over-expression and gender on Ca(2+)-handling proteins (RyR2, SERCA2a/PLB, and NCX), and on pro-(CaMKII, Cn/NFAT) and anti-hypertrophic (GSK3β) signalling pathways in a thoracic aortic constriction (TAC) mouse model. Wild type mice (WT) and mice over-expressing FKBP12.6 of both genders underwent TAC or sham-operation (Sham). FKBP12.6 over-expression ameliorated post-TAC survival rates in both genders. Over time, FKBP12.6 over-expression reduced the molecular signature of left ventricular hypertrophy (LVH) and the transition to HF (BNP and β-MHC mRNAs) and attenuated Cn/NFAT activation in TAC-males only. The gender difference in pro- and anti-hypertrophic LVH signals was time-dependent: TAC-females exhibited earlier pathological LVH associated with concomitant SERCA2a down-regulation, CaMKII activation, and GSK3β inactivation. Both genotypes showed systolic dysfunction, possibly related to down-regulated RyR2, but only FK-TAC-males exhibited preserved diastolic LV function. Although FKBP12.6 over-expression did not impact the vicious cycle of TAC-induced HF, this study reveals some subtle sequential and temporal gender differences in Ca(2+)-signalling pathways of pathological LVH.

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis., (Copyright © 2011 Wiley Periodicals, Inc.)

The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB(+1), a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB(+1) properties shift from a proteasomal ubiquitin-fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB(+1). In these mice, UBB(+1) protein is scarcely detectable in the neuronal cell population. Accumulation of UBB(+1) commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB(+1) protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB(+1) is continuously degraded and disease-related UBB(+1) accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB(+1) and its role in neurodegenerative disease., ((c) 2010 Wiley-Liss, Inc.)

Ca(2+) is a second messenger in pathways that transduce external signals and activate cellular processes in plants and animals. Ca(2+)-mediated signal transduction is involved in key pathways that contribute to a variety of fundamental physiological processes in eukaryotic cells. However, little is known about the molecular mechanisms of Ca(2+)-mediated signal transduction in filamentous fungi. In this study, the MoCMK1 gene, encoding a putative Ca(2+)/calmodulin-dependent kinase, was identified in the rice blast fungus Magnaporthe oryzae. Three MoCMK1 deletion mutants were obtained by a targeted gene replacement. Colonies of the MoCMK1 mutants had sparse aerial hyphae and fewer conidia than the wild-type strain on complete medium. Conidial germination and appressorial formation were delayed in the DeltaMocmk1 mutants. In spray inoculation tests, DeltaMocmk1 mutants exhibited a weakened ability to infect the susceptible rice cultivar CO-39, compared to the wild-type strain Guy11. These results showed that MoCMK1 plays key roles in the pathogenicity of the rice blast fungus., (Copyright 2009 Elsevier GmbH. All rights reserved.)

NMDA receptors (NMDAR) contribute to neuronal development throughout the CNS. However, their mode(s) of activation preceding synaptic maturation is unclear, as they are not co-localized with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) which normally provide sufficient depolarization to relieve voltage-dependent blockade by Mg(2+). We used cerebellar granule neurons (CGNs) cultured at a near-physiological KCl concentration to examine maturation-dependent changes in NMDAR responses. In contrast, most studies use KCl-supplemented medium to promote survival. At 2-4 days in vitro CGNs: (i) express developmental markers resembling the in vivo migratory phenotype; (ii) maintain a basal amount of calcium responsive element-binding protein phosphorylation that requires NMDARs and calcium/calmodulin-dependent kinases, but not AMPARs; (iii) exhibit NMDA-mediated Ca(2+) influx not effectively blocked by ambient Mg(2+) (0.75 mM) or AMPARs; (iv) maintain a more depolarized resting membrane potential and increased resistance compared to synaptically-connected CGNs. Moreover, migrating CGNs in explant cultures demonstrate NMDA-mediated Ca(2+) influx not effectively blocked by 0.75 mM Mg(2+), and NMDAR but not AMPAR antagonists slow migration. These data suggest the biophysical properties of immature CGNs render NMDARs less sensitive to Mg(2+) blockade, enhancing the likelihood of activation in the absence of AMPAR depolarization.

Death-associated protein kinase (DAPK) is a serine/threonine kinase that participates in the modulation of apoptosis and tumor suppression. Our previous study revealed high levels of DAPK protein expression in differentiated endometrial adenocarcinoma cells. To clarify the role of DAPK in human endometrial adenocarcinomas, we down-regulated endogenous DAPK expression in HHUA cells, a well-differentiated endometrial adenocarcinoma cell line, using specific small-interfering RNAs (siRNAs). The suppression of endogenous DAPK expression triggered apoptosis in HHUA cells, as evidenced by an increase in the sub-G1 DNA content in flow cytometric analyses. The apoptosis induced by the DAPK siRNA transfections was caspase-dependent, as characterized by the activations of caspase-3, -8 and -9. RNase protection assays detected higher levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), DR4 and DR5 transcripts in the DAPK siRNA-transfected HHUA cells than in the control siRNA-transfected cells. Consistent with these findings, enzyme-linked immunosorbent assays revealed that the DAPK siRNA transfections significantly increased the secretion of TRAIL protein from the cells. Treatment with recombinant human TRAIL protein dose-dependently suppressed the cell viability of HHUA cells. The present findings reveal that down-regulation of endogenous DAPK expression in HHUA cells induces caspase-dependent apoptosis, possibly through increased TRAIL, DR4 and DR5 signaling, thereby suggesting that DAPK expression is essential for HHUA cell survival. Consequently, endogenous DAPK mRNA may represent a potential candidate for molecularly targeted anticancer therapies.

Outside of the TLR paradigm, there is little understanding of how pathogen recognition at the cell surface is linked to functional responses in cells of the innate immune system. Recent work in this area demonstrates that the yeast particle zymosan, by binding to the beta-glucan receptor Dectin-1, activates an ITAM-Syk-dependent pathway in dendritic cells, which is required for optimal cytokine production and generation of an oxidative burst. It remains unclear how activation of Syk is coupled to effector mechanisms. In human macrophages, zymosan rapidly activated a calcium-dependent pathway downstream of Dectin-1 and Syk that led to activation of calmodulin-dependent kinase II and Pyk2. Calmodulin-dependent kinase and Pyk2 transduced calcium signals into activation of the ERK-MAPK pathway, CREB, and generation of an oxidative burst, leading to downstream production of IL-10. These observations identify a new calcium-mediated signaling pathway activated by zymosan and link this pathway to both inflammatory and anti-inflammatory responses in macrophages.

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

Death-associated protein kinase (DAPK) is a key player in multiple cell death signaling pathways. We report that DAPK is regulated by DANGER, a partial MAB-21 domain-containing protein. DANGER binds directly to DAPK and inhibits DAPK catalytic activity. DANGER-deficient mouse embryonic fibroblasts and neurons exhibit greater DAPK activity and increased sensitivity to cell death stimuli than do wild-type control cells. In addition, DANGER-deficient mice manifest more severe brain damage after acute excitotoxicity and transient cerebral ischemia than do control mice. Accordingly, DANGER may physiologically regulate the viability of neurons and represent a potential therapeutic target for stroke and neurodegenerative diseases.

Death-associated protein kinase (DAPK) is a stress-regulated protein kinase that mediates a range of processes, including signal-induced cell death and autophagy. Although the kinase domain of DAPK has a range of substrates that mediate its signalling, the additional protein interaction domains of DAPK are relatively ill defined. This review will summarize our current knowledge of the DAPK interactome, the use of peptide aptamers to define novel protein-protein interaction motifs, and how these new protein-protein interactions give insight into DAPK functions in diverse cellular processes, including growth factor signalling, the regulation of autophagy, and its emerging role in the regulation of immune responses.

Our previous studies have found that protein kinase C (PKC) alpha and epsilon take part in the regulation of calcium sensitization and vascular reactivity after hemorrhagic shock. However, the regulatory mechanism is still unclear. The aim of this study was to investigate the role of PKC-potentiated inhibitory protein for protein phosphatase 1 of 17 kDa (CPI-17) and zipper-interacting protein kinase (ZIPK) in the regulation of PKC-alpha and PKC-epsilon on vascular calcium sensitivity after hemorrhagic shock. Using superior mesenteric artery from hemorrhagic shock rats (at 40 mmHg for 2 h), the effects of PKC-alpha and PKC-epsilon agonists on calcium sensitivity and the phosphorylation of 20-kDa myosin light chain (MLC20), and the modulating effects of CPI-17 and ZIPK in this process were observed. In hypoxia-treated vascular smooth muscle cells (VSMCs), the effects of PKC-alpha and PKC-epsilon agonists on protein expression and activity of CPI-17 and ZIPK and the modulating effects of CPI-17 and ZIPK on the regulation of PKC-alpha and PKC-epsilon on the activity of myosin light chain phosphatase (MLCP) were observed. The results indicated that PKC-alpha and PKC-epsilon agonists thymelea toxin and carbachol seemed to increase the calcium sensitivity and MLC20 phosphorylation of superior mesenteric artery after hemorrhagic shock and antagonize the increase of MLCP activity in VSMC after hypoxia. The effects of PKC-alpha and PKC-epsilon agonists were abolished by CPI-17 and ZIPK neutralizing antibody. The protein expression and activity of CPI-17 and ZIPK in VSMC were suppressed after hypoxia but increased by PKC-alpha and PKC-epsilon agonists. Immunoprecipitation experiment showed that PKC-alpha and PKC-epsilon, especially PKC-epsilon, may directly bind with ZIPK but not CPI-17 in VSMC, and ZIPK may directly bind with CPI-17. These results suggest that CPI-17 and ZIPK participate in the regulation of PKC-alpha and PKC-epsilon on vascular calcium sensitivity after hemorrhagic shock. Protein kinase C alpha and epsilon regulation of MLCP-MLC20 phosphorylation pathway in the process of VSMC contraction is mainly through ZIPK; CPI-17 may play an indirect modulating role.

In this review, we first describe the mechanisms by which the epidermal growth factor receptor generates a Ca(2+) signal and, subsequently, we compile the available experimental evidence regarding the role that the Ca(2+)/calmodulin complex, formed after the rise in cytosolic free Ca(2+) concentration, exerts on the receptor. We focus not only on the indirect action that Ca(2+)/calmodulin exerts on the epidermal growth factor receptor, as a result of the activation of distinct calmodulin-dependent kinases, but also, and more extensively, on the direct interaction of Ca(2+)/calmodulin with the receptor. We also describe several mechanistic models that could account for the Ca(2+)/calmodulin-mediated regulation of epidermal growth factor receptor activity. The control exerted by calmodulin on distinct epidermal growth factor receptor-mediated cellular functions is also discussed. Finally, the phosphorylation of this Ca(2+) sensor by the epidermal growth factor receptor is highlighted.

Death-associated protein kinase (DAPK) regulates many distinct signalling events, including apoptosis, autophagy and membrane blebbing. The role of DAPK in the blebbing process is only beginning to be understood and, in this review, we will first summarize what is known about the cytoskeletal proteins and signalling cascades that participate in bleb growth and retraction and then highlight how DAPK integrates with these processes. Membrane blebs are quasispherical cellular protrusions that have a lifetime of approximately 2 min. During expansion, blebs are initially devoid of actin, although actomyosin contractions provide the motive force for growth. Once growth slows, an actin cortex reforms and actin-bundling and contractile proteins are recruited. Finally, myosin contraction powers bleb retraction into the cell body. Blebbing occurs in a variety of cell types, from cancerous cells to embryonic cells, and can be seen in cellular phenomena as diverse as cell spreading, movement, cytokinesis and cell death. Although the machinery that executes this is still undefined in detail, the conservation of blebbing phenomenon suggests a fundamental role in metazoans and DAPK offers a door to further dissect this fascinating process.

Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

Autophagy is an evolutionarily conserved lysosomal pathway used to degrade and recycle long-lived proteins and cytoplasmic organelles. This homeostatic ability makes autophagy an important pro-survival mechanism in response to several stresses, such as nutrient starvation, hypoxia, damaged mitochondria, protein aggregation and pathogens. However, several recent studies have highlighted that autophagy also acts as a pro-death mechanism. What on the surface seem like conflicting roles of autophagy may be explained by the fact that the decision between pro-survival and pro-death is determined by the level of activation. A better understanding of autophagy signaling pathways will be helpful to elucidate how the level of autophagy is precisely regulated under different conditions and eventually how the final outcome is decided. In this review, we briefly discuss the pro-survival and pro-death roles of autophagy, and then discuss the mechanism by which autophagy is regulated, mainly focusing on death-associated protein kinase in the nematode Caenorhabditis elegans.

The extracellular environment and tissue architecture contribute to proper cell function and growth control. Cells growing in monolayers on standard polystyrene tissue culture plates differ in their shape, growth rate and response to external stimuli, compared with cells growing in vivo. Here, we showed that the EGFR (epidermal growth factor receptor) ligand heparin-binding EGF-like growth factor (HB-EGF) strongly stimulated cell growth in nude mice, but not in cells cultured in vitro. We explored the effects of HB-EGF on cell growth under various cell culture conditions and found that growth promotion by HB-EGF was needed in three-dimensional (3D) or two-dimensional (2D) culture systems in which cell-matrix adhesion was reduced. Under such conditions, cell growth was extremely suppressed in the absence of HB-EGF, but markedly potentiated in the presence of HB-EGF. When the integrin signal was reduced using antibodies or knockout of either integrin beta1 or focal adhesion kinase (FAK), cells showed HB-EGF-dependent growth. We also showed that EGF, transforming growth factor-alpha (TGFalpha) or ligands of other receptor tyrosine kinases (RTKs) stimulated cell growth in 3D culture, but not in tissue culture plates. These results indicate that the integrin signal was sufficient to support cell growth in 2D tissue culture plates without addition of the growth factor, whereas stimulation by growth factors was clearly demonstrated in culture systems in which integrin signals were attenuated.

The nucleotide receptor P2Y(1) regulates a variety of physiological processes and is involved in platelet aggregation. Using human P2Y(1)-receptors C-terminally fused with a fluorescent protein, we studied the role of potential receptor phosphorylation sites in receptor internalization and beta-arrestin-2 translocation by means of confocal microscopy. Three receptor constructs were generated that lacked potential phosphorylation sites in the third intracellular loop, the proximal C terminus, or the distal C terminus. The corresponding receptor constructs were expressed in human embryonic kidney (HEK)-293 cells and stimulated with 100 muM ADP. Rapid receptor internalization was observed for the wild-type receptor and from those constructs mutated in the third intracellular loop and the proximal C terminus. However, the construct lacking phosphorylation sites at the distal C terminus did not show receptor internalization upon stimulation. The microscopic data were validated by HA-tagged receptor constructs using a cell surface enzyme-linked immunosorbent assay. P2Y(1)-receptor stimulated beta-arrestin-2-yellow fluorescent protein (YFP) translocation followed the same pattern as receptor internalization. Hence, no beta-arrestin-2-YFP translocation was observed when the distal C-terminal phosphorylation sites were mutated. Individual mutations indicate that residues Ser352 and Thr358 are essential for receptor internalization and beta-arrestin-2-YFP translocation. In contrast, protein kinase C (PKC)-mediated receptor desensitization was not affected by mutation of potential phosphorylation sites in the distal C terminus but was prevented by mutation of potential phosphorylation sites in the proximal C terminus. P2Y(1)-receptor internalization in HEK-293 cells was not blocked by inhibitors of PKC and calmodulin-dependent protein kinase. Thus, we conclude that P2Y(1)-receptor desensitization and internalization are mediated by different phosphorylation sites and kinases.

Nurr1 is a transcription factor specific for the development and maintenance of the midbrain dopamine (DA) neurons. Exogenous Nurr1 in neural precursor (NP) cells induces the differentiation of DA neurons in vitro that are capable of reversing motor dysfunctions in a rodent model for Parkinson disease. The promise of this therapeutic approach, however, is unclear due to poor cell survival and phenotype loss of DA cells after transplantation. We herein demonstrate that Nurr1 proteins undergo ubiquitin-proteasome-system-mediated degradation in differentiating NP cells. The degradation process is activated by a direct Akt-mediated phosphorylation of Nurr1 proteins and can be prevented by abolishing the Akt-target sequence in Nurr1 (Nurr1(Akt)). Overexpression of Nurr1(Akt) in NP cells yielded DA neurons in which Nurr1 protein levels were maintained for prolonged periods. The sustained Nurr1 expression endowed the Nurr1(Akt)-induced DA neurons with resistance to toxic stimuli, enhanced survival, and sustained DA phenotypes in vitro and in vivo after transplantation.

Background: The cardiomyocyte is a prime example of inherently complex biological system with inter- and cross-connected feedback loops in signalling, forming the basic properties of intracellular homeostasis. Functional properties of cells and tissues have been studied e.g. with powerful tools of genetic engineering, combined with extensive experimentation. While this approach provides accurate information about the physiology at the endpoint, complementary methods, such as mathematical modelling, can provide more detailed information about the processes that have lead to the endpoint phenotype., Results: In order to gain novel mechanistic information of the excitation-contraction coupling in normal myocytes and to analyze sophisticated genetically engineered heart models, we have built a mathematical model of a mouse ventricular myocyte. In addition to the fundamental components of membrane excitation, calcium signalling and contraction, our integrated model includes the calcium-calmodulin-dependent enzyme cascade and the regulation it imposes on the proteins involved in excitation-contraction coupling. With the model, we investigate the effects of three genetic modifications that interfere with calcium signalling: 1) ablation of phospholamban, 2) disruption of the regulation of L-type calcium channels by calcium-calmodulin-dependent kinase II (CaMK) and 3) overexpression of CaMK. We show that the key features of the experimental phenotypes involve physiological compensatory and autoregulatory mechanisms that bring the system to a state closer to the original wild-type phenotype in all transgenic models. A drastic phenotype was found when the genetic modification disrupts the regulatory signalling system itself, i.e. the CaMK overexpression model., Conclusion: The novel features of the presented cardiomyocyte model enable accurate description of excitation-contraction coupling. The model is thus an applicable tool for further studies of both normal and defective cellular physiology. We propose that integrative modelling as in the present work is a valuable complement to experiments in understanding the causality within complex biological systems such as cardiac myocytes.

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca2+ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca2+ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca2+ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca2+ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca2+ spiking decoding.

Background: Low plasma high-density lipoprotein (HDL) is associated with elevated cardiovascular risk and aspects of the metabolic syndrome. We hypothesized that HDL modulates glucose metabolism via elevation of plasma insulin and through activation of the key metabolic regulatory enzyme, AMP-activated protein kinase, in skeletal muscle., Methods and Results: Thirteen patients with type 2 diabetes mellitus received both intravenous reconstituted HDL (rHDL: 80 mg/kg over 4 hours) and placebo on separate days in a double-blind, placebo-controlled crossover study. A greater fall in plasma glucose from baseline occurred during rHDL than during placebo (at 4 hours rHDL=-2.6+/-0.4; placebo=-2.1+/-0.3 mmol/L; P=0.018). rHDL increased plasma insulin (at 4 hours rHDL=3.4+/-10.0; placebo= -19.2+/-7.4 pmol/L; P=0.034) and also the homeostasis model assessment beta-cell function index (at 4 hours rHDL=18.9+/-5.9; placebo=8.6+/-4.4%; P=0.025). Acetyl-CoA carboxylase beta phosphorylation in skeletal muscle biopsies was increased by 1.7+/-0.3-fold after rHDL, indicating activation of the AMP-activated protein kinase pathway. Both HDL and apolipoprotein AI increased glucose uptake (by 177+/-12% and 144+/-18%, respectively; P<0.05 for both) in primary human skeletal muscle cell cultures established from patients with type 2 diabetes mellitus (n=5). The mechanism is demonstrated to include stimulation of the ATP-binding cassette transporter A1 with subsequent activation of the calcium/calmodulin-dependent protein kinase kinase and the AMP-activated protein kinase pathway., Conclusions: rHDL reduced plasma glucose in patients with type 2 diabetes mellitus by increasing plasma insulin and activating AMP-activated protein kinase in skeletal muscle. These findings suggest a role for HDL-raising therapies beyond atherosclerosis to address type 2 diabetes mellitus.

Background: The initial development of pulsed electromagnetic field (PEMF) therapy and its evolution over the last century for use in clinical surgery has been slow, primarily because of lack of scientifically-derived, evidence-based knowledge of the mechanism of action., Objective: Our objective was to review the major scientific breakthroughs and current understanding of the mechanism of action of PEMF therapy, providing clinicians with a sound basis for optimal use., Methods: A literature review was conducted, including mechanism of action and biologic and clinical studies of PEMF. Using case illustrations, a holistic exposition on the clinical use of PEMF in plastic surgery was performed., Results: PEMF therapy has been used successfully in the management of postsurgical pain and edema, the treatment of chronic wounds, and in facilitating vasodilatation and angiogenesis. Using scientific support, the authors present the currently accepted mechanism of action of PEMF therapy., Conclusions: This review shows that plastic surgeons have at hand a powerful tool with no known side effects for the adjunctive, noninvasive, nonpharmacologic management of postoperative pain and edema. Given the recent rapid advances in development of portable and economical PEMF devices, what has been of most significance to the plastic surgeon is the laboratory and clinical confirmation of decreased pain and swelling following injury or surgery.

Certain characteristics of tumor cells make it possible to develop rational strategies for targeting tumors without harming normal cells. These include the presence of cell surface molecules that characterize the current state of the tumor (e.g. CD30 on Hodgkin lymphoma cells) and the genetic and epigenetic changes that activate oncogenes and inactivate tumor suppressor genes (e.g. the inactivation of tumor suppressor gene DAPK2 in Hodgkin lymphoma cells, which blocks apoptosis). We have developed a novel tumor-targeting fusion protein by combining a selective ligand (CD30L) with a constitutively active version of DAPK2 (DAPK2'-CD30L), thus increasing tumor specificity and reducing systemic toxicity. We showed that this immunokinase fusion protein induces apoptosis specifically in CD30(+)/DAPK2(-) tumor cells in vitro and significantly prolonged overall survival in a disseminated Hodgkin lymphoma xenograft SCID mouse model. Therapeutic strategies based on the cell-specific restoration of a defective, tumor-suppressing kinase demonstrate the feasibility of targeted therapy using recombinant immunokinases.

This article highlights studies published during the past year that represent significant scientific achievements in the world of calmodulin kinase cascades. Calmodulin is the primary receptor for calcium present in all cells. The binding of its calcium ligand results in a conformational change in calmodulin, which allows the calcium-calmodulin complex to interact with many different targets. In the studies to be summarized in this review, the particular calmodulin cascade involved is shown to be the pathway responsible for important biological responses, including long-term memory formation, dendritic cell survival, hypercapnia, neuronal migration, synapse formation, autophagy, fatty acid oxidation, and energy balance. In some cases, the pathway was previously unknown, and the identification of the calmodulin cascade represents the definition of roles. In other cases, manipulating the cascade has suggested therapeutic approaches to certain diseases, most significantly, type 2 diabetes and obesity.

Oral glucose uptake alters the function of immune cells and an elevation of systemic CXCL8 was described. Monocytes secrete high amounts of CXCL8 and therefore it was analyzed whether glucose or insulin upregulate monocytic CXCL8 release. Incubation of monocytes with insulin for 2h induced CXCL8 mRNA and secretion whereas glucose had no effect. Inhibition of the phosphatidylinositol 3-kinase by wortmannin or the mammalian target of rapamycin by rapamycin did not influence insulin-mediated CXCL8 induction. In contrast, blockage of the ERK-specific MAP kinase MEK with PD98059, that prevents phosphorylation of ERK1/ERK2, abrogated insulin-induced CXCL8 release in primary monocytes. To investigate the in vivo effect of oral glucose uptake, monocytes of healthy probands were isolated in the fasted state and 2h after glucose ingestion and CXCL8 mRNA and protein were increased in the latter. CXCL8 was also higher when determined in the cell lysate of leukocytes 2h after glucose uptake whereas plasma CXCL8 levels were significantly reduced. In summary, these data indicate that oral glucose uptake in insulin-sensitive adults is associated with elevated monocytic and reduced systemic CXCL8.

In the nervous system, many intracellular responses to elevated calcium are mediated by CaM kinases (CaMKs), a family of protein kinases whose activities are initially modulated by binding Ca(2+)/calmodulin and subsequently by protein phosphorylation. One member of this family, CaMKII, is well-established for its effects on modulating synaptic plasticity and learning and memory. However, recent studies indicate that some actions on neuronal development and function attributed to CaMKII may instead or in addition be mediated by other members of the CaMK cascade, such as CaMKK, CaMKI, and CaMKIV. This review summarizes key neuronal functions of the CaMK cascade in signal transduction, gene transcription, synaptic development and plasticity, and behavior. The technical challenges of mapping cellular protein kinase signaling pathways are also discussed.

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca(2+)/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5'-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5' of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

Endothelial cell lipotoxicity mediated by accumulation of free fatty acids is an early event in the pathogenesis of atherosclerosis. The energy-sensor AMP-activated protein kinase (AMPK) is a key regulator of endothelial cell lipid metabolism. To test the hypothesis that bradykinin (BK) regulates AMPK and fatty acid oxidation in endothelium, stimulations of bovine aortic endothelial cells (BAECs) with bradykinin were performed. BK stimulation caused a 2.3-fold increase in AMPK activity (p<0.05). Activation of AMPK by BK in BAECs was inhibited by STO-609, an inhibitor of calmodulin-dependent kinase kinase (CaMKK), which is a known kinase upstream of AMPK. BK stimulation of BAECs also increased phosphorylation of acetyl-CoA carboxylase and this was inhibited by both STO-609 and over expression of an adenovirus encoded AMPK dominant negative (Ad-AMPK-DN). Furthermore, BK caused a 1.7-fold increase in palmitate oxidation in BAECs (p<0.05) and this increase was completely inhibited by the Ad-AMPK-DN (p<0.005). Inhibition of AMPK activation in response to BK by STO-609 had no effect on activating phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1177), consistent with CaMKK and AMPK not being required for phosphorylation of eNOS in response to BK. In conclusion, BK stimulates endothelial cell fatty acid oxidation by CaMKK-dependent activation of AMPK. The effect of BK on endothelial lipid metabolism represents a novel pathway for targeting fatty acid mediated endothelial cell dysfunction.