CYP2B6 gene is subjected to a single-nucleotide polymorphism (G516T) with heterozygotes (GT) and homozygotes (TT) presenting decreased enzymatic activity. This case-control study aimed to investigate the association of CYP2B6 G516T polymorphism with the susceptibility of AML and its cytogenetic and clinical characteristics. Genotyping was performed on 619 AML patients and 430 healthy individuals using RCR-RFLP and a novel LightSNip assay. The major finding was a statistically higher frequency of the variant genotypes (GT and TT) in patients compared to the controls (GT:38.8% vs 29.8% and TT:9.3% vs 5.3% respectively) (p<0.001). More specifically, a significantly higher frequency of GT+TT genotypes in de novo AML patients (46.6%) and an immensely high frequency of TT in secondary AML (s-AML) (20.5%) were observed. The statistical analysis showed that the variant T allele was approximately 1.5-fold and 2.4-fold higher in de novo and s-AML respectively than controls. Concerning FAB subtypes, the T allele presented an almost 2-fold increased in AML-M2. Interestingly, a higher incidence of the TT genotype was observed in patients with abnormal karyotypes. In particular, positive correlations of the mutant allele were found in patients carrying specific chromosomal aberrations , complex or monosomal karyotypes. Finally, a strikingly higher frequency of TT genotype was also observed in patients stratified to the poor risk group. In conclusion, our results provide evidence for the involvement of the CYP2B6 polymorphism in AML susceptibility and suggest a possible role of the CYP2B6 genetic background on the development of specific chromosomal aberrations.The etiology of acute myeloid leukemia (AML) underlies the influence of genetic variants in candidate genes. The CYP2B6 enzyme detoxifies many genotoxic xenobiotics, protecting cells from oxidative damage. The  This non-sense polymorphism affects metabolic activity by altering substrate binding TT) have a lower enzymatic activity than individuals homozygous for the wild type G allele (GG), while heterozygotes (GT) display intermediate activity CYP2B6 G516T polymorphism exhibits ethnic variation. In Caucasians, the frequency of the both mutant genotypes (TT+GT) is reported to be 21.6%–28.9% TT) is limited to 3%–6% The G516T CYP2B6 polymorphism in myeloid malignancies concerning 36 AML Turkey patients and 164 AML Chinese patients, respectively GT and TT genotypes were found to be increased in patients with AML compared to the controls, demonstrating an important role of the T variant allele in AML susceptibility. However, only in one of them 516T CYP2B6 polymorphism was associated with chromosomal abnormalities and more specifically the recurrent genetic abnormalities restricted to the category of WHO 2008 classification. In that study, the investigated polymorphism was found to be increased in 13 AML patients with AML1-ETO fusion gene indicating an association between the t and the presence of the mutant T allele Recent studies have shown a strong association between the presence of this inactivating polymorphism not only in hematological malignancies but also in breast cancer 516T CYP2B6 polymorphism in AML susceptibility, we studied the distribution of the G516T CYP2B6 genotypes and allele frequencies in a large cohort of Greek patients (n = 619) with de novo or secondary AML and in healthy individuals (n = 430). The CYP2B6 genotype was also evaluated in respect to patients’ demographic and clinical characteristics and specific chromosomal abnormalities.In the present study, in order to evaluate the potential impact of the Gde novo AML and 116 had s-AML. Healthy donors were age and sex matched unrelated individuals with a negative history of previous malignancies and normal peripheral blood cell counts. Both cases and controls enrolled in the study came from different areas of Greece, having thus, a homogeneous ethnic background. The project was in accordance with the declaration of Helsinki and the protocol of the study was approved by the Ethical Committee of the NSCR “Demokritos”. Informed consent was provided from all AML patients and donors included in the study.The study included 619 AML patients and 430 healthy individuals. Diagnosis was established in Greek hospitals between 2008 and 2012, based on the WHO requirements with ≥20% bone marrow or peripheral blood blasts, except cases carrying the recurrent cytogenetic abnormalities t, t, inv(16) or t. Among patients, 503 had Bone marrow (BM) samples were obtained at the time of diagnosis and submitted for cytogenetic analysis at the Cytogenetics Unit of NCSR “Demokritos”. The cytogenetic analysis was performed on trypsin G-banded chromosome preparations, from unstimulated BM cultures. Karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN) 2013 Total genomic DNA was extracted from bone marrow cells and/or peripheral blood of patients with AML and from peripheral blood leukocytes of healthy donors using QIAamp DNA Blood Mini Kit  following standard procedures according to the manufacturer’s instructions. Extracted DNA was used as template for the subsequent genotypic analysis.CYP2B6 genotype analysis was performed using a conventional PCR-RFLP method for all AML and control samples. Additionally, we genotyped 186 patients and 186 control samples using a novel Real-Time PCR assay. The two analyses provided identical results for all the samples studied by the two methods.The The PCR-RFLPs assay was performed using Taq DNA polymerase  with primers and conditions previously described by Lang et?al. vs T]. Thus, after amplification, the two CYP2B6 alleles are distinguished by determination of melting curves with the wild type G allele resulted in a melting peak at 50°C and mutant T allele in a melting peak at 58°C , using novel LightSNiP assay (rs3745274), based on SimpleProbe®melting curve analysis . To determine melting profiles, sample’s fluorescence decreases during heating, giving that the signal from the probe is quenched as the probe is displaced. The fluorescent data are converted to derivative melting curves by plotting the negative derivative of the fluorescence (F) with respect to temperature (T) versus temperature [-(dF/dT)  at 58°C .p–values less than 0.05 were considered as statistically significant. Odds ratios (ORs) are given with 95% confidence interval (CI). Statistical analysis for multiple testing was performed using the Bonferroni’s correction. Hardy-Weinberg equilibrium was evaluated for our control group using the Chi-squared test. Statistical analysis was performed using SPSS  version 20 software.For the statistical significance of differences in genotype distributions and allele frequencies between AML patients and controls or among other parameters under study, the Pearson Chi-square test with continuity Yates’ correction was employed. de novo AML and 116 s-AML patients with a median age of 59.5 and 68.9 years at diagnosis, respectively.de novo AML patients according to FAB classification showed that the most common FAB subtype was M4 in 23.6% of patients followed by M2 in 22.7%, M3 in 19.8%, M5 in 15.3%, M1 in 8.4%, M0 in 5.7%, M6 in 3.3% and M7 in 1.2%. A successful karyotypic analysis was achieved in 605 out of 619 (97.7%) patients. Among them, abnormal karyotypes were found in 422 (69.8%) patients; 325 with de novo AML (66.3%) and 97 with s-AML (84.3%). Complex karyotypes were presented in 24.1% of AML patients (21.6% in de novo and 34.8% in s-AML), while MKs were found in 19.5% of AML patients (17.3% in de novo and 28.6% in s-AML). The most common chromosomal abnormalities in our cohort of de novo AML patients were -7/del(7q) (15.7%), -5/del(5q) (11.2%), isolated +8 (9.0%), abnormalities of 11q23 (6.5%), t (5.9%), inv(16) (5.9%), t (5.7%) and loss of the Y chromosome (4.3%). As a sole change, loss of the Y chromosome was considered as disease-associated clonal abnormality when it was found in more than 75% of metaphase cells s-AML the most common abnormalities were -7/del(7q) (28.7%), -5/del(5q) (28.7%), t (6.1%) and +21 (6.1%). A total of 211 of 605 AML patients carrying more than one recurrent aberration were included in more than one corresponding cytogenetic category. None of patients showed abnormalities on 19q31.2 chromosomal region (CYP2B6 gene locus) The cytogenetic characteristics of AML cases are also summarized in CYP2B6 genotypes and allele frequencies in AML patients and healthy individuals are summarized in CYP2B6 genotype was distributed as follows: 64.9% wild-type (GG), 29.8% heterozygous (GT), and 5.3% homozygous mutants (TT). The observed frequencies were in accordance with the Hardy-Weinberg laws of equilibrium , with the homozygous TT variant genotype frequency to fall into the range of other Caucasian population studies CYP2B6 genotyping was successfully performed in 572 out of 619 AML patients (92.9%). The genotypic distribution between AML patients and healthy individuals was significantly different, showing a higher frequency of the variant genotypes (heterozygotes GT and homozygotes TT) in patients compared to the controls (38.8% vs 29.8% and 9.3% vs 5.3% respectively) . In particular, significantly higher frequencies of the variant genotypes were observed in de novo AML patients compared to the controls . Similarly, increased frequencies of variant genotypes were observed in s-AML patients, with the most marked difference to be the immensely high frequency of homozygotes for the mutant allele compared to the controls . It is noteworthy that in 12 out of 618 patients with available BM and PB samples, the genotypic analysis on both samples revealed the same CYP2B6 genotype.The vs 0.202, respectively). Interestingly, the highest T allele frequency was observed in s-AML patients (0.379 in s-AML vs 0.266 in de novo AML). The variant T allele was approximately 1.6-fold higher in AML patients  and 2.4-fold in s-AML  than controls.Distribution of allele frequencies revealed that patients with AML exhibited increased variant T allele frequency compared to the controls  and homozygotes TT into the M6 FAB subtype  as compared with the control group. Allele frequency distribution analysis confirmed the above differences, showing a higher T allele frequency among AML-M2 and AML-M6 patients . The CYP2B6 variant allele was approximately 2-fold higher in M2  and an almost 4-fold in M6 than expected . However, the sample size of M6 category was too small to point out any possible association.CYP2B6 genotype and allele frequency distribution in AML patients according to the karyotypic results and the risk group based on cytogenetic findings are presented in TT) was observed in patients with abnormal karyotypes, compared to those with normal karyotypes . Allele distribution analysis showed that the CYP2B6 variant allele was 1.7-fold higher in AML with abnormal karyotypes than our controls . Particularly, the mutant T allele was found to be 2.5-fold higher in s-AML patients with abnormal karyotype, and 1.5-fold in de novo AML patients with abnormal karyotype than our healthy donors . This difference was presented mainly due to the increased incidence of homozygous mutant (TT) genotypes in patients with -7/del(7q)  and -5/del(5q) , as well as heterozygotes (GT) in patients carrying trisomy 8 , trisomy 21  and t . The mutant T allele frequencies were 0.446, 0.436, 0.394, 0.348 and 0.293 in patients carrying t, -5/del(5q), -7/del(7q), +21 and +8 in their karyotype, respectively. Thus, patients showing the abnormalities t, -5/del(5q), -7/del(7q) have an almost 3-fold increased frequency of carrying the variant T allele compared to the control population ; patients showing +21 and +8 have an almost 2-fold risk . The increased frequency of T allele was confirmed in both de novo and s-AML groups of patients  was observed, with a striking higher frequency of the homozygotes for the variant T allele (TT) in patients belonging to the poor risk group compared to the other prognostic groups (17.7% vs 5.5% in intermediate and 1.6% in good risk group) . Particularly, the mutant allele was 2-fold higher in patients belonging to the poor prognostic group than expected. This finding was confirmed in both de novo and secondary AML patients showing an almost 2- fold and 3-fold increased risk of carrying the mutant allele, respectively  . The allCYP2B6 has been shown to catalyse the oxidation of a number of structurally diverse xenobiotics. Its metabolic activity depends on single-nucleotide polymorphisms (SNPs) 516T germline variation that reduces the enzymatic activity and blocks the biotransformation of carcinogen substrates to harmless metabolites CYP2B6 G516T polymorphism and AML susceptibility, we performed a case-control study including 572 AML patients and 430 healthy donors. Moreover, the CYP2B6 polymorphism was also investigated in respect to gender, age, FAB subtype, karyotype and risk classification groups based on cytogenetic findings.Several reports suggest that genetic predisposition along with exposure in environmental genotoxic compounds may play a pivotal role in the pathogenetic pathways implicated in AML development de novo AML. Our results revealed that the most common subtype was M4 followed by M2, M3 and M5. According to the literature, M0, M6 and particularly M7 subtypes are consistently rare, whereas the frequencies of the more common subtypes present variability: M1 , M2 , M4  and M5  s-AML than de novo. The stratification of de novo AML patients into good (13.1%), intermediate (57.1%) and poor (29.8%) risk groups was found to be comparable to well-defined representative groups of European AML patients s-AML patients into the above prognostic groups based only on chromosomal abnormalities revealed an increased frequency of the poor prognostic group (49.6%), which is consistent with the literature Concerning FAB classification, our patients’ group was found to be comparable to those of prior reports comprising large series of patients with 516T CYP2B6 gene polymorphism. The observed frequency of the homozygous variant genotype among the Greek healthy donors fell into the range previously reported in Caucasian populations p<0.001). This finding was confirmed for both de novo and s-AML patients; the variant T allele was found to be 1.5- and 2.4-fold higher in de novo and s-AML respectively than the control group, indicating a possible effect of the G516T CYP2B6 germline polymorphism on AML susceptibility.The present study comprises the largest series of AML patients ever evaluated for the GTT or GT) can not efficiently metabolize genotoxic compounds resulting in the accumulation of cell lesions and consequently in the development of AML. According to the above, toxicity related to inefficient detoxification of chemotherapeutic agents used for treatment of a previous hematological malignancy or other cancer, such as cyclophosphamide and ifosfamide 516T CYP2B6 polymorphism in AML susceptibility and highlighted an increased T allele frequency in AML. In detail, the first study reported a higher frequency of GT variant genotype in 36 AML patients from Turkey GT heterozygotes in 164 AML Chinese patients The diminished CYP2B6 enzymatic activity, as a result of the presence of the variant T allele, could predispose individuals to be more susceptible to develop AML. This could mean that individuals carrying the variant T allele in homozygous or heterozygous state ; 1.5-fold in de novo and 2.5-fold in s-AML. Further stratification of patients with abnormal karyotypes according to their chromosomal aberrations showed a higher frequency of CYP2B6 T allele in AML patients with t, -5/del(5q), -7/del(7q), +21 and +8. This finding concerns both de novo and s-AML patients. CYP2B6 induction by products of oxidative stress in BM comprises a protective mechanism against genetic damage that could contribute to leukemogenesis. Individuals carrying the T allele of CYP2B6 gene present diminished enzymatic activity and decreased ability to metabolize and inactivate various carcinogens, such as benzene metabolites, alkylating agents, naphthalene, trichloroethylene and aflatoxic B1 in?vitro studies have shown a significant increased frequencies of −5 and −7 in human lymphoblast cell lines after exposure to hydroquinone (HQ) CYP2B6 gene status should be taken into account for treatment optimization with alkylating agents, given that CYP2B6 deficiency could alter the metabolic capacity against these agents Higher incidence of the variant T allele was observed in AML patients with abnormal karyotype  and/or -7/del(7q) (p<0.001). Moreover, s-AML patients with monosomal and/or complex karyotypes, known to be related with a poor prognosis, presented also higher frequencies of variant mutant allele as well as variant genotypes. The associations between CYP2B6 genotype, cytogenetic aberrations, disease course and outcome would be an area of further research.Another interesting finding was the higher frequency of the mutant T allele observed in patients with poor prognosis based on cytogenetic findings. This indicates that the presence of the variant allele in homozygous or heterozygous state is probably related with specific chromosomal abnormalities conferring a poor prognosis. Indeed, statistical analysis revealed a strong positive association between the variant 516T CYP2B6 polymorphism on AML susceptibility suggesting that inherited defective function of the CYP2B6 detoxification pathway may be an important genetic determinant of AML risk. The higher frequency of the mutant allele found in patients with specific chromosomal abnormalities or in patients with monosomal and complex karyotypes indicates a strong association between the decreased CYP2B6 enzymatic activity and the occurrence of certain chromosomal abnormalities in AML. Further studies on this polymorphism in association with the patients’ response to treatment with alkylating agents or inhibitors of topoisomerases II may provide valuable information for the prediction of treatment response in relation to the CYP2B6 genotypes.In conclusion, our results provide evidence for a pathogenetic role of the GFile S1Figures S1 and S2 and Tables S1-S3. G-banded bone marrow karyotypes of AML patients showing a) 46,XX,del(7)(q22q32) b) 46,XX,del(5)(q13q33) c) 47,XY,+8 d) 45,X,-X,t. Figure S2. Gel electrophoresis of G516T CYP2B6 genotyping by BsrI PCR-RFLP on a 2% (w/v) agarose gel. The digestion of the 526-bp PCR product of CYP2B6 yields three bands of 241-bp, 268-bp and 17-bp* for the G/G genotype (wild-type) (lanes 1 and 2), two bands of 509-bp and 17-bp for the T/T homozygous mutant genotype (lane 5), and four bands at 509-bp, 241-bp, 268-bp and 17-bp for the heterozygous G/T genotype (lanes 3 and 4). Lane 6: negative BsrI digestion sample (no target DNA). Lane M: DNA ladder N3236S . *The small restriction fragment of 17bp is not appeared.(DOC)Click here for additional data file.


There is a light-induced fall in intracellular Ca2+. As Ca2+ dissociates from GCAPs in the 20–200 nM range, ROS-GC activity rises to quicken the photoresponse recovery. GCAPs then progressively turn down ROS-GC activity as Ca2+ and cyclic GMP levels return to baseline. To date, GCAPs mediate the only known mechanism of ROS-GC regulation in the photoreceptors. However, in mammalian cone outer segments, cone synapses and ON bipolar cells, another Ca2+ sensor protein, S100B, complexes with ROS-GC1 and senses the Ca2+ signal with a K1/2 of 400 nM. Unlike GCAPs, S100B stimulates ROS-GC activity when Ca2+ is bound. Thus, the ROS-GC system in cones functions as a Ca2+ bimodal switch; with rising intracellular Ca2+, its activity is first turned down by GCAPs and then turned up by S100B. This presentation provides a historical perspective on the role of S100B in the photoreceptors, offers a pictorial model for the “bimodal” operation of the ROS-GC switch and projects future tasks that are needed to understand its operation. Some accounts of this review have been adopted from the original publications of these authors.Photoreceptor rod outer segment membrane guanylate cyclase (ROS-GC) is central to visual transduction; it generates cyclic GMP, the second messenger of the photon signal. Photoexcited rhodopsin initiates a biochemical cascade that leads to a drop in the intracellular level of cyclic GMP and closure of cyclic nucleotide gated ion channels. Recovery of the photoresponse requires resynthesis of cyclic GMP, typically by a pair of ROS-GCs, 1 and 2. In rods, ROS-GCs exist as complexes with guanylate cyclase activating proteins (GCAPs), which are Ca The str2+ ; however2+ ,b. Yet a2+ .Later, the true photoreceptor ROS-guanylate cyclase (ROS-GC) was purified directly from bovine outer segments  and its 2+ binding protein, recoverin, that appeared to regulate guanylate cyclase activity  or at high (sub-micromolar) ranges of [Ca2+]. An even more exciting possibility exists for the simultaneous expression of both Ca2+-regulatory subunits in the same cell. ROS-GC could then operate as a Ca2+-bimodal transduction switch, stimulated at low and at high free [Ca2+] by Ca2+ -free GCAPs and by Ca2+-bound S100B, respectively. A theoretical Ca2+-BIMODAL ROS-GC transduction model embodying these features was proposed . Bimodal Ca2+ sensing could be an elegant general mechanism for neural transmission.The capacity for ROS-GC to couple to GCAPs or to CD-GCAP allows for a cell to sculpt its response to Ca2+-binding protein on SDS-PAGE  for half-maximal stimulation of guanylate cyclase. Clearly, CD-GCAP was not quite the same as the commercially obtained S100B.Despite being structurally identical proteins, biochemical analyses revealed two surprising functional discrepancies. As expected, S100A1–S100B and S100ββ dimer (S100B) from commercial sources stimulated cloned recombinant and wt-photoreceptor ROS-GC . But the2+, while S100B purification included zinc affinity chromatography . To investigate, CD-GCAP was subjected to zinc affinity chromatography (CD-GCAP-to-S100B) and S100B was heated to 75°C in Ca2+ (S100B-to-CD-GCAP). Guanylate cyclase activation, calcium–sensitivity, and hydroxylamine lability measurements demonstrated that CD-GCAP-to-S100B was identical to S100B and that S100B-to-CD-GCAP was identical to CD-GCAP. Apparently zinc inhibits the native S100B potency toward stimulation of ROS-GC. An additional finding was that S100B monomer is about twice as effective as the dimer in activating the ROS-GC catalytic activity. Hence, CD-GCAP and commercially obtained S100B are conformational isomers. The native form of S100B present in the retinal neurons is CD-GCAP.In the search for the explanation for these functional differences, the proteins were analyzed for post-translational modifications and higher order structures . After t50 of 395 nM, measured using surface plasmon spectroscopy, was comparable to the EC50 of 800 nM for stimulation of ROS-GC1 by S100B in a biochemical assay (Direct interaction between ROS-GC1 and S100B was verified by cross-linking experiments with bis-(sulfosuccinimidyl)suberate. A cross-linked complex, consisting of ROS-GC1 dimer and S100B, was detected by both ROS-GC1 and S100B antibodies on Western blots . Biochemal assay .2+-dependent stimulation by S100B, but the region aa 965–1016 appears to be the most critical , the Δ965–1054 mutant failed to respond to GCAP2, the Δ972–1045 responded with Vmax of ~50% of that of the wild-type ROS-GC1, and the response of the Δ1016–1054 mutant matched that of the wild-type cyclase, indicating that the functional GCAP2-modulated domain resided within aa 965–1016. Detailed peptide competition assays narrowed down the GCAP2-modulated site to the sequence motif Y965-N981 . As expected, the S100B-KO retina showed no S100B immunoreactivity  is now easy to understand. In ROS-GC1-/- retinas, cones degenerate but the rods remain intact  retina in which 33% of the photoreceptors are cones, in contrast to the 3% cone population in the mouse , Ca2+ will not bind to S100B and activate the switch if [Ca2+]i never exceeds 250 nM. However, the value of 250 nM for [Ca2+]i in darkness was taken from mouse rods . Indeed, genetic mutations that result in a hyperactive ROS-GC complex cause the photoreceptors to degenerate , bovine retinal synaptosomes (P1) were prepared and analyzed. Purity of the preparation was ascertained by Western blotting with rhodopsin kinase antibody. No immunoreactivity for rhodopsin kinase was detected, assuring that the preparation was not contaminated with ROSs, the original residential site of the ROS-GC1. Western blots were positive for ROS-GC1, GCAP1 and S100B demonstrating that indeed the synaptosomal fraction contained all the components of the ROS-GC1 bimodal system and was suitable for functional analyses.Immunohistochemical studies also place ROS-GC1, GCAP1 and S100B in the synaptic layers of the retina . To testTo be physiologically relevant, the components of the ROS-GC1 bimodal system, ROS-GC1, GCAP1 and S100B must reside in proximity and not, e.g., in different populations of synaptosomes. Because the Western analyses described above did not provide information about the proximity of ROS-GC1, GCAP1 and S100B molecules, immunohistochemical analyses  and crosCryosections of bovine retina were doubly labeled with antibodies against GCAP1 or ROS-GC1 and SV2, a synaptic vesicle protein that strongly stains photoreceptor termini . The resCross-linking experiments with a non-cleavable imidoester confirmed the proximity of ROS-GC1 and GCAP1 in the synaptosomal fraction . Without2+ concentrations ranging from 10 nM to 10 μM. Below 200 nM, Ca2+ elicited a dose-dependent decrease in guanylate cyclase activity with an “inhibitory” value (IC50) for Ca2+ of 100 nM. But at higher concentrations, ROS-GC activity began to climb with an EC50 of 0.8 μM. Side by side analysis of photoreceptor outer segment and P1 membranes demonstrated that the cyclic GMP synthetic activities were inhibited identically by free Ca2+ with an IC50 of 100 nM.Functionality of the ROS-GC1 bimodal switch in the P1 fraction was determined by measuring guanylate cyclase activity at Ca503–I522 in ROS-GC1 is critical and specific for the cyclase activation by GCAP1 -dependent inhibition upon the addition of an excess of GCAP1  A suitable mammalian model with abundant cones needs to be selected. No such mouse model is available at present. Through genetic manipulation, development of the rods has been suppressed in Nrl-/- mice and their retinas are cone-exclusive. But these cones may not be exact copies of the natural, although biochemically and physiologically, they appear to be very close (A. ansorgei), whose retina contains about 30% cones in contrast to the 3% population in the mouse  Ca2+-modulated signal transduction by the ROS-GC complex needs to be defined at a molecular level. For the S100B native to cones, what are the kinetic parameters of the S100B transduction switch? By what mechanism does S100B sense Ca2+ and how does it effect a change in the catalytic domain of ROS-GC? (4) The physiological role of S100B in the cone photoresponse needs to be assessed. Does it accelerate the recovery of cone response and increase the dark current? What limits S100B stimulated ROS-GC activity at high Ca2+? Does S100B increase the rate of vesicular release of neurotransmitter at the synapse? S100A1 is also expressed in outer segments, what is its function? (5) S100B is expressed in inner retinal neurons but nothing is known about what it does there. (6) S100B protein does not fit in the presently classified NCS protein family, sharing only 15% sequence identity and containing only two EF-hands. Therefore, the NCS family needs to be expanded to include S100B as a branch.The composition and the operational principles of cones and rods differ in ways that support their specialized visual functions: extremely high sensitivity in rods, rapid responsiveness over a wide dynamic range in cones. In addition to the GCAPs, S100B serves as an additional Cary close , 2006. Ohe mouse . (2) TheThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Immunoglobulin E (IgE) plays a central role in allergic disease. The B-cell receptor CD23 is pivotal in regulating IgE synthesis.Results: Ca2+-dependent structural changes in CD23 enable additional interactions with IgE.Conclusion: Mechanism for Ca2+-induced increase in affinity is revealed.Significance: Ca2+ binding brings an extra degree of modulation to CD23 function. 2+-free and Ca2+-bound forms, as well as the crystal structure of the Ca2+-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca2+ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca2+ binds at the principal and evolutionarily conserved binding site in CD23. Ca2+ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca2+-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca2+ brings an extra degree of modulation to CD23 function.Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type  lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca The prevalence of allergy is increasing worldwide . AllergiDK ∼10−10m). It is responsible for allergic sensitization and the immediate type I hypersensitivity reaction in which minute amounts of allergen cross-link receptor-bound IgE on mast cells and basophils, triggering cellular degranulation Der p I generates a soluble monomeric form of CD23 consisting of just the lectin head domain, termed derCD23  and a less well conserved “auxiliary” binding site (site 1)  .2+ is not required , indicates that a single Ca2+ ion binds at the principal binding site. Surprisingly, the crystal structure revealed that “loop 4,” known to contribute to IgE binding (2+ and ordered in its absence. We recently solved the structure of derCD23 in complex with Fcϵ3-4 (a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains) and found loop 4 partially disordered in the absence of Ca2+ . The resulting vectors were transformed into BL21 (DE3) competent cells.Mutations  were introduced into the derCD23 gene using the QuikChange Lightning site-directed mutagenesis kit (Stratagene). The wild-type derCD23 gene, in a pET5a vector , was use1H NMR at 500 MHz .Wild-type and six mutant derCD23 proteins were all expressed, refolded, and purified according to protocols previously described , 17. CorIgE-Fc  and Fcϵ3-4 were prepared as described previously .m HEPES, pH 7.4, 150 mm NaCl, 4 mm CaCl2, 0.01% (v/v) surfactant P-20) or HBS/EDTA  surfactant P-20) and injected over the sensor chip at a flow rate of 25 μl min−1 at concentrations of 4000, 2000, 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8, and 3.9 nm. Each data set was collected at 5, 15, 25, and 35 °C. Analyte concentrations were injected in duplicate, a titration series of “low-to-high” analyte concentration followed by a “high-to-low” series. A 2-min association phase was followed by a 5-min dissociation phase, which allowed complete dissociation of analyte from the senor surface. Standard double-referencing data subtraction methods were applied . Specific binding surfaces were prepared by coupling derCD23 to a CM5 sensor chip via amine coupling. Coupling density was limited to <100 RU. Samples of Fcϵ2-4 (IgE-Fc) were dialyzed into either HBS/Ca  and 0.44 mm derCD23  diluted with an equal volume of 0.2 m sodium acetate trihydrate, 0.1 m Tris, pH 8.5, 16% PEG 4000, and 10 mm CaCl2, and the drops were then micro-seeded with crystals of the “apo” Fcϵ3-4-derCD23 complex  or Daresbury SRS  synchrotrons. Indexing, integration, and merging of data were carried out with the MOSFLM , 22 or H(HKL2000  suite of2+-bound derCD23- Fcϵ3-4 complex structures were solved by molecular replacement with PHASER (2+-free derCD23 (PDB 2H2R) (4EZM) (The derCD23 and Cah PHASER  using thDB 2H2R)  and the ) (4EZM) , respectRfree set in thin resolution shells . Data were analyzed using the Microcal Origin software supplied with the machine using a 1:1 binding model under “low c-value” conditions where the stoichiometry is fixed at 1:1 , and the DK and ΔH values were floated.Wild-type and mutant derCD23 protein samples were initially dialyzed against 10 mm apo-derCD23 mixed against 20 mm CaCl2 with or without preincubation with stoichiometric amounts of the prolyl isomerase cyclophilin A. Tryptophan fluorescence was excited at 296 nm and all fluorescence emission collected above 305 nm. Typically, 6–10 runs were averaged before fitting to a double exponential  with a 1:1 mixing ratio. Experiments were carried out at 25 °C in the same buffer as for the ITC (see above) with 2 μonential  using th2+. The difference in binding affinity is highly temperature-dependent. At 5 °C, the difference in affinity is less than 2-fold (1.6 and 3.0 μm), but at 35 °C there is a 30-fold difference in binding affinity (2 and 58 μm) . The thermodynamic parameters of the two interactions are also notably different, as shown in a van't Hoff plot of the binding thermodynamics . The structure consists of two α-helices and two β-sheets formed by eight β-strands with four disulfide bonds . Unlike previously solved derCD23 crystal structures  , electrand 2H2T . These land 2H2T , 18.2+-free and Ca2+-bound derCD23 structures reveals that the only conformational differences are in loop 4 and, to a lesser degree, in loop 1  are in a similar position to that observed in the Ca2+-bound structure, although the orientation of the Thr-251 side chain is different. In Ca2+-free “conformation 2,” only Glu-249 remains in a similar position when compared with the Ca2+-bound structure, although marked differences are seen for both Thr-251  are distinctly different between the Car copies , and the2+ have an effect upon adjacent residues. The trans configuration of Pro-250 forms two hydrogen bonds with Arg-253, whereas cis Pro-250 cannot; consequently, both Arg-253 and Ser-254 are disordered when Pro-250 is cis (conformation 1), but are ordered, with Arg-253 pointing into a cavity between loops 1 and 4 , when Pro-250 is trans (conformation 2). In the presence of Ca2+, Pro-250 is effectively locked into the cis configuration.These two possible configurations of Pro-250 in the absence of Ca2+ ions, respectively, Ca2+-soaked human derCD23 crystals reveal that a single Ca2+ ion binds at the principal binding site in all four molecules of the asymmetric unit  and the Ca2+ ion itself is well defined in three of the derCD23 molecules; electron density for the three water molecules and Thr-251 in the fourth molecule is unclear. Although the site of Ca2+ binding is consistent with the observations of Wurzburg et al. (2+ differ from that reported by them for the double mutant (H213R/G256S) human derCD23 crystal structure (PDB code 2H2T).Unlike MBL  and DC-Sric unit . The eleg et al. , the det2+-bound derCD23 crystal structure presented here, three residues  provide four oxygen atoms, and three water molecules provide a further three for the binding of Ca2+ .To verify that the principal site is indeed the location of Caresidues  were mutDK for Ca2+ of 1.5 mm. This agrees with a previously calculated affinity determined by NMR perturbation studies  .The crystal structures of the derCD23 principal and auxiliary Ca2+-free derCD23. Similarly, Pro-250 is trans in two copies and cis in the others. D258A (auxiliary) and D270A  crystallized differently, and in all eight and seven molecules within the asymmetric unit, respectively, Pro-250 is trans. In contrast the E249A  mutant, which crystallized in yet another form, has cis Pro-250 in all three independent copies. Here also, loop 4 adopts a similar conformation to that observed in the Ca2+-bound derCD23 structure (and conformation 2 of the Ca2+-free and S252A structures). Mutation of the adjacent Glu-249, part of the EPT motif, to alanine appears to have locked Pro-250 into the cis configuration. Thus, of the 30 independent copies of derCD23 presented here, 19 are trans; of the remaining 11, four are cis as a result of the Ca2+ present, and 3 are cis as a result of mutation of the adjacent Glu-249. Thus, trans is apparently favored in the absence of Ca2+ or other factors.The S252A auxiliary binding site mutant crystallized in the same conditions and same crystal form as wild-type Ca2+-binding sites upon introduction of the point mutations that could inadvertently affect the binding of Ca2+, with the exception of D258A. Mutation of Asp-258, which faces Pro-250 at the base of loop 4, to alanine results in a conformational change in loop 4 and a reorientation of Arg-253 compared with the Ca2+-free derCD23 structure; this was seen in all eight molecules within the asymmetric unit. The side chain of Arg-253 flips ∼180° and projects into the principal Ca2+-binding site cavity, occupying the Ca2+ ion position . The re-positioned Arg-253 side chain forms two salt bridges with the carboxylate group of Glu-249. Occupation of the Ca2+-binding site by Arg-253 explains why the D258A mutant fails to bind Ca2+.No major conformational changes were observed in either the principal or auxiliary Ca2+-free crystal structure of the double-mutant derCD23 , as seen for Ca2+ binding to MBL  are bound to six derCD23 molecules (labeled G–L). A single derCD23-Fcϵ3-4 complex  is depicted in 2+-bound derCD23-Fcϵ3-4 complex differ by only 5°, similar to the range observed in the Ca2+-free complex structure. This closed conformation allosterically precludes binding to FcϵRI . Each of the six derCD23 molecules has a Ca2+ ion bound at the principal binding site; the electron density for the ion is clear in four of the molecules but is ambiguous in copies H and K. Comparison of the derCD23 molecules from the Ca2+-free and Ca2+-bound complexes shows that the only substantial difference involves residues Ser-256 and Glu-257 of loop 4, for which no electron density was observed in the Ca2+-free complex . All four salt bridges and two of the four additional hydrogen bonds observed in the Ca2+-free complex  and Arg-440 (Fcϵ3-4). However, in the Ca2+-bound complex, there are two additional salt bridges (both Asp-227 and Asp-258 with Arg-440) and two new hydrogen bonds formed (Gln-255 to Arg-440 and Cys-273 to Ser-437) (B). Arg-440, in the Cϵ3-Cϵ4 linker region, is key to these additional interactions with Asp-227 (loop 1) and Asp-258 (loop 4) following Ca2+-induced ordering and conformational change in loop 4.Turning to the interface between derCD23 and Fcϵ3-4, which is almost identical in all six copies in the Ca complex , all inv complex  are retaSer-437) B. Arg-44supplemental Fig. S8). Thus, although Ca2+ binds only at the principal binding site, it induces conformational changes that bring into play Asp-258 from the auxiliary site, which then makes additional contacts with IgE to enhance the affinity.This involvement of Asp-258 is particularly intriguing because it is the only conserved residue of the auxiliary binding site that remains in human CD23 . Only the principal Ca2+ site is fully conserved in human CD23, but previous structural studies on soluble derCD23 had identified conflicting locations for the binding of a single Ca2+ ion. The NMR solution structure of derCD23, together with chemical shift perturbation measurements, suggested that Ca2+ binds only at the auxiliary binding site  are coning site , but subpal site .2+-bound wild-type human derCD23 was solved. Several single point mutations in both the principal and auxiliary binding sites of derCD23 were also generated; their crystal structures were solved, and their ability in solution to bind calcium was assessed by ITC. The structure of Ca2+-bound derCD23 reported here shows a single ion bound at the principal calcium site, liganded by Glu-249, Thr-251, Asp-270, and three water molecules; the DK is 1.5 mm. Ca2+ binding to the mutant derCD23 molecules  was consistent with binding only to the principal site, with the exception of D258A, which showed no Ca2+ binding .  of MBL is coupled to a conformational change in loop 4 that involves the trans-cis isomerization of Pro-191, the structural equivalent of Pro-250 in CD23. In the absence of Ca2+, MBL exists as a mixture of two conformers, but binding of Ca2+ traps loop 4 and Pro-191 in the cis form  and not when Ca2+ binds only to the auxiliary site (site 1 in MBL).This is consistent with the extensive studies by Ng et al. , 37 show2+ binding to MBL using stopped-flow spectrofluorimetry. This revealed a characteristic biphasic fluorescence change as follows: a fast (∼1 s−1) burst phase followed by a slower conformational change, identified as the trans/cis prolyl isomerization accompanying Ca2+ binding because the observed rate was accelerated in the presence of cyclophilin A.Weis and co-workers  compleme2+ binding to derCD23, and we discovered striking similarities to the MBL results. First, there is a biphasic fluorescence change with observed rates for the two steps close to those seen for MBL . These additional salt bridges, Asp-227 and Asp-258 with Arg-440, which lie in the Cϵ3-Cϵ4 linker region, undoubtedly make the principal contribution to the enhanced affinity of CD23 for IgE in the presence of Ca2+.The binding of Caen bonds . Loop 4, ordered . The conare lost B. These 2+ binding on loop 4. In the presence of calcium, the interaction is entropically favorable, and without calcium it is entropically unfavorable. Although there are undoubtedly significant differences in how water and counter ions behave in the Ca2+-bound and Ca2+-free structures, the most obvious structural effect is on the dynamics of loop 4. The greater entropic contribution to binding in the presence of Ca2+ therefore at least partly reflects the smaller entropic penalty upon forming the complex with a more ordered loop 4. The earlier NMR study of derCD23 showed that this loop exhibits substantial mobility when free in solution , has been conserved in CD23 and “co-opted” into making an additional interaction with IgE (B). In human CD23, the other auxiliary Ca2+ site residues have not been conserved, but in murine CD23 , the auxiliary site ligands are conserved, and binding of a second Ca2+ ion is predicted. It is unclear how this might affect IgE binding, but the effect of Ca2+ will certainly differ; indeed, the early report of a requirement for Ca2+ in the murine system is pertinent (2+, may inform attempts to develop modulatory molecules for therapeutic purposes.CD23 stands out among the Ig receptors as a member of a different superfamily, and although binding to human IgE does not require either carbohydrate or Cawith IgE B. In humertinent . Human C


Pubmed for literature, on links between calcium malnutrition and risk of chronic diseases, we found the highest degree of evidence for osteoporosis, colorectal and breast cancer, as well as for hypertension, as the only major cardiovascular risk factor. Low calcium intake apparently has some impact also on cardiovascular events and disease outcome. Calcium malnutrition can causally be related to low activity of the extracellular calcium-sensing receptor (CaSR). This member of the family of 7-TM G-protein coupled receptors allows extracellular Ca2+ to function as a “first messenger” for various intracellular signaling cascades. Evidence demonstrates that Ca2+/CaSR signaling in functional linkage with vitamin D receptor (VDR)-activated pathways (i) promotes osteoblast differentiation and formation of mineralized bone; (ii) targets downstream effectors of the canonical and non-canonical Wnt pathway to inhibit proliferation and induce differentiation of colorectal cancer cells; (iii) evokes Ca2+ influx into breast cancer cells, thereby activating pro-apoptotic intracellular signaling. Furthermore, Ca2+/CaSR signaling opens Ca2+-sensitive K+ conductance channels in vascular endothelial cells, and also participates in IP3-dependent regulation of cytoplasmic Ca2+, the key intermediate of cardiomyocyte functions. Consequently, impairment of Ca2+/CaSR signaling may contribute to inadequate bone formation, tumor progression, hypertension, vascular calcification and, probably, cardiovascular disease.Through a systematic search in  Calcium malnutrition can be linked to the pathogenesis of various chronic diseases . et al.  = 0.02). This association was even stronger (RR = 0.67) in women with estrogen receptor-positive tumors. Consumption of dairy products was also inversely associated with risk  in this study. In a recent case-control study conducted among Chinese women by Zhang et al.  = 0.029 and 0.014, respectively). It was, however, not clear whether calcium intake itself was an independent risk factor for prostate cancer. This issue seemed to be clarified by Allen et al. [p [trend] = 0.02), but not calcium from other foods. The situation elsewhere in the world appears to be less clear. Huncharek et al. [et al. [p [trend] = 0.165) with low-grade cancer but inversely associated (p [trend] = 0.034) with high-grade cancer. A preventive effect of high calcium intake against high-grade cancers was also found in a recent case-control study conducted by Williams et al. o is attenuated by the systemic actions of calcium-regulating hormones, 1,25-dihydroxyvitamin D3 and parathyroid hormone (PTH). This allows physiological variations in [Ca2+]o to occur only within a narrow range. How these can be translated into modulation of cellular functions became clear, when Brown and colleagues [2+]o, i.e., in the range of 0.05 mM  in the plasma and in other extracellular fluid compartments. Hence, the CaSR plays a key role not only in systemic Ca2+ homeostasis but also in local control of cellular functions, such as cartilage and bone formation o gradient that, according to Whitfield o the receptor transduces signals preferentially along the PLC pathway causing intracellular accumulation of IP3 . In a h [et al.  the CaSRCa2+ see . in vitro and in vivo studies that calcimimetic compounds upregulate the CaSR in vascular cells and thereby attenuate vascular mineralization associated with reduced kidney function .Juxtaglomerular cells: According to Atchison and Beierwaltes [2+/CaSR in juxtaglomerular cells inhibits a calcium-inhibitable isoform of adenylate cyclase  mediates the anti-hypertensive effect of elevated extracellular calcium in two ways: (i) CaSR signaling via PKC results in up-regulation of COX-2 and enhanced synthesis of the natriuretic prostaglandin E2 [2, CaSR signaling targets apical electrolyte transporters and channels participating in the control of electrolyte reabsorption from the lumen [andin E2 ,163; (iihe lumen . The imphe lumen . Taken t2+/CaSR may therefore contribute to the adverse health effects of low habitual calcium intake. Because of the synergistic action of Ca2+/CaSR-activated and vitamin D receptor-mediated signaling cascades, one should bear in mind that, for optimal health outcome, restoration of both an adequate calcium and vitamin D status is necessary. There is evidence that efficient disease prevention does not require intake of more calcium and vitamin D than currently recommended for maintaining optimal bone health [There is no doubt that the CaSR plays an important role in local control of cellular homeostasis and function in many tissues and organs. Impaired or malfunctioning signaling from Cae health .


Cyanea capillata, could cause a dose-dependent increase of systolic blood pressure, which seemed to be the result of direct constriction of vascular smooth muscle (VSM). The aim of this study is to investigate whether TE could induce vasoconstriction in vitro and to explore its potential mechanism. Using isolated aorta rings, a direct contractile response of TE was verified, which showed that TE could induce concentration-dependent contractile responses in both endothelium-intact and -denuded aortas. Interestingly, the amplitude of contraction in the endothelium-denuded aorta was much stronger than that in the endothelium-intact one, implying that TE might also bring a weak functional relaxation in addition to vasoconstriction. Further drug intervention experiments indicated that the functional vasodilation might be mediated by nitric oxide, and that TE-induced vasoconstriction could be attributed to calcium influx via voltage-operated calcium channels (VOCCs) from the extracellular space, as well as sarcoplasmic reticulum (SR) Ca2+ release via the inositol 1,4,5-trisphosphate receptor (IP3R), leading to an increase in [Ca2+]c, instead of activation of the PLC/DAG/PKC pathway or the sympathetic nerve system.Our previous studies demonstrated that tentacle extract (TE) from the jellyfish,  In recent years, there has been growing evidence that jellyfish blooms are increasing globally in frequency and persisting longer than usual in response to anthropogenic disturbance and climate change ,2. With Cyanea capillata. Our result suggested that TE may serve as a potential alternative to the nematocyst venom for isolating and purifying cardiovascular toxicity proteins, because they share similar cardiovascular activity in vivo c are the main events that regulate the contractile state of VSMC. Furthermore, usually, an increase in [Ca2+]c results in VSM contraction, and a decrease in [Ca2+]c results in VSM relaxation [2+]c may arise from the opening of calcium channels in the plasma membrane, allowing the influx of extracellular Ca2+, or from the release of Ca2+ stores, such as SR c.It has been well established that changes in c; (3) the vasoconstrictive effects of TE are not due to the activation of the PLC/DAG/PKC pathway; and (4), finally, the sympathetic nerve system is not involved in TE-induced vasoconstriction.In conclusion, a direct vasoconstrictive effect of TE from the jellyfish,


However, the impact of aging on Ca2+ homeostasis in neurons remains largely unknown. Here, we survey the molecular mechanisms that mediate neuronal Ca2+ homeostasis and discuss the impact of aging on their efficacy. To address the question of how aging impinges on Ca2+ homeostasis, we consider potential nodes through which mechanisms regulating Ca2+ levels interface with molecular pathways known to influence the process of aging and senescent decline. Delineation of this crosstalk would facilitate the development of interventions aiming to fortify neurons against age-associated functional deterioration and death by augmenting Ca2+ homeostasis.The nervous system becomes increasingly vulnerable to insults and prone to dysfunction during aging. Age-related decline of neuronal function is manifested by the late onset of many neurodegenerative disorders, as well as by reduced signaling and processing capacity of individual neuron populations. Recent findings indicate that impairment of Ca Here, we briefly overview the main mechanisms neurons use in order to achieve an intricate regulation of the intracellular concentration of Ca2+. In addition, we discuss the accumulating evidence on the potential role of deregulated Ca2+ homeostasis in aging and disease of the nervous system.Fluctuations in intracellular calcium concentration act as signals for a variety of processes in neurons. Most notably, Ca decades . Moreovetability , integratability , synaptitability , gene extability , metaboltability , and protability . Given i2+ channels allow the passive influx of calcium ions down their electrochemical gradient. These channels are categorized into two major groups depending on the mechanism controlling their transition between the open and closed conformations: channels gated by voltage , and channels gated by ligand binding, in neurons usually L-glutamate .Plasma membrane Ca2+ channels are multi-protein complexes comprising several different subunits: α1, α2δ, β1-4, and γ , a glycosylated membrane protein expressed throughout the brain, was identified as the pore-forming subunit of a unique plasma membrane Ca2+-permeable voltage-gated ion channel  interacts with two plasma membrane-associated t-SNARE proteins, SNAP-25 and syntaxin-1  component and a slow asynchronous (tonic) component  receptors, the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, and the N-methyl-D-aspartate (NMDA) receptors. The channels formed by AMPA and KA receptors are primarily permeable to Na+ and K+ and exhibit a rather low conductance to Ca2+  and synaptic plasticity, a process associated with memory and learning as discussed later.In addition to voltage-gated channels, a number of Ca to Ca2+ . By contand Ca2+ . These rrization . Thus, t2+ from cells . One is the ATP-driven plasma membrane Ca2+ pump (PMCA) and the other is the Na+/Ca2+ exchanger (NCX), a complex similar to that discussed later for the removal of Ca2+ from the mitochondrial matrix into the cytoplasm .Two major plasma membrane mechanisms are responsible for the extrusion of Caytoplasm . Unlike 2+ pump has a higher affinity for Ca2+ (Kd = 100 nM) but a very slow turnover, whereas NCX has a much lower affinity (Kd = 1000 nM) but a higher turnover. Both types of transporters are co-expressed in neurons and in astrocytes  of the presynaptic terminals, whereas NCX is excluded from these sites and present in a more dispersed fashion on the rest of the neuron . Moreover, the discovery of a multitude of PMCA isoforms and alternative splice variants  residues in the C-terminal tail . In general, Ca2+ exits the ER through several types of Ca2+ release channels, such as inositol 3-phosphate (InsP3) receptors, ryanodine receptors (RyR), nicotinic acid adenine dinucleotide phosphate (NAADP) receptors, and polycystin-2 channels [the relative of transient receptor potential (TRP) proteins]. In neurons, the NAADP receptors were reported to exist in brain microsome preparations  and the Ca2+ activated RyR, both forming large tetrameric channel proteins. Both receptor families are comprised of multiple members that display distribution patterns that are both temporally and spatially regulated in neurons. For example, there are three RyRs, all of which can be activated by Ca2+ on the cytosolic side with differential sensitivities (RyR1 > RyR2 > RyR3). All three members have been detected in neurons, with distinct patterns that change during development and postnatal growth. For example, postnatally, RyR1 is highly expressed in cerebellar Purkinje cells, RyR3 in the hippocampus, striatum, and diencephalon, while many neurons co-express more than one RyR isoform  mode, where elevated cytoplasmic Ca2+ induces further Ca2+ release. Ca2+ waves in neurons were described more recently, after the notion had first been established using Xenopus oocytes plasmic reticulum Ca2+ ATPase (SERCA) family. This family includes three members (SERCA1–3), as well as two splice isoforms of SERCA2. While SERCA2b is ubiquitously expressed, SERCA2a and SERCA3 are found almost exclusively in cerebellar Purkinje neurons. Inhibition of the SERCA pumps results in a relatively slow emptying of ER Ca2+ stores, with Ca2+ exiting the ER through poorly described pathways . The SPCAs in addition supply the Golgi lumen with Mn2+, which is needed for many enzymatic reactions in this compartment. Mammalian SPCA was originally cloned from rat using a probe derived from sequences of the ATP-binding site of SERCA1 and SERCA2 (ATP2C1) was described by two independent groups (ATP2C1 results in four SPCA1 proteins with C-termini differing in length and specific amino acid sequence (ATP2C2) was independently described in 2005 by two groups , in conditions of low ATP demand and oxygen consumption, or through a mitochondrial proton/Ca2+ exchanger (two or more H+ per Ca2+). Indirect experiments with isolated mitochondria under pathological conditions or Ca2+ overload suggest an additional, higher conductance route, through the transient opening of the mitochondrial permeability transition pore (mPTP). However, the physiological relevance of mPTP in Ca2+ homeostasis remains controversial and is not supported by genetic ablation studies . A large family of these Ca2+-binding proteins is the one containing EF-hand Ca2+ binding domains. These motifs consist of two 10–12 residue long alpha helices, oriented perpendicularly against each other, separated by a 12-residue long loop region. EF-hand domains often exist as multiple pairs generating a wide structural and functional variability within this large family of proteins , reticulocalbin is localized in the lumen of the ER  interaction. The Ca2+-myristoyl switch could be a mechanism that affects the compartmentation of signaling cascades in neurons and/or the transmission of Ca2+ signals to their membranes , in which patients lose vision due to degeneration of photoreceptors , the postsynaptic increase in calcium is mediated through the activation of the NMDA receptor. As already mentioned earlier, NMDA receptor activation allows the influx of calcium only when the receptor is occupied by L-glutamate and concomitantly the postsynaptic membrane is depolarized. Emerging evidence indicates that the synaptic plasticity shift during aging results from changes in the source of Ca reduced  and Ca2+ncreased . The incncreased , or phoschannels . Interes rabbits , rodents rabbits , non-hum rabbits , and eld rabbits .2+ homeostasis, including enhanced release of Ca2+ from the ER , one of the most abundant proteins in neurons comprising 1–2% of the total protein. Although it is expressed both pre- and postsynaptically, its expression is particularly high in the postsynaptic density, where it is ideally located to respond to changes in calcium concentration. There are more than 30 isoforms of CaMKII and numerous substrates, many of which are located in the postsynaptic density  affects the stability of synapses and general neuronal physiology, ultimately culminating in altered function of olfactory neurons  diseases, amyotrophic lateral sclerosis (ALS), and other neurodegenerative disorders  are the most common genetic factors responsible for about 20% of familial ALS cases . Oxidative stress caused by the damaging effect of ROS to proteins, lipids, and DNA, is a common feature of aging-related diseases, including ALS . The mechanism by which presenilin mutations cause AD involves increased production of Aβ1–42 which aggregates and damages neurons. This view has been recently expanded by emerging findings suggesting that perturbed ER Cans in AD . For exael of AD . This can the ER . Intereseceptors . Future 2+ homeostasis is also likely to contribute to the initiation or progression of the neurodegenerative process by enhancing neuronal vulnerability to metabolic and other stressors , an inhibitor of the mitochondrial respiratory chain complex I, causes Parkinson-like symptoms , are associated with recessive early onset forms of PD, whereas mutations in α-synuclein (PARK1–4) and LRRK2 (PARK8) are responsible for dominant forms of familial PD. Mitochondrial dysfunction has been described for mutants of all these genes (Additional genetic factors could increase the rate at which mitochondrial Cams of PD . Mutatiose genes .2+ handling by the PD-related proteins. DJ-1 is a multitask protein that, in addition to its main role as an antioxidant (2+ concentration values to permit depolarization-induced Ca2+ release from the sarcoplasmic reticulum in muscle cells (2+-induced mitochondrial uncoupling and ROS production during physiological pacemaking (Recent papers have started to explore in more detail the possibility of Caioxidant , is alsole cells . Moreovecemaking .2+ influx from the extracellular milieu by enhancing the plasma membrane ion permeability (2+ permeability (2+ dysfunction influence each other are not clear, however, a functional interplay is unambiguous: Increased intracellular Ca2+ promotes α-synuclein aggregation, which in turn could promote intracellular Ca2+ increase (in vitro using non-neuronal cell lines, their relevance to DA neuron physiology and pathology remains to be examined.Regarding α-synuclein, it has been described that it can modulate Caeability  either teability  or throueability . The actincrease . A recenincrease . As thes2+ fluxes, has been recently proposed starting with the observation that the co-expression of mutant PINK1 in a cellular model of PD-expressing mutated α-synuclein exacerbated the observed mitochondrial defects, that is, increased mitochondrial size with loss of cristae and reduced ATP levels (2+ influx. As by blocking mitochondrial Ca2+ uptake, it was possible to restore the original phenotype (2+ metabolism, but the results are controversial. In one case, it was proposed that PINK1 absence caused an impairment of mitochondrial Ca2+ efflux, probably affecting the mitochondrial Na+/Ca2+ exchanger activity and thus resulting in mitochondrial Ca2+ overload, ROS production, and impaired respiration (2+ uptake and consequently to affect energy metabolism (2+ vulnerability (As to PINK1, its direct role in regulating cellular, and most specifically mitochondrial CaP levels . The prohenotype , thus supiration . In anottabolism . Howevertabolism , as wellrability .2+ homeostasis in the biology of all cells, it is not completely surprising that more and more studies suggest that deregulated Ca2+ is actively involved in the course of normal aging and in diverse pathological conditions. A general message arising from these studies is that in the nervous system Ca2+ signaling and homeostasis should be examined in view of the amazing cellular diversity exhibited by the nervous system. The machinery controlling Ca2+ homeostasis is similarly diverse among neurons, uniquely suited to the needs of each neuronal subtype. Taken together, the intrinsic differences of neurons in morphology, connectivity, proteome and Ca2+ homeostatic machinery are very likely to collectively and synergistically contribute to the selective vulnerability of distinct neuronal populations to different causes of senescence. The more we understand the interplay of Ca2+ homeostatic mechanisms with the intrinsic qualities of different neurons, the closer we will get to developing cell-specific therapies.Given the fundamental importance of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potentialconflict of interest.


The aim of this study was to assess the effects of resveratrol on hydrogen peroxide  and intracellular Ca2+ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.INa.L in myocytes both in the absence and presence of H2O2 (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin , INa.L inhibitors, decreased INa.L in cardiomyocytes in the presence of 300 µM H2O2. H2O2 (300 µM) increased the reverse INCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H2O2 of the diastolic intracellular Ca2+ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca2+ transient rise and decay. As expected, 2 µM TTX had no effect on ICa.L.Resveratrol  decreased 2O2-induced augmentation of INa.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.Resveratrol protects the cardiomyocytes by inhibiting the H INa.L) has been recognized as an important factor contributing to the abnormal repolarization in ischemic and failured hearts INa.L plays an important role in determining the action potential duration (APD) + concentration ([Na+]i) INa.L in rat ventricular myocytes + inflow during hypoxia increased [Na+]i which in turn rose the intracellular Ca2+ concentration ([Ca2+]i) via the Na+-Ca2+ exchanger (NCX) resulting in a Na+-dependent intracellular Ca2+ overload induced by INa.L2+]i caused cardiac arrhythmias and irreversible cell damage INa.L caused arrhythmic activity and contractile dysfunction INa.L is considered to be a new potential target for therapeutic intervention in patients with myocardial ischaemia and heart failure Despite intensive research has been conducted in recent years, cardiac arrhythmias remain a serious problem. Late sodium current , a polyphenol in various vegetables and fruits, is abundant in grapes. The root extracts of Polygonum cuspidatum, a constituent of Chinese and Japanese folk medicine, is also a good source of resveratrol INa.L. Previous investigation showed that 50 µM of resveratrol reduced INa.L in a recombinant expression system with the R1623Q LQT3 mutation INa.L in ventricular myocytes with increased H2O2 has not been reported. Therefore, this study was designed to address the impact of resveratrol on the Na+-dependent Ca2+ overload induced by H2O2-induced increase in INa.L in ventricular myocytes, with the intention to shed some light on its potential clinical application in the future.To further clarify the pharmacological mechanisms and the scope of application of the agent, it is critical to determine the effect of resveratrol on  −1 i.v.) and xylazine (7.5 mg kg−1 i.m.). Hearts were excised rapidly and perfused retrogradely on a Langendorff apparatus for 5 min with a Ca2+-free Tyrode’s solution containing (in mM): NaCl 135, KCl 5.4, MgCl2 1, NaH2PO4 0.33, HEPES 10 and glucose 10 , and then a Tyrode’s solution containing enzyme  and bovine serum albumin  for 40–50 min. The perfusate was finally switched to KB solution containing (in mM): KOH 70, taurine 20, glutamic acid 50, KCl 40, KH2PO4 20, MgCl2 3, EGTA 0.5, HEPES 10, and glucose 10 (pH 7.4), for 5 min. All perfusates were bubbled with 100% O2 and maintained at 37°C. The left ventricles were then cut into small chunks and gently agitated in KB solution. The cells were filtered through nylon mesh and stored in KB solution at 25°C. The use of animals in this investigation was approved by the Institutional Animal Care and Use Committee of Wuhan University of Science and Technology and conformed to the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health  and the Guide for the Care and Use of Laboratory Animals of Hubei Province, China.Adult New Zealand white rabbits (body weight 1.7–2 kg) of either sex were heparinized (2000 U) and anesthetized with ketamine (30 mg kg2 (control) and were then exposed to Tyrode's solution containing 300 µM H2O2 for 7 min. Next, isolated cells were perfused with Tyrode's solution containing both 300 µM H2O2 and one of the following, resveratrol  for 10 min, 4 µM tetrodotoxin (TTX) for 10 min, or 4 µM ranolazine for 5 min.Isolated cells were perfused with Tyrode's solution saturated oxygenated with 100% OINa.L, the intracellular pipette solution contained (mM): CsCl 120, CaCl2 1, MgCl2 5, Na2ATP 5, TEACl 10, EGTA 11, HEPES 10 (pH 7.3). The extracellular solution contained (mM): NaCl 135, CsCl 70, CaCl2 1,MgCl2 1, CdCl2 0.05, glucose 5, HEPES 5 (pH 7.4). In addition, 1 µM nicardipine was used to block the L-type Ca2+ channels.To record +-Ca2+ exchanger current (INCX), the intracellular pipette solution contained (mM): NaCl 20, CaCl2 10, aspartic acid 50, MgCl2 3, EGTA 20, HEPES 10, MgATP 5 and CsOH 120 (pH 7.3). The bath solution contained (mM): NaCl 140,CaCl2 2, MgCl2 2, HEPES 5, and glucose 10 (pH 7.4). In addition, 20 µmol L−1 ouabain, 1 mmol L−1 BaCl2, 2 mmol L−1 CsCl and 1 µmol L−1 nicardipine were used to block the Na+-K+ pump, K+ channels and L-Ca2+ channels, respectively. INCX was measured as the Ni2+ sensitive current that could be blocked by NiCl2 5.0 mmol L−1.To record Na2+ fluorescence measurement, the bath solution contained (mM): NaCl 131, KCl 4, CaCl2 1, MgCl2 1, glucose 10, and HEPES 10 (pH 7.4).Krebs-Henseleit bicarbonate (KHB) buffer for intracellular CaICa.L), the intracellular pipette solution contained (mM): CsCl 80, CsOH 60, aspartic acid 40, CaCl2 0.65, HEPES 5, EGTA 10, MgATP 5 and disodium creatine phosphate 5 (pH 7.2). The bath solution contained (mM): NaCl 135, KCl 5.4, MgCl2 0.5, CaCl2 1.8, NaH2PO4 0.33, HEPES 10, glucose 10 (pH 7.4).To record L-type calcium current . All other chemicals were purchased from Sigma. Stock solutions of drugs were prepared in water. Each of the stocks was diluted to the required concentrations in the external recording solution immediately before use.H−1. INa.L, INCX and ICa.L were recorded in voltage clamp mode by using whole-cell patch-clamp techniques in rabbit ventricular myocytes. Patch electrodes were pulled with a two-stage puller . Their resistances were in the range of 1–1.5 MΩ. Capacitance and series resistances were adjusted to obtain minimal contribution of the capacitive transients. A 60% to 80% compensation of the series resistance was usually achieved without ringing. Currents were obtained with an EPC 9 amplifier  and a Multiclamp 700B amplifier , filtered at 2 kHz, digitized at 10 kHz, and stored on a computer hard disk for further analysis.Experiments were performed at room temperature (22–24°C). Rabbit ventricular myocytes were placed into a recording chamber that was bathed with normal extracellular solution, in the absence and presence of drug (s), at a rate of 2 ml min2+ level were inferred from the ratio of the fura-fluorescence intensity (FFI) at both wavelengths. Intracellular Ca2+ fluorescence measurements were assessed using the following indices: diastolic intracellular Ca2+ level (diastolic FFI) (340/380 ratio), electrically stimulated rise in intracellular Ca2+ (△FFI) (340/380 ratio), maximal velocity of Ca2+ rise and Ca2+ decay (340/380 ratio).Myocytes were loaded with fura-2-AM (0.5 µM) for 10 min at 25°C, and fluorescence measurements were recorded with a dual excitation fluorescence photomultiplier system (Ionoptix). Myocytes were imaged through an Olympus IX-70 Fluor 40 × oil objective. The cells were field stimulated with a suprathreshold (150%) voltage and at a frequency of 0.5 Hz, 3-ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator . The polarity of the stimulatory electrodes was reversed frequently to avoid possible build up of electrolyte by-products. Cells were exposed to light emitted by a 75-W lamp and passed through either a 340- or 380-nm filter (bandwidths were ±15 nm) while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 340 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular CaINa.L were tested at 200 ms in depolarization testing pulse to eliminate the influence of transient sodium current (INa.T). Statistical significance between two groups and multiple groups were evaluated by Student’s t-test and one-way analysis of variance (ANOVA), respectively. All values were expressed as mean ± SD, and the number of cells (n) in each group was given. P<0.05 was considered to be statistically significant.Whole-cell recordings were analyzed using clampfit 9.0  and PulseFit . Figures were plotted by Origin . All amplitudes of INa.L, the current was recorded first in the absence and then in the presence of 4 µM TTX with 300 ms voltage steps from a holding potential (HP) of −120 to −20 mV. The values of current recorded before (control condition) and after application of TTX were −0.400±0.050 and −0.154±0.038 pA pF−1 , respectively, indicating that this TTX-sensitive current recorded was INa.L. When INa.L was recorded under normal condition using depolarizing pulses with a duration of 300 ms applied at 0.25 Hz from a HP of −120 mV in 10 mV increments between −70 and −20 mV, administration of 10, 20, 40 and 80 µM resveratrol resulted in decreased amplitudes of INa.L in a concentration dependent manner  increased the amplitudes of INa.L but 10, 20, 40 and 80 µM resveratrol decreased the amplitudes of INa.L in the continued presence of H2O2  induced by 300 µM H2O2 with an IC50 of 26.192 µM. Ranolazine  attenuated the increased INa.L in the presence of 300 µM H2O2 in a concentration dependent manner  reversed the increased INa.L caused by 300 µM H2O2. The values of INa.L under the control conditions, after application of 300 µM H2O2 and 4 µM TTX were −0.323±0.087, −0.878±0.071 and −0.258± −0.045 pA pF−1 , respectively.Currents were recorded using depolarizing pulses with a duration of 300 ms at a rate of 0.25 Hz from a HP of −120 mV, in 10 mV increments between −70 and −20 mV. Administration of resveratrol at concentrations of 10, 20, 40 and 80 µM resulted in a decrease in the amplitudes of  to H2O2 . H2O2  before returning to −40 mV. The current-time relationship was constructed from the declining slope of the ramp pulse (2+-sensitive (NCX) current obtained by subtracting the data in the trace d, h or l from the data in the trace a, e, i, b, f, j, c, g or k in the panel 4A, 4D or 4G. INCX measured at voltage levels of +50 mV and −100 mV, respectively, as the Ni2+-sensitive current by subtracting the current recorded in the presence from that in the absence of 5 mM NiCl2.Electrogenic mp pulse . Figure INCX was recorded after 7 minutes of exposure of H2O2. The mean current density of the inward INCX had little change, while the reverse INCX increased significantly , fura-fluorescence intensity change (△FFI), maximal velocity of Ca2+ rise and Ca2+ decay were all enhanced by 150 µM H2O2  TTX is relatively a selective INa.L blocker. Using depolarizing pulses with a duration of 300 ms applied at 0.5 Hz from a HP of −80 mV, in 10 mV increments between −40 and +40 mV, ICa.L was recorded in the absence  and ultimately causing cell damage INa.L by H2O2 and hypoxia INa and INa.L in the R1623Q LQT3 mutation in a recombinant expression system INa.L and reverse INCX under proarrhythmic conditions (H2O2) in rabbit ventricular myocytes has not been investigated yet. The data from this study addressed the impact of resveratrol on the Na+-dependent Ca2+ overload.The mechanisms underlying the genesis of ischemia- and reperfusion-induced arrhythmias are notoriously complex and controversial. There has been an interest in the concept that oxygen free radicals play a role in the pathogenesis of myocardial ischemia and infarction. It has been reported that a burst of HINa.L was increased by H2O2  2+ levels. Ranolazine is currently approved as an antianginal agent that reduces the Na+-dependent Ca2+ overload via inhibition of the INa.L and thus improves diastolic tone and oxygen handling during myocardial ischemia INa.L is an important contributing factor to intracellular Ca2+ overload in the pathogenesis of myocardial ischemia and infarction. In rabbit ventricular myocytes, low concentrations of TTX (1.5–4.0 µM) did not alter the L-type Ca2+ current  attenuated the increase in the reverse INCX by H2O2. Thus, we concluded that the effect of resveratrol to inhibit the increased reverse INCX caused by H2O2 was from its inhibition of INa.L.Recently, it has been reported that reverse ficantly . Differe2O2 significantly increased the amplitude of calcium transients and diastolic calcium concentration in the ventricular cell which could be reversed by TTX (2 µM). The intracellular Ca2+ overload caused by ROS was due to an increase in [Na+]i followed with an increase in Ca2+ influx via the reverse mode of the NCX 2+ into the cell will cause intracellular Ca2+ overload 2+ channel with an IC50 value of 55±2 µM INa.L and restrained Ca2+ overload induced by H2O2 but not affected L-type Ca2+ channels  also restrained the increased calcium transients amplitude and the diastolic calcium concentration induced by H2O2 (150 µM). Therefore the effects of resveratrol on the Na+-dependent Ca2+ overload induced by enhanced INa.L were similar to 2 µM TTX, suggesting that the reduction of Ca2+ overload by resveratrol may have similar mechanism to TTX, i.e., inhibition of INa.L. Indeed, resveratrol has also been suggested to inhibit the ryanodine receptor-induced intracellular Ca2+ increase 2+]i. The results in this study indicated that resveratrol reduced both INa.L and reverse INCX which was responsible for the reversal of intracellular Ca2+ overload in the presence of H2O2. Resveratrol may inhibit both the ryanodine receptor-induced intracellular Ca2+ overload and INa.L-induced increase in reverse INCX to attenuate the intracellular Ca2+ overload. Further research will be needed to clarify the contribution of the two pathways by resveratrol in the absence and presence of H2O2.In this study, 150 µM Hchannels , supportINa.L is an important target for resveratrol to prevent or treat ventricular arrhythmias. INa.L increased by H2O2 induces intracellular Ca2+ overload  through the increase in the reverse INCX. The inhibitive effect of resveratrol on H2O2-induced INa.L may reduce the concentration of [Na+]i, lower [Ca2+]i by attenuating reverse NCX to eliminate Ca+ overload, and ultimately inhibit the electrical abnormalities.


The most ubiquitous and abundant protein that serves as a receptor to sense changes in Ca2+ concentrations is Calmodulin (CaM), thus mediating the role as second messenger of this ion. The Ca2+/CaM complex initiates a plethora of signaling cascades that culminate in alteration of cell functions. Among the many Ca2+/CaM binding proteins, the multifunctional protein kinases CaMKII and CaMKIV play pivotal roles in the cell.Calcium Ca+ is a un I.The general structure of CaMKs includes an N-terminal kinase domain, an autoregulatory domain, an overlapping CaM-binding domain and, in phosphorylase kinase and CaMKII, a C-terminal association domain that is essential for multimerization and targeting.2+/CaM2+/CaM-independent activity2+ levels are reduced, the kinase is fully active until CaM dissociates CaMKIV shows very strong nuclear localization2+ signaling pathway to be either antagonistic or additive with the cAMP pathway for activation of CREB, depending on the relative activity of specific CaM kinases.These observations provide a mechanism that would permit the CaIII.Bok et alIV.1 phase or proceeds to S phase. The progression through G1 into the DNA synthesizing S phase is driven by cyclin-dependent kinase (CDK)4 and CDK6, that interact with the cyclin D family of proteins, and CDK2, that interacts with cyclins A/E 1 restriction point and regulate cyclin D1 expression during mid-G1 phase 2+ signaling and the ERK pathway has been documented 2+-induced ERK activation Cell proliferation is regulated by converging signals on the cell cycle machinery that determine whether the cell stays in the GV.The multifunctional CaMKs family proteins are involved in the control of differentiation and survival of neurons and hematopoietic stem cells +ScaI+Lin−/low cells (KLScells), a cell population that includes long-term and short term hematopoietic stem cells as well as other multipotent progenitor cells −/−KLS cells display enhanced proliferation as well as increased apoptosis, in vivo and in vitro, compared with wild type (WT) cells and have decreased levels of phospho-CREB (pCREB), CBP, Bcl-2 mRNA and Bcl-2 protein. Re-expression of CaMKIV in Camk4−/−KLS cells restores Bcl-2 and CBP levels and rescues the proliferation defects.The hematopoietic stem cell (HSC) gives rise to all mature, terminally differentiated cells of the blood. CaMKIV is involved in early hematopoietic development, and the absence of CaMKIV results in a reduction in the number of c-Kit2+ signaling in DC. For example, apoptotic body engulfment and processing are accompanied by a rise in intracellular Ca2+ and are dependent on external Ca2+2+ increases in DC, 2+-dependent pathway in the regulation of DC migration. The role of a Ca2+-dependent pathway in the mechanism regulating DC maturation is suggested by the opposite effects induced by Ca2+ ionophores or chelation of extracellular Ca2+ on this processCamk4 / DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive.Many critical biological functions involve Ca2+ regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII phosphorylates sarcoplasmic reticulum2+ homeostasis CaMKII has been implicated in several key aspects of acute cellular CaVI.Sepsis is a special type of host inflammatory response to bacterial infection that originates from massive and widespread release of pro-inflammatory mediators. Bacterial endotoxins, such as LPS, are the major offending factors in sepsis that activate TLR-mediated signaling to generate inflammatory response that is amplified in a self-sustaining manner. There are meny evidences of a correlation between multifunctional CaM kinases and TLR-4 signaling. CaMKII directly phosphorylates components of TLR signaling, and promotes cytokine production in macrophagesVII.2+-CaM-CaMKK-CaMKs pathways in effecting proliferation, survival, differentiation and associated molecular events. Intriguing new findings also indicate that, although the two kinases might share some substrates, there is specificity in the pathways they contribute, thus reflecting both shared and unique properties. The emergence of ERK as a critical CaMKII regulatory target for cell proliferation has united membrane proximal regulatory events orchestrated by the Ras activated cascade with key transcriptional CaMKs targets.CaMKs define a family of ser-thr kinases that direct a wide range of cellular processes and cell fate decisions. Since their discovery, much of the focus has been on their regulation of memory and learning. In recent years, studies on CaMKII and CaMKIV signaling in a number of cell models have established the importance of the Ca2+ is ubiquitously present in the cells, hence its compartimentalization and the regulation of its downstream kinases need to be finely tuned, in order to efficiently regulate biological functions. The involvement of CaMKII and CaMKIV in pathways that regulate functions as different as proliferation, survival and differentiation imply numerous cross-talks and their harmonization. Both kinases require Ca2+ increases to be activated, although other events are required to support their differential activation. Subcellular compartimentalization provides another tool to distinctively activate CaMKII and CaMKIV depending upon the cell’s needs. It is possible, though, to hypothesize a further mechanism of counter-regulation between the two kinases: insights into the regulation and impact of a crosstalk between CaMKII and CaMKIV signaling might bring in new highlights for biological functions, and their disruption in human diseases.Ca