Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII a in epileptic hippocampal neurons

Purpose: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II a expression in a model of epileptic neurons were investigated. Method: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg2+ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg2+ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II a protein expression was assessed by Western-blot. Ca2+ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca2+ turnover was observed under a laser scanning confocal microscope. Results: The CaMK II a expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca2+ fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes. Conclusion: GLP may inhibit calcium overload and promote CaMK II a expression to protect epileptic neuron

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr286-α-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr286 autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory

Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging.

Localization of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE' splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca(2+)/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction

Improving a Natural CaMKII Inhibitor by Random and Rational Design

CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ∼100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not.These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction

Duty, desire or indifference? A qualitative study of patient decisions about recruitment to an epilepsy treatment trial

BACKGROUND: Epilepsy is a common neurological condition, in which drugs are the mainstay of treatment and drugs trials are commonplace. Understanding why patients might or might not opt to participate in epilepsy drug trials is therefore of some importance, particularly at a time of rapid drug development and testing; and the findings may also have wider applicability. This study examined the role of patient perceptions in the decision-making process about recruitment to an RCT (the SANAD Trial) that compared different antiepileptic drug treatments for the management of new-onset seizures and epilepsy. METHODS: In-depth interviews with 23 patients recruited from four study centres. All interviews were tape-recorded and transcribed; the transcripts were analysed thematically using a qualitative data analysis package. RESULTS: Of the nineteen informants who agreed to participate in SANAD, none agreed for purely altruistic reasons. The four informants who declined all did so for very specific reasons of self-interest. Informants' perceptions of the nature of the trial, of the drugs subject to trial, and of their own involvement were all highly influential in their decision-making. Informants either perceived the trial as potentially beneficial or unlikely to be harmful, and so agreed to participate; or as potentially harmful or unlikely to be beneficial and so declined to participate. CONCLUSION: Most patients applied 'weak altruism', while maintaining self-interest. An emphasis on the safety and equivalence of treatments allowed some patients to be indifferent to the question of involvement. There was evidence that some participants were subject to 'therapeutic misconceptions'. The findings highlight the individual nature of trials but nonetheless raise some generic issues in relation to their design and conduct

Public health in community pharmacy: a systematic review of pharmacist and consumer views

BACKGROUND The increasing involvement of pharmacists in public health will require changes in the behaviour of both pharmacists and the general public. A great deal of research has shown that attitudes and beliefs are important determinants of behaviour. This review aims to examine the beliefs and attitudes of pharmacists and consumers towards pharmaceutical public health in order to inform how best to support and improve this service. METHODS Five electronic databases were searched for articles published in English between 2001 and 2010. Titles and abstracts were screened by one researcher according to the inclusion criteria. Papers were included if they assessed pharmacy staff or consumer attitudes towards pharmaceutical public health. Full papers identified for inclusion were assessed by a second researcher and data were extracted by one researcher. RESULTS From the 5628 papers identified, 63 studies in 67 papers were included. Pharmacy staff: Most pharmacists viewed public health services as important and part of their role but secondary to medicine related roles. Pharmacists' confidence in providing public health services was on the whole average to low. Time was consistently identified as a barrier to providing public health services. Lack of an adequate counselling space, lack of demand and expectation of a negative reaction from customers were also reported by some pharmacists as barriers. A need for further training was identified in relation to a number of public health services. Consumers: Most pharmacy users had never been offered public health services by their pharmacist and did not expect to be offered. Consumers viewed pharmacists as appropriate providers of public health advice but had mixed views on the pharmacists' ability to do this. Satisfaction was found to be high in those that had experienced pharmaceutical public health. CONCLUSIONS There has been little change in customer and pharmacist attitudes since reviews conducted nearly 10 years previously. In order to improve the public health services provided in community pharmacy, training must aim to increase pharmacists' confidence in providing these services. Confident, well trained pharmacists should be able to offer public health service more proactively which is likely to have a positive impact on customer attitudes and health

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved

Efficacy of Synaptic Inhibition Depends on Multiple, Dynamically Interacting Mechanisms Implicated in Chloride Homeostasis

Chloride homeostasis is a critical determinant of the strength and robustness of inhibition mediated by GABAA receptors (GABAARs). The impact of changes in steady state Cl− gradient is relatively straightforward to understand, but how dynamic interplay between Cl− influx, diffusion, extrusion and interaction with other ion species affects synaptic signaling remains uncertain. Here we used electrodiffusion modeling to investigate the nonlinear interactions between these processes. Results demonstrate that diffusion is crucial for redistributing intracellular Cl− load on a fast time scale, whereas Cl−extrusion controls steady state levels. Interaction between diffusion and extrusion can result in a somato-dendritic Cl− gradient even when KCC2 is distributed uniformly across the cell. Reducing KCC2 activity led to decreased efficacy of GABAAR-mediated inhibition, but increasing GABAAR input failed to fully compensate for this form of disinhibition because of activity-dependent accumulation of Cl−. Furthermore, if spiking persisted despite the presence of GABAAR input, Cl− accumulation became accelerated because of the large Cl− driving force that occurs during spikes. The resulting positive feedback loop caused catastrophic failure of inhibition. Simulations also revealed other feedback loops, such as competition between Cl− and pH regulation. Several model predictions were tested and confirmed by [Cl−]i imaging experiments. Our study has thus uncovered how Cl− regulation depends on a multiplicity of dynamically interacting mechanisms. Furthermore, the model revealed that enhancing KCC2 activity beyond normal levels did not negatively impact firing frequency or cause overt extracellular K− accumulation, demonstrating that enhancing KCC2 activity is a valid strategy for therapeutic intervention

A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

Autophosphorylation of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca(2+)/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca(2+)/CaM-stimulated CaMKII activity has been described, but remains controversial.In order to determine the contribution of T286 autophosphorylation to Ca(2+)/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca(2+)/CaM-stimulated phosphorylation for two different peptide substrates (to ~75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca(2+)/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.These results indicate that Ca(2+)/CaM-stimulated CaMKII activity is mildly (~1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity