Modulation of ATP-induced LTP by cannabinoid receptors in rat hippocampus

Cannabinoids exert powerful action on various forms of synaptic plasticity. These retrograde messengers modulate GABA and glutamate release from presynaptic terminals by acting on presynaptic CB1 receptors. In particular, they inhibit long-term potentiation (LTP) elicited by electrical stimulation of excitatory pathways in rat hippocampus. Recently, LTP of the field excitatory postsynaptic potential (fEPSP) induced by exogenous ATP has been thoroughly explored. The present study demonstrates that cannabinoids inhibit ATP-induced LTP in hippocampal slices of rat. Administration of 10 μM of ATP led to strong inhibition of fEPSPs in CA1/CA3 hippocampal synapses. Within 40 min after ATP removal from bath solution, robust LTP was observed (fEPSP amplitude comprised 130.1 ± 3.8% of control, n = 10). This LTP never appeared when ATP was applied in addition to cannabinoid receptor agonist WIN55,212-2 (100 nM). Selective CB1 receptor antagonist, AM251 (500 nM), completely abolished this effect of WIN55,212-2. Our data indicate that like canonical LTP elicited by electrical stimulation, ATP-induced LTP is under control of CB1 receptors.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-012-9296-5) contains supplementary material, which is available to authorized users.     

Protein kinase CK2 is required for dorsal axis formation in Xenopus embryos

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of beta-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including beta-catenin. Here, we have examined the role of CK2 in dorsal axis formation. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2alpha was able to block ectopic axis formation induced by XWnt8 and beta-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis.     

Protein kinases in Toxoplasma gondii

Toxoplasma gondii is an obligatory intracellular pathogen that causes life threatening illness in immunodeficient individuals, miscarriage in pregnant woman, and blindness in newborn children. Similar to any other eukaryotic cell, protein kinases play critical and essential roles in the Toxoplasma life cycle. Accordingly, many studies have focused on identifying and defining the mechanism of function of these signalling proteins with a long-term goal to develop anti-Toxoplasma therapeutics. In this review, we briefly discuss classification and key components of the catalytic domain which are critical for functioning of kinases, with a focus on domains, families, and groups of kinases within Toxoplasma. More importantly, this article provides a comprehensive, current overview of research on kinase groups in Toxoplasma including the established eukaryotic AGC, CAMK, CK1, CMGC, STE, TKL families and the apicomplexan-specific FIKK, ROPK and WNG family of kinases. This work provides an overview and discusses current knowledge on Toxoplasma kinases including their localization, function, signalling network and role in acute and chronic pathogenesis, with a view towards the future in probing kinases as viable drug targets.     

Downregulation of KCC2 following LTP contributes to EPSP-spike potentiation in rat hippocampus

GABAergic synaptic inhibition plays a critical role in regulating long-term potentiation (LTP) of glutamatergic synaptic transmission and circuit output. The K(+)-Cl(-) cotransporter 2 (KCC2) is an important factor in determining inhibitory GABAergic synaptic strength besides the contribution of GABA(A) receptor. Although much knowledge has been gained regarding activity-dependent downregulation of KCC2 in many pathological conditions, the potential change and contribution of KCC2 in LTP expression is still unknown. In this study, we found that downregulation of KCC2 was accompanied with the occurrence of LTP but not that of long-term depression in hippocampal CA1 region. Meanwhile, KCC2 level in CA3/DG and adjacent cortex was stable in the process of LTP expression in Schaffer collateral synapses. Blockade of NMDA receptor with APV not only prevented LTP induction also abolished the reduction of KCC2. Furthermore, the inhibition of KCC2 function with furosemide directly induced EPSP-spike (E-S) potentiation, an important component of LTP in hippocampus. The present data suggest a novel mechanism that LTP formation is accompanied by the downregulation of KCC2, which is underlying GABAergic strength and most likely contributes to the E-S potentiation following LTP.     

Protein kinases and protein phosphatases in prokaryotes: a genomic perspective

For many years, the regulation of protein structure and function by phosphorylation and dephosphorylation was considered a relatively recent invention that arose independently in each phylogenetic domain. Over time, however, incidents of apparent domain trespass involving the presence of 'eukaryotic' protein kinases or protein phosphatases in prokaryotic organisms were reported with increasing frequency. Today, genomics has provided the means to examine the phylogenetic distribution of 'eukaryotic' protein kinases and protein phosphatases in a comprehensive and systematic manner. The results of these genome searches challenge previous conceptions concerning the origins and evolution of this versatile regulatory mechanism.     

Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases

With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.     

Expression of raf serine/threonine protein kinases in the cell bodies of primary sensory neurons of the adult rat

We have used immunocytochemistry to examine the distribution of raf protein kinases in sensory neurons of the adult rat. In lumbar and trigeminal sensory ganglia, cells of all size ranges appeared to be raf immunoreactive and this was confirmed by double labeling using subpopulation specific markers. Almost all cells labeled with Griffonia simplicifolia IB4 (a small cell marker) or immunostained by using a large cell marker (RT97) showed raf immunoreactivity. These two markers label cells known to differ in their expression of neurotrophin receptors (trk). Thus raf kinases are not confined to cells expressing only certain trk subtypes. In the dorsal horn of the spinal cord, raf immunoreactivity was present in scattered neurons. However, sensory axons identified by IB4 histochemistry were devoid of raf immunostaining. Lectin-labeled nerve fibers in the cornea, lower lip and glans penis were also not immunoreactive. Ligation of the sciatic nerve did not produce any accumulation of raf immunoreactivity, confirming that raf kinases are not axonally transported to the peripheral processes of sensory neurons. Surgical dissection of the sciatic nerve caused the normal homogeneous cytoplasmic raf immunoreactivity to be replaced in some cells by a staining concentrated predominantly under the plasma membrane. One possibility is that this represents activation of raf in these cells. Received: 2 October 1995 / Accepted: 4 March 1996     

Anxiolytic-like effects of substance P administration into the dorsal, but not ventral, hippocampus and its influence on serotonin

Substance P (SP) is known to be involved in processes related to learning and memory, fear, anxiety and stress. SP and NK1 receptors are localized in the hippocampus, a brain structure involved in learning and memory as well as emotional processes. As there is evidence for differential functions of the ventral (VH) and dorsal (DH) hippocampus in a variety of behaviors, we here evaluated the effects of injections of SP into the VH and DH in rats submitted to the elevated plus-maze (EPM) and open field (OF) tests. The results obtained showed that infusions of 100 and 1000 ng of SP into the DH, but not VH, increased open arm activity in the EPM and in the central zone of the OF, indicative of anxiolytic-like action. These effects were observed in the absence of significant changes in general motor activity. In an additional experiment to examine whether these effects of SP are mediated by local serotoninergic mechanisms, extracellular concentrations of this monoamine were assessed by use of in vivo microdialysis. Infusions of SP into the DH did not influence the extracellular concentration of serotonin. These data indicate that neurokinins in the DH, but not VH, are involved in mechanisms associated with anxiety and that the mediation of SP in anxiety-related behaviors is independent of local serotonergic mechanisms.     

Protein tyrosine kinases in neutrophil activation and recruitment

Migration of leukocytes into tissue is a key element of innate and adaptive immunity. The first contact of leukocytes with endothelial cells is mediated by engagement of selectins with their counter-receptors which results in leukocyte rolling. During rolling, leukocytes collect different inflammatory signals that activate intracellular signaling pathways. Integration of these signals induces leukocyte activation, firm arrest, post-adhesion strengthening, intravascular crawling, and transmigration. In neutrophils, like in T-cells and platelets, both G-protein-coupled receptor-dependent and -independent activation pathways exist that lead to integrin activation. Accumulating evidence suggests that different protein tyrosine kinases play key roles in signal transduction pathways regulating neutrophil activation and recruitment to inflammatory sites. This review focuses on the role of protein tyrosine kinases of the Src, Syk, and Tec families for neutrophil activation and recruitment.     

电刺激右后背海马诱导双侧前背海马网络癫痫

目的 :急性强直电刺激右侧后背HPC诱导双侧HPC癫痫电网络形成的细胞机制。方法 :强直电刺激 (6 0Hz,2s,0 .4～ 0 .6mA)大鼠右后背HPCCA1基树突区 ,每隔 10min刺激一次 ,施加 10个刺激串。结果 :①分别抑制双侧CA1神经元单位放电频率 ,对侧的抑制效应更明显 (对侧 :6 2 .94 %± 3.6 8% ;同侧 :36 .6 1%± 3.14 % ,P <0 .0 1) ,出现抑制后爆发式放电。随着刺激串数的增加 ,抑制作用逐渐减弱。②同步原发性网络和单位后放电 ,以同侧CA1多见 (P<0 .0 1)。③ 90Hz或 12 0Hz原发性或继发性网络后放电仅仅累及同侧CA1。④对侧CA3基树突区网络与下托神经元单位放电出现同步继发性后放电 ,反复发作 ,持续约数小时。结论 :电刺激诱导的对侧HPC抑制后爆发式放电和长时程、反复发作的网络与单个神经元同步继发性后放电可能是跨半球癫痫网络形成的重要表现形式。     

喉癌中差异表达的蛋白激酶及其抑制剂的生物信息学筛选

目的：筛选喉癌中差异表达的激酶基因以及调控这些差异表达激酶的激酶抑制剂,为喉癌的分子治疗提供新的靶点。方法： 利用PubMed 数据库和SAM 软件筛选喉癌中差异表达的激酶基因。体外培养人喉癌Hep-2和FaDu 细胞。实时定量聚合酶链反 应（Real-time PCR）检测目的激酶在喉癌细胞系中的表达,以验证全基因表达谱结果的准确性。利用KEGG数据库获得激酶调控 的通路。文献挖掘和人工筛选获得差异表达激酶的激酶抑制剂。结果：①在喉癌基因组表达谱中,共筛选出差异表达基因207个, P<0.05,其中激酶基因4 个,分别为CDC42、CDK7、SLK 和TXK。②Real-time PCR 结果显示在人喉癌Hep-2 和FaDu 细胞中这四 个差异基因也出现差异表达,P<0.05,证明全基因组的结果准确。③通路分析的结果显示4 个差异激酶共调控25 个通路。④文献 挖掘和人工筛选的结果显示共有10 个激酶抑制剂调控CDC42,7 个激酶抑制剂调控CDK7,1 个激酶抑制剂调控SLK,2 个激酶 抑制剂调控TXK。并且再次文献挖掘的结果显示在这20 个激酶抑制剂中,有9 个在癌症方面的研究较少,文献<10 篇。结论： 喉癌中共有四个激酶CDC42、CDK7、SLK 和TXK发生差异表达,并发挥促癌作用。它们的激酶抑制剂可能有潜在的抗癌作用, 为喉癌的分子治疗提供新的切入点。     

Protein kinases in elicitor signal transduction in plant cells

Plants have the ability to respond to pathogen invasion by specific defense reactions. Components of mammalian signal transduction chains have been identified in plants, and several lines of evidence have implicated such components in elicitor signal transmission in defense responses. In particular, it has been assumed that elicitor signals are transduced via a protein kinase cascade, although the identity of the protein kinases and the function of the phosphorylated proteins remain to be determined. The purpose of this review is to discuss the roles of protein kinases in elicitor signal transduction pathways in plant cells based on recent progress in this field.     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.     

电刺激大鼠右后背海马诱导前背海马神经网络癫痫样电活动

本文旨在探讨单侧海马(hippocampus,HPC)内神经网络与HPC癫痫发生的关系及其细胞机制。实验在45只SSprague-Dawley大鼠上完成。急性强直电刺激大鼠右侧后背HPC CAl基树突区(acute tetanizatio of the posterior dorsal hippocampus,ATPDH;60Hz,2s,0.4-0.6mA)诱发HPC癫痫模型,同步记录同侧前背HPC CAl顶树突区单位放电和基树突区深部电图。结果,ATPDH可以沿长铀向前1．8mm处对前背MIC神经网络产生下述效应：(1)同步或非同步原发性单位与深部电图后放电,在同步性后放电锁时(time-lock)关系明显。非同步性后放电的深部电图癫痫样电活动具有宽频带特征(5-90Hz);(2)原发性单位后放-后抑制效应可以引发低频原发性电图后放电,长时程爆发式单位放电可以诱发高频原发性电图后放电;(3)短束原发性电图后放电也可以诱发原发性单位后放电;(4)原发性电图后放电和神经元单位放电的抑制效应具有明显可塑性特征。以上结果提示,重复施加ATPDH可以引起前背HPC癫痫相关性病理生理性神经网络的重建;而单个神经元与神经网络的异常电活动之间具有明显的互动作用和突触传递可塑性特征;沿HPC长铀内在抑制性通路的过度活动也可以诱发电图癫痫样电活动,导致HPC网络癫痫的发生。     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80–90% was found in the intermembrane fraction, while the rest was associated with mitoplast. The intermembrane prostimulated kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplast. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinase, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Protein kinase modulators interfere with bud formation in Hydra vulgaris

The fresh water polyp Hydra can reproduce asexually by forming buds. These buds separate from the parent animal due to the development of foot tissue in a belt-like region and the formation of a constriction basal to that region. A single pulse treatment with activators of protein kinase C, including 1,2-dioctanoyl-rac-glycerol and 12-o-tetradecanoylphorbol-13-acetate, and inhibitors of various protein kinases, including staurosporine, H-7 and genistein, interfered with foot and constriction formation. The buds did not separate. Therewith, branched animals were formed, some of which bore a lateral foot patch. Simultaneous treatments with an activator and inhibitor led to a higher amount of branched animals than treatments with one of these agents alone. Based on the different specificities of the activators and inhibitors used we propose that activation of a protein kinase C and/or inhibition of a probably non-C-type protein kinase interfere with the decrease of positional value at the bud's base, a process necessary to initiate the pattern forming system leading to foot formation. Correspondence to: F. Perez     

Lithium Decreases Membrane-Associated Protein Kinase C in Hippocampus: Selectivity for the α Isozyme

We investigated the effects of lithium on alterations in the amount and distribution of protein kinase C (PKC) in discrete areas of rat brain by using [³H]phorbol 12, 13-dibutyrate quantitative autoradiography as well as western blotting. Chronic administration of lithium resulted in a significant decrease in membrane-associated PKC in several hippocampal structures, most notably the subiculum and the CA1 region. In contrast, only modest changes in [³H]phorbol 12, 13-dibutyrate binding were observed in the various other cortical and subcortical structures examined. Immunoblotting using monoclonal anti-PKC antibodies revealed an isozyme-specific 30% decrease in hippocampal membrane-associated PKC α, in the absence of any changes in the labeling of either the β_(I/II) or γ isozymes. These changes were observed only after chronic (4 week) treatment with lithium, and not after acute (5 days) treatment, suggesting potential clinical relevance. Given the critical role of PKC in regulating neuronal signal transduction, lithium's effects on PKC in the limbic system represent an attractive molecular mechanism for its efficacy in treating both poles of manic-depressive illness. In addition, the decreased hippocampal membrane-associated PKC observed in the present study offers a possible explanation for lithium-induced memory impairment.     

Combined effects of LTP/LTD and synaptic scaling in formation of discrete and line attractors with persistent activity from non-trivial baseline

In this article, we analyze combined effects of LTP/LTD and synaptic scaling and study the creation of persistent activity from a periodic or chaotic baseline attractor. The bifurcations leading to the creation of new attractors have been detailed; this was achieved using a mean field approximation. Attractors encoding persistent activity can notably appear via generalized period-doubling bifurcations, tangent bifurcations of the second iterates or boundary crises, after which the basins of attraction become irregular. Synaptic scaling is shown to maintain the coexistence of a state of persistent activity and the baseline. According to the rate of change of the external inputs, different types of attractors can be formed: line attractors for rapidly changing external inputs and discrete attractors for constant external inputs.     

Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface

Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na₃VO₄, a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na₃VO₄, and transfection with Lyn restores the cortical actin cap in the presence of Na₃VO₄. Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.