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

Dcc Regulates Asymmetric Outgrowth of Forebrain Neurons in Zebrafish

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates

The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis

The proto-oncogene RAS, coding for a 21 kDa protein (p21), is mutated in 20% of lung cancer. However, the literature remains controversial on its prognostic significance for survival in lung cancer. We performed a systematic review of the literature with meta-analysis to assess its possible prognostic value on survival. Published studies on lung cancer assessing prognostic value of RAS mutation or p21 overexpression on survival were identified by an electronic search. After a methodological assessment, we estimated individual hazard ratios (HR) estimating RAS protein alteration or RAS mutation effect on survival and combined them using meta-analytic methods. In total, 53 studies were found eligible, with 10 concerning the same cohorts of patients. Among the 43 remaining studies, the revelation method was immunohistochemistry (IHC) in nine and polymerase chain reaction (PCR) in 34. Results in terms of survival were significantly pejorative, significantly favourable, not significant and not conclusive in 9, 1, 31, 2, respectively. In total, 29 studies were evaluable for meta-analysis but we aggregated only the 28 dealing with non-small-cell lung cancer (NSCLC) and not the only one dealing with small-cell-lung cancer (SCLC). The quality scores were not statistically significantly different between studies with or without significant results in terms of survival, allowing us to perform a quantitative aggregation. The combined HR was 1.35 (95% CI: 1.16–1.56), showing a worse survival for NSCLC with KRAS2 mutations or p21 overexpression and, particularly, in adenocarcinomas (ADC) (HR 1.59; 95% CI 1.26–2.02) and in studies using PCR (HR 1.40; 95% CI 1.18–1.65) but not in studies using IHC (HR 1.08; 95% CI 0.86–1.34). RAS appears to be a pejorative prognostic factor in terms of survival in NSCLC globally, in ADC and when it is studied by PCR

The role of HER-2/neu expression on the survival of patients with lung cancer: a systematic review of the literature

C-erbB-2 prognostic value for survival in patients with lung cancer remains controversial. We performed a systematic review of the literature to clarify its impact. Studies were identified by an electronic search in order to aggregate the survival results, after a methodological assessment using the scale of the European Lung Cancer Working Party. To be eligible, a study had to deal with c-erbB-2 assessment in lung cancer patients and to analyse survival according to c-erbB-2 expression. In total, 30 studies were eligible: 24 studies dealt with non-small-cell lung carcinoma (NSCLC), five with adenocarcinoma and one study dealt with small-cell carcinoma. In all, 31% of the patients were positive for c-erbB-2. According to c-erbB-2 expression, 13 studies were 'negative' (significant detrimental effect on survival), one 'positive' (significant survival improvement) and 16 not significant. Significant studies had a better subscore relative to analysis and results report than nonsignificant studies. In total, 86% of the significant studies and only 56% of the nonsignificant studies were evaluable for the meta-analysis. This suggests a possible bias in our aggregated results. For NSCLC, the hazard ratio was 1.55 (95% CI: 1.29-1.86) in favour of tumours that do not express c-erbB-2. In conclusion, the overexpression of c-erbB-2 might be a factor of poor prognosis for survival in NSCLC, but there is a potential bias in favour of the significant studies with an overestimation risk of the magnitude of the true effect of c-erbB-2 overexpression.Journal ArticleResearch Support, Non-U.S. Gov'tReviewinfo:eu-repo/semantics/publishe

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

Inhibitory Effect of TNF-α on Malaria Pre-Erythrocytic Stage Development: Influence of Host Hepatocyte/Parasite Combinations

BACKGROUND: The liver stages of malaria parasites are inhibited by cytokines such as interferon-gamma or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-alpha possibly because of differences in the models used. We have reassessed the role of TNF-alpha in the different cellular systems used to study the Plasmodium pre-erythrocytic stages. METHODS AND FINDINGS: Human or mouse TNF-alpha were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-alpha treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-alpha inhibition was mediated by a soluble factor present in the supernatant of TNF-alpha stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-alpha prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. CONCLUSIONS: Treatment TNF-alpha prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection

Epigenetic Analysis of KSHV Latent and Lytic Genomes

Epigenetic modifications of the herpesviral genome play a key role in the transcriptional control of latent and lytic genes during a productive viral lifecycle. In this study, we describe for the first time a comprehensive genome-wide ChIP-on-Chip analysis of the chromatin associated with the Kaposi's sarcoma-associated herpesvirus (KSHV) genome during latency and lytic reactivation. Depending on the gene expression class, different combinations of activating [acetylated H3 (AcH3) and H3K4me3] and repressive [H3K9me3 and H3K27me3] histone modifications are associated with the viral latent genome, which changes upon reactivation in a manner that is correlated with their expression. Specifically, both the activating marks co-localize on the KSHV latent genome, as do the repressive marks. However, the activating and repressive histone modifications are mutually exclusive of each other on the bulk of the latent KSHV genome. The genomic region encoding the IE genes ORF50 and ORF48 possesses the features of a bivalent chromatin structure characterized by the concomitant presence of the activating H3K4me3 and the repressive H3K27me3 marks during latency, which rapidly changes upon reactivation with increasing AcH3 and H3K4me3 marks and decreasing H3K27me3. Furthermore, EZH2, the H3K27me3 histone methyltransferase of the Polycomb group proteins (PcG), colocalizes with the H3K27me3 mark on the entire KSHV genome during latency, whereas RTA-mediated reactivation induces EZH2 dissociation from the genomic regions encoding IE and E genes concurrent with decreasing H3K27me3 level and increasing IE/E lytic gene expression. Moreover, either the inhibition of EZH2 expression by a small molecule inhibitor DZNep and RNAi knockdown, or the expression of H3K27me3-specific histone demethylases apparently induced the KSHV lytic gene expression cascade. These data indicate that histone modifications associated with the KSHV latent genome are involved in the regulation of latency and ultimately in the control of the temporal and sequential expression of the lytic gene cascade. In addition, the PcG proteins play a critical role in the control of KSHV latency by maintaining a reversible heterochromatin on the KSHV lytic genes. Thus, the regulation of the spatial and temporal association of the PcG proteins with the KSHV genome may be crucial for propagating the KSHV lifecycle

Genome-Wide Association Study SNPs in the Human Genome Diversity Project Populations: Does Selection Affect Unlinked SNPs with Shared Trait Associations?

Genome-wide association studies (GWAS) have identified more than 2,000 trait-SNP associations, and the number continues to increase. GWAS have focused on traits with potential consequences for human fitness, including many immunological, metabolic, cardiovascular, and behavioral phenotypes. Given the polygenic nature of complex traits, selection may exert its influence on them by altering allele frequencies at many associated loci, a possibility which has yet to be explored empirically. Here we use 38 different measures of allele frequency variation and 8 iHS scores to characterize over 1,300 GWAS SNPs in 53 globally distributed human populations. We apply these same techniques to evaluate SNPs grouped by trait association. We find that groups of SNPs associated with pigmentation, blood pressure, infectious disease, and autoimmune disease traits exhibit unusual allele frequency patterns and elevated iHS scores in certain geographical locations. We also find that GWAS SNPs have generally elevated scores for measures of allele frequency variation and for iHS in Eurasia and East Asia. Overall, we believe that our results provide evidence for selection on several complex traits that has caused changes in allele frequencies and/or elevated iHS scores at a number of associated loci. Since GWAS SNPs collectively exhibit elevated allele frequency measures and iHS scores, selection on complex traits may be quite widespread. Our findings are most consistent with this selection being either positive or negative, although the relative contributions of the two are difficult to discern. Our results also suggest that trait-SNP associations identified in Eurasian samples may not be present in Africa, Oceania, and the Americas, possibly due to differences in linkage disequilibrium patterns. This observation suggests that non-Eurasian and non-East Asian sample populations should be included in future GWAS

Genetic Determination and Linkage Mapping of Plasmodium falciparum Malaria Related Traits in Senegal

Plasmodium falciparum malaria episodes may vary considerably in their severity and clinical manifestations. There is good evidence that host genetic factors contribute to this variability. To date, most genetic studies aiming at the identification of these genes have used a case/control study design for severe malaria, exploring specific candidate genes. Here, we performed a family-based genetic study of falciparum malaria related phenotypes in two independent longitudinal survey cohorts, as a first step towards the identification of genes and mechanisms involved in the outcome of infection. We studied two Senegalese villages, Dielmo and Ndiop that differ in ethnicity, malaria transmission and endemicity. We performed genome-scan linkage analysis of several malaria-related phenotypes both during clinical attacks and asymptomatic infection. We show evidence for a strong genetic contribution to both the number of clinical falciparum malaria attacks and the asymptomatic parasite density. The asymptomatic parasite density showed linkage to chromosome 5q31 (LOD = 2.26, empirical p = 0.0014, Dielmo), confirming previous findings in other studies. Suggestive linkage values were also obtained at three additional chromosome regions: the number of clinical malaria attacks on chromosome 5p15 (LOD = 2.57, empirical p = 0.001, Dielmo) and 13q13 (LOD = 2.37, empirical p = 0.0014 Dielmo), and the maximum parasite density during asymptomatic infection on chromosome 12q21 (LOD = 3.1, empirical p<10−4, Ndiop). While regions of linkage show little overlap with genes known to be involved in severe malaria, the four regions appear to overlap with regions linked to asthma or atopy related traits, suggesting that common immune related pathways may be involved

A Genome-Wide Association Scan on the Levels of Markers of Inflammation in Sardinians Reveals Associations That Underpin Its Complex Regulation

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals—5 of which were identified only with the custom arrays—and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10−29); for ESR, at the HBB (rs4910472, p = 2.31×10−11) and UCN119B/SPPL3 (rs11829037, p = 8.91×10−10) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10−13) and in CADM3 (rs3026968, p = 7.63×10−13); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10−21), near DARC (rs3845624, p = 1.43×10−10), UNC119B/SPPL3 (rs11829037, p = 1.50×10−14), and ICOSLG/AIRE (rs113459440, p = 1.54×10−08) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process