Schwann cell lines derived from malignant peripheral nerve sheath tumors respond abnormally to platelet-derived growth factor-BB

Neurofibromatosis type 1 (NF1) is a genetic disease caused by the loss of neurofibromin, which can lead to formation of highly invasive malignant peripheral nerve sheath tumors (MPNST). We characterized platelet-derived growth factor-beta (PDGF-beta) receptor expression levels and signal transduction pathways in NF1 MPNST cell lines and compared them with the expression of PDGF-beta receptors in normal human Schwann cells (nhSC). As examined by Western blotting, PDGF-beta receptor expression levels were similar in nhSC and NF1 MPNST cell lines. MAPK and Akt also were phosphorylated in both cell types to a similar degree in response to PDGF B chains (PDGF-BB). However, increased intracellular calcium (Ca2+) levels in response to PDGF-BB were observed only in the NF1 MPNST cell lines; nhSC did not show any increase in intracellular calcium when stimulated with PDGF-BB. The calcium response in NF1 MPNST cell lines was blocked with thapsigargin, suggesting that the PDGF-BB-stimulated increases in intracellular calcium originated in the internal compartment of the cell rather than reflecting influx of calcium from the extracellular compartment. Calmodulin kinase II (CAMKII) is phosphorylated in response to PDGF-BB in the NF1 MPNST cell lines, whereas no phosphorylation of CAMKII was observed in nhSCs. The decreased growth of NF1 MPNST cell lines after treatment with a CAMKII inhibitor is consistent with the view that aberrant activation of the calcium-signaling pathway by PDGF-BB contributes to the formation of MPNST in NF1 patients.     

Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis

The key physiological event essential to the establishment of nitrogen-fixing bacteria and phosphate-delivering arbuscular mycorrhizal symbioses is the induction of nuclear calcium oscillations that are required for endosymbioses. These regular fluctuations in nucleoplasmic calcium concentrations are generated by ion channels and a pump located at the nuclear envelope, including the CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15). However, how the CNGC15s are regulated in planta to sustain a calcium oscillatory mechanism remains unknown. Here, we demonstrate that the CNGC15s are regulated by the calcium-bound form of the calmodulin 2 (holo-CaM2), which, upon release of calcium, provides negative feedback to close the CNGC15s. Combining structural and evolutionary analyses of CaM residues with bioinformatic analysis, we engineered a holo-CaM2 with an increased affinity for CNGC15s. In planta, the expression of the engineered holo-CaM2 accelerates the calcium oscillation frequency, early endosymbioses signaling and is sufficient to sustain over time an enhanced root nodule symbiosis but not an increased arbuscular mycorrhization. Together, these results reveal that holo-CaM2 is a component of endosymbiosis signaling required to modulate CNGC15s activity and the downstream root nodule symbiosis pathway.

Nutrient acquisition is fundamental to life. Plants have evolved strategies to overcome soil phosphate limitation and gain access to atmospheric dinitrogen by developing beneficial associations with arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria, respectively. Unlike other crops, the vast majority of legumes have mastered associations with both endosymbionts, positioning them as key crops to develop sustainable agricultural practices in both developed and developing countries (1).The entry of nitrogen-fixing bacteria, known as rhizobia, and AM fungi into legume roots is initiated by the recognition of the endosymbiont. Host plants have plasma-membrane receptor-like kinases (2–6) that recognize rhizobial elicitors, lipochitooligosaccharides (LCOs), also known as Nod factors (7), and mycorrhizal factors composed of derivatives of LCOs and shorter chain chitooligosaccharides (8, 9). Although rhizobial and AM elicitors are recognized by different complexes of receptor-like kinases (10, 11), both symbionts require the activation of calcium oscillations in root epidermal nuclei (9, 12, 13) to set off the endosymbiosis program. In the model legume Medicago truncatula, two types of nuclear envelope localized ion channels are required to generate the calcium oscillation; the DOESN’T MAKE INFECTIONS1 (DMI1) channel and paralogs of CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15) (14), and the calcium pump, MCA8 (15). Similar to the animal CNGCs, plant CNGCs are tetrameric ion channels that can include different CNGC units (16, 17). In M. truncatula, CNGC15a, CNGC15b, and CNGC15c are all involved in nuclear calcium oscillation in the root epidermis, nodulation, and arbuscular mycorrhization, suggesting that the three units could assemble into a heterocomplex at the nuclear envelope (14). However, how CNGC15s are regulated in planta to sustain a calcium oscillatory mechanism remains unknown.In this study, we demonstrate that CNGC15s are regulated by the calcium-bound form of the calmodulin 2 (holo-CaM2) in planta, which shapes the oscillatory pattern of nucleoplasmic calcium concentration by providing negative feedback on CNGC15s to cause its closure. By engineering CaM2 to generate CaM2^R91A, which specifically increased holo-CaM2 binding affinity to each CNGC15 unit, we accelerated closure of CNGC15s and increased the calcium oscillation frequency. We further show that accelerating the calcium oscillation frequency was sufficient to accelerate the early endosymbiosis signaling and that the expression of CaM2^R91A resulted in an enhanced root nodule symbiosis but not enhanced AM colonization. Our data reveal differential regulation of rhizobia and AM endosymbioses by CaM2^R91A and suggest that modulating calcium signaling can be used as a strategy to positively impact symbiosis with nitrogen-fixing bacteria.     

Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension

Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca(2+)](i), Ca(2+)-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca(2+)](i) and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca(2+)-dependent and Ca(2+)-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca(2+) antagonist-resistant forms of hypertension.     

针刺对大鼠钙调素活性影响的实验研究

目的 :进一步探索针刺对钙调素活性的影响 ,研究钙调素在经络活动、针刺效应中的作用。方法 :在前期有关研究的基础上 ,采用磷酸二酯酶法 (PDE)测定针刺“下脘”穴后 ,“下脘”穴局部及其同经的“巨阙”穴处的钙调素 (CaM)活性的变化。结果 :针刺“下脘”穴后 ,其局部钙调素的活性明显高于同经“巨阙”穴的活性 (P <0 0 1 ) ,且两穴的活性明显高于针刺前任脉组织的活性 (P<0 0 1 )。结论 :针刺可能对钙调素有一定的激活作用 ;针刺对钙调素的激活作用可能沿着经络系统传递     

Brain ischemia induces serine phosphorylation of neuronal nitric oxide synthase by Ca^2＋ ／ calmodulin-dependent protein kinase II in rat hippocampus

INTRODUCTIONConstitutively expressed nitric oxide synthase(NOS) has three isoforms, including neuronal NOS(nNOS), inducible NOS (iNOS), and endothelial NOS(eNOS), which has been isolated and cloned[1]. NOScatalyzed L-arginine to generate L-citruline and nitricoxide (NO), a gaseous messenger molecule, the over-production of which plays a critical role in glutamate-induced neurotoxicity after ischemia[2]. In mutant micedeficient in nNOS and subsequent NO production, inf-arct s…     

The effect of divalent cations on neuronal nitric oxide synthase activity.

Neuronal nitric oxide synthase (NOS I) is a Ca(2+)/calmodulin-binding enzyme that generates nitric oxide (NO*) and L-citrulline from the oxidation of L-arginine, and superoxide (O(2)*(-)) from the one-electron reduction of oxygen (O(2)). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca(2+), which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca(2+) binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I-mediated catalysis. As in the case of Ca(2+), we demonstrate that Ni(2+), Ba(2+), and Mn(2+) can activate NOS I to metabolize L-arginine to L-citrulline and NO*, and afford O(2)*(-) in the absence of L-arginine. In contrast, Cd(2+) did not activate NOS I to produce either NO* or O(2)*(-), and the combination of Ca(2+) and either Cd(2+), Ni(2+), or Mn(2+) inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO*, O(2)*(-) and products derived from these free radicals.     

CaMKIIδ and cardiomyocyte Ca2+ signalling new perspectives on splice variant targeting

Control of cardiomyocyte cytosolic Ca²⁺ levels is crucial in determining inotropic status and ischemia/reperfusion stress response. Responsive to fluctuations in cellular Ca²⁺, Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a serine/threonine kinase integral to the processes regulating cardiomyocyte Ca²⁺ channels/transporters. CaMKII is primarily expressed either in the δ_B or δ_C splice variant forms, which may mediate differential influences on cardiomyocyte function and pathological response mechanisms. Increases in myocyte Ca²⁺ levels promote the binding of a Ca²⁺/calmodulin complex to CaMKII, to activate the kinase. Activity is also maintained through a series of post‐translational modifications within a critical region of the regulatory domain of the protein. Recent data indicate that the post‐translational modification status of CaMKIIδ_B/δ_C variants may have an important influence on reperfusion outcomes. This study provided the first evidence that the specific type of CaMKII post‐translational modification has a role in determining target selectivity of downstream Ca²⁺ transporters. The study was also able to demonstrate that the phosphorylated form of CaMKII closely co‐localizes with CaMKIIδ_B in the nuclear/myofilament fraction, contrasting with a co‐enrichment of oxidized CaMKII in the membrane fraction with CaMKIIδ_C. It has also been possible to conclude that a hyper‐phosphorylation of CaMKII (Thr287) in reperfused hearts represents a hyper‐activation of the CaMKIIδ_B, which exerts anti‐arrhythmic actions through an enhanced capacity to selectively increase sarcoplasmic reticulum Ca²⁺ uptake and maintain cytosolic Ca²⁺ levels. This suggests that suppression of global CaMKIIδ may not be an efficacious approach to developing optimal pharmacological interventions for the vulnerable heart.     

Ca~(2+)/钙调蛋白依赖的蛋白激酶Ⅱ信息通路在DPDPE长时程作用的NG108-15细胞中的作用

目的　观察阿片类依赖时Ca2 + /钙调蛋白依赖的蛋白激酶Ⅱ (CaMKⅡ )信息通路的变化 ,以及Ca2 + /CaMKⅡ信息通路与cAMP水平之间的关系。方法　以NG10 8 15细胞作为体外的细胞模型 ,分别采用竞争性蛋白结合法及放射免疫法、PDE法、γ 3 2 P参入法以及RT PCR测定cAMP水平、钙调蛋白 (CaM)活性、CaMKⅡ活性和mDOR的mRNA表达水平的变化。结果　DPDPE长时程作用NG10 8 15细胞 ,cAMP水平升高 ,形成阿片依赖 ;细胞CaM活性和CaMKⅡ活性也升高 ,该升高可被CaM拮抗剂W 7所抑制 ,而CaMKⅡ活性的升高可被CaMKⅡ特异性抑制剂KN 6 2所抑制。W 7或KN 6 2可抑制阿片依赖导致的细胞cAMP水平的升高。阿片依赖时 ,加入纳洛酮诱发戒断 ,CaM活性、CaMKⅡ活性进一步增高。而在阿片依赖时 ,δ阿片受体的mRNA表达水平无明显变化。结论　Ca2 + /钙调蛋白依赖的蛋白激酶Ⅱ信息通路可通过调节cAMP水平参与阿片依赖的机制。