Calcium Calmodulin Research Articles

Dynamic Substrate Gating in CaMKII by Autophosphorylation

Calcium/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates diverse substrates in cellular processes ranging from metabolism and cell cycle control to calcium homoeostasis, excitable cell activity and plasticity. CaMKII is a multimeric holoenzyme composed of 8–14 subunits from four closely related isoforms (α, β, γ, δ). The calcium sensor CaM binds to a target sequence within the autoregulatory domain (ARD) of each CaMKII subunit. Coincident CaM binding within the holoenzyme supports intersubunit autophosphorylation (T286 α-isoform and T287 β-, γ-, and δ-isoforms) within the ARD; a process enabling calcium-spike frequency encoding in excitable cells. We now show that T287 autophosphorylation tunes substrate phosphorylation to couple the activation state of CaMKII to substrate selectivity. Specifically, unlike high-affinity substrates, weak substrates require T286 autophosphorylation. Substrate switching may allow many of CaMKII substrates with weak non-consensus sites to be coupled to T287 autophosphorylation. The mechanism of this substrate filtering appears linked to the ARD as a catalytic fragment devoid of the ARD (and T286) exhibits broad substrate specificity as seen with T287 autophosphorylation (and phosphomimetic substitutions). Consistently, we find that substrates differential compete for access to the catalytic domain as indicated by differential ARD displacement as measured by associative changes in CaM binding. Graded CaMKII activity/autophosphorylation within the holoenzyme produces differential substrate phosphorylation profiles consistent with calcium-spike frequency encoding. We provide a structural model to explain T287 autophosphorylation-associated regulation of substrate selectivity and hypothesize that calcium-spike frequency encoding into T286 autophosphorylation tunes CaMKII's substrate selectivity to yield diverse cellular responses.

Calcium Calmodulin Regulates Zinc Mediated Changes in the Structure, Self-Association, and Activity of CaMKII

Ca2+-Calmodulin-dependent protein kinase II (CaMKII) is an interpreter of Ca2+ signaling and has been shown to be a critical component of learning and memory. This dodecameric oligomer also functions in part as a scaffolding molecule, serving to promote the formation of higher order signaling complexes within synapses. We set to study the effect that divalent cations, known to be critically important in neuronal signaling, have on the structure and function CaMKII. First, we computed the electrostatic surface potential of the dodecameric complex as a means to predict possible binding sites for divalent cations. A discrete set of high probability binding sites were identified at the interface between catalytic and association domains of neighboring subunits, far from the Mg2+ binding sites required to coordinate ATP in the catalytic cleft. Next, we experimentally characterized the effects of divalent cations on CaMKII structure using fluorescence, light scattering and electron microscopy. We found that some, but not all, divalent ions including Zn2+ promote the reversible formation of self-assembled CaMKII fibers that can extend for several μm. When associated in fibers, CaMKII activity is compromised; however, Ca2+/CaM activation of the enzyme disassembles fibers and the loss of activity is reversed. Interestingly, fiber assembly does not interfere with the autophosphorylation of CaMKII at T286, an autoregulatory modification, which confers autonomous activity in monodisperse CaMKII. We compile our results with structural and mechanistic data from literature to propose a model of Zn2+-mediated CaMKII fiber formation, where Ca2+/CaM regulates fiber formation through allosteric modification of a divalent ion site identified in our electrostatic map.

Proteomic analysis of transgenic rice overexpressing a calmodulin calcium sensor reveals its effects on redox signaling and homeostasis

Calmodulin1-1 (Cam1-1) gene in rice (Oryza saliva L.) is highly induced by salt stress and transgenic rice lines overexpressing OsCam1-1 are more salt-tolerant. To identify potential proteins and cellular processes that may be regulated by CaM in rice, a comparative proteomic analysis between wild type and transgenic rice was conducted, which revealed 72 differentially expressed proteins. A Gene Ontology analysis showed that several of these differentially expressed proteins were involved in the response to stimuli and signal transduction. Compared with wild type, the transgenic rice had a higher level of a putative GTPase-activating protein (GAP), a regulatory protein that binds to activated GTP-binding proteins and stimulates their GTPase activity. Real-time RT-PCR analyses verified that GAP transcript levels were higher in all transgenic rice lines than in wild type. A co-expression analysis indicated that GAP was positively and negatively co-expressed with genes encoding peroxidases and glutathione transferases. Under salt stress (150 mM NaCl), guaiacol peroxidase activity was higher in transgenic rice than in wild type. In contrast, compared with wild type, transgenic rice exhibited lower and delayed induction of glutathione transferase activity under salt stress. Under normal conditions, the H2O2 levels were lower in transgenic rice than in wild type. After 24 h of salt stress, H2O2 levels had slightly increased in both wild type and transgenic rice, leading to slightly lower H2O2 levels in transgenic rice than in wild type. Together, these results indicate that OsCam1-1 overexpression may affect redox signaling and homeostasis, resulting in an altered antioxidant system in rice plants under salt stress.

Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure.

Disruption in subcellular targeting of Ca(2+) signaling complexes secondary to changes in cardiac myocyte structure may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure (HF) and certain arrhythmias. To explore microdomain-targeted remodeling of ventricular L-type Ca(2+) channels (LTCCs) in HF. Super-resolution scanning patch-clamp, confocal and fluorescence microscopy were used to explore the distribution of single LTCCs in different membrane microdomains of nonfailing and failing human and rat ventricular myocytes. Disruption of membrane structure in both species led to the redistribution of functional LTCCs from their canonical location in transversal tubules (T-tubules) to the non-native crest of the sarcolemma, where their open probability was dramatically increased (0.034±0.011 versus 0.154±0.027, P<0.001). High open probability was linked to enhance calcium-calmodulin kinase II-mediated phosphorylation in non-native microdomains and resulted in an elevated ICa,L window current, which contributed to the development of early afterdepolarizations. A novel model of LTCC function in HF was developed; after its validation with experimental data, the model was used to ascertain how HF-induced T-tubule loss led to altered LTCC function and early afterdepolarizations. The HF myocyte model was then implemented in a 3-dimensional left ventricle model, demonstrating that such early afterdepolarizations can propagate and initiate reentrant arrhythmias. Microdomain-targeted remodeling of LTCC properties is an important event in pathways that may contribute to ventricular arrhythmogenesis in the settings of HF-associated remodeling. This extends beyond the classical concept of electric remodeling in HF and adds a new dimension to cardiovascular disease.

Beneficial Effects of a CaMKIIα Inhibitor TatCN21 Peptide in Global Cerebral Ischemia.

Aberrant calcium influx is a common feature following ischemic reperfusion (I/R) in transient global cerebral ischemia (GCI) and causes delayed neuronal cell death in the CA1 region of the hippocampus. Activation of calcium-calmodulin (CaM)-dependent protein kinase IIα (CaMKIIα) is a key event in calcium signaling in ischemic injury. The present study examined the effects of intracerebroventricular (icv) injection of tatCN21 in ischemic rats 3h after GCI reperfusion. Cresyl violet and NeuN staining revealed that tatCN21 exerted neuroprotective effects against delayed neuronal cell death of hippocampal CA1 pyramidal neurons 10days post-GCI. In addition, TatCN21 administration ameliorated GCI-induced spatial memory deficits in the Barnes maze task as well as anxiety-like behaviors and spontaneous motor activity in the elevated plus maze and open field test, respectively. Mechanistic studies showed that the administration of tatCN21 decreased GCI-induced phosphorylation, translocation, and membrane targeting of CaMKIIα. Treatment with tatCN21 also inhibited the level of CaMKIIα-NR2B interaction and NR2B phosphorylation. Our results revealed an important role of tatCN21 in inhibiting CaMKIIα activation and its beneficial effects in neuroprotection and memory preservation in an ischemic brain injury model.

Developmental changes in plasticity, synaptic, glia and connectivity protein levels in rat dorsal hippocampus

Thus far the identification and functional characterization of the molecular mechanisms underlying synaptic plasticity, learning, and memory have not been particularly dissociated from the contribution of developmental changes. Brain plasticity mechanisms have been largely identified and studied using in vitro systems mainly derived from early developmental ages, yet they are considered to be general plasticity mechanisms underlying functions -such as long-term memory- that occurs in the adult brain. Although it is possible that part of the plasticity mechanisms recruited during development is then re-recruited in plasticity responses in adulthood, systematic investigations about whether and how activity-dependent molecular responses differ over development are sparse. Notably, hippocampal-dependent memories are expressed relatively late in development, and the hippocampus undergoes and extended developmental post-natal structural and functional maturation, suggesting that the molecular mechanisms underlying hippocampal neuroplasticity may actually significantly change over development. Here we quantified the relative basal expression levels of sets of plasticity, synaptic, glia and connectivity proteins in rat dorsal hippocampus, a region that is critical for the formation of long-term explicit memories, at two developmental ages, postnatal day 17 (PN17) and PN24, which correspond to a period of relative functional immaturity and maturity, respectively, and compared them to adult age. We found that the levels of numerous proteins and/or their phosphorylation, known to be critical for synaptic plasticity underlying memory formation, including immediate early genes (IEGs), kinases, transcription factors and AMPA receptor subunits, peak at PN17 when the hippocampus is not yet able to express long-term memory. It remains to be established if these changes result from developmental basal activity or infantile learning. Conversely, among all markers investigated, the phosphorylation of calcium calmodulin kinase II α (CamKII α and of extracellular signal–regulated kinases 2 (ERK-2), and the levels of GluA1 and GluA2 significantly increase from PN17 to PN24 and then remain similar in adulthood, thus representing correlates paralleling long-term memory expression ability.

Molecular and biochemical analysis of supplementation of calcium under in vitro condition on tuberization in potato ( Solanum tuberosum L.)

Abstract Calcium plays an important role in plant physiology and various plant cell signaling pathways and alters several biochemical processes by activating specific enzymes. Cytosolic calcium (Ca2+) regulates the activity of Ca2+ dependent protein kinases, which phosphorylate various key metabolic enzymes. In this study, expression of the calcium (Ca2+) dependent proteins, calmodulin (CaM1) and Calcium dependent Protein Kinase (StCDPK), as well as the other important tuberizing pathway enzyme, the lipoxygenase (LOX; EC 1.13.11.12) were studied upon Ca2+ application to the single node segments of potato used for tuberization studies. Calcium at higher levels (9 mM) significantly improved the tuber number, growth and tuber yield under in vitro conditions. The expression level of the CaM1 and StCDPK were significantly higher in stolons and initial tubers showing positive correlation with supplemental Ca2+ and tuberization response. Similar trends were also observed with LOX enzyme activity, which increased by 18% and 25% with the supplementation of Ca2+ at 6 and 9 mM, respectively to the tuber induction medium, when compared to explants tuberizing on the medium without the Ca2+ control. Here we report the increase in tuberization, tuber growth and tuber yield with the supplementation of Ca2+ in tuberization induction medium could be attributed to the increased expression of the Ca2+ dependent proteins and enhanced lipoxygenase activity.

Quercetin targets the interaction of calcineurin with LxVP-type motifs in immunosuppression.

Calcineurin (CN) is a unique calcium/calmodulin (CaM)-activated serine/threonine phosphatase. To perform its diverse biological functions, CN communicates with many substrates and other proteins. In the physiological activation of T cells, CN acts through transcriptional factors belonging to the NFAT family and other transcriptional effectors. The classic immunosuppressive drug cyclosporin A (CsA) can bind to cyclophilin (CyP) and compete with CN for the NFAT LxVP motif. CsA has debilitating side effects, including nephrotoxicity, hypertension and tremor. It is desirable to develop alternative immunosuppressive agents. To this end, we first tested the interactions between CN and the LxVP-type substrates, including endogenous regulators of calcineurin (RCAN1) and NFAT. Interestingly, we found that quercetin, the primary dietary flavonol, can inhibit the activity of CN and significantly disrupt the associations between CN and its LxVP-type substrates. We then validated the inhibitory effects of quercetin on the CN-NFAT interactions in cell-based assays. Further, quercetin also shows dose-dependent suppression of cytokine gene expression in mouse spleen cells. These data raise the possibility that the interactions of CN with its LxVP-type substrates are potential targets for immunosuppressive agents.

Effect of pH on the structure, function, and stability of human calcium/calmodulin-dependent protein kinase IV: combined spectroscopic and MD simulation studies.

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium-calmodulin dependent signal through a secondary messenger, Ca(2+), which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca(2+) concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0-11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0-4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV.

Endosulfan Induces CYP1A1 Expression Mediated through Aryl Hydrocarbon Receptor Signal Transduction by Protein Kinase C.

CYP1A1 is a phase I xenobiotic-metabolizing enzyme whose expression is mainly driven by AhR. Endosulfan is an organochlorine pesticide used agriculturally for a wide range of crops. In this study, we investigated the effect of endosulfan on CYP1A1 expression and regulation. Endosulfan significantly increased CYP1A1 enzyme activity as well as mRNA and protein levels. In addition, endosulfan markedly induced XRE transcriptional activity. CH-223191, an AhR antagonist, blocked the endosulfan-induced increase in CYP1A1 mRNA and protein expression. Moreover, endosulfan did not induce CYP1A1 gene expression in AhR-deficient mutant cells. Furthermore, endosulfan enhanced the phosphorylation of calcium calmodulin (CaM)-dependent protein kinase (CaMK) and protein kinase C (PKC). In conclusion, endosulfan-induced up-regulation of CYP1A1 is associated with AhR activation, which may be mediated by PKC-dependent pathways.

Structure guided design of potential inhibitors of human calcium-calmodulin dependent protein kinase IV containing pyrimidine scaffold.

Calmodulin dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family and considered as an encouraging target for the development of novel anticancer agents. The interaction and binding behavior of three designed inhibitors of human CAMKIV, containing pyrimidine scaffold, was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. In silico docking studies were performed to screen several compounds containing pyrimidine scaffold against CAMKIV. Molecular docking calculation predicted the binding of these ligands in active-site cavity of the CAMKIV structure correlating such interactions with a probable inhibition mechanism. Finally, three active pyrimidine substituted compounds (molecules 1-3) have been successfully synthesized and characterized by (1)H and (13)C NMR. Molecule 3 is showing very high binding-affinity for the CAMKIV, with a binding constant of 2.2×10(8), M(-1) (±0.20). All three compounds are nontoxic to HEK293 cells up to 50 μM. The cell proliferation inhibition study showed that the molecule 3 has lowest IC50 value (46±1.08 μM). The theoretical and experimental observations are significantly correlated. This study reveals some important observations to generate an improved pyrimidine based compound that holds promise as a therapeutic agent for the treatment of cancer and neurodegenerative diseases.

Abstract 17263: Cardiac Myosin Binding Protein-C Phosphorylation Mitigates Pressure-Overload Heart Failure Despite Alterations in Calcium Handling

Heart failure with reduced ejection (HFrEF) fraction exhibits both systolic and diastolic dysfunctions; therefore, treatments that improve contractility and lusitropy will be helpful. Cardiac myosin binding protein-C (cMyBP-C), which is a thick filament protein, can increase rate of cross-bridge cycling by its phosphorylation status to enhance contractility and lusitropy. Consequently, we hypothesized that cMyBP-C phosphorylation mitigates HFrEF by preserving systolic and diastolic functions. We tested our idea by challenging WT cMyBP-C(tWT), phosphorylation-deficient cMyBP-C(t3SA):S273A/S282A/S302A, and phosphorylation-mimetic cMyBP-C(t3SD):S273D/S282D/S302D mouse models with trans-aortic constriction (TAC) to induce pressure-overload HFrEF. Survival at 5 weeks showed notable differences (tWT 75.6±6.7% n=41, t3SA 53.4±6.7% n=68, t3SD 92.1±4.4% n=38, p=0.00037). Serial echocardiography showed that cardiac structure and function deteriorated for all mouse models. In comparison to tWT at post-TAC week-5, t3SA demonstrated ventricular dilation and reduced ejection fraction (EF); conversely, t3SD exhibited better preservation of diastolic function (smaller E/Ea, where E=blood flow Doppler and Ea=tissue Doppler of peak myocardial relaxation velocity). Electrically paced intact papillary muscles from t3SA and t3SD demonstrated slower intracellular calcium decay (smaller decay rate constant kCa). Western blotting showed: (1) decreased calcium-ATPase (SERCA2a) expression in t3SA and t3SD, (2) lowest ryanodine receptor (RyR2) expression in t3SD, and (3) decreased calcium calmodulin kinase-2 phosphorylation (pCaMK2) only in t3SD. These calcium handling protein alterations likely prevented stronger rescue by cMyBP-C phosphorylation mimetic. Thus, cMyBP-C phosphorylation mimetic mitigated TAC-induced HFrEF (better survival and preservation of diastolic function) despite depression of calcium handling proteins.

RETRACTED: Calcium and SOL Protease Mediate Temperature Resetting of Circadian Clocks

SummaryCircadian clocks integrate light and temperature input to remain synchronized with the day/night cycle. Although light input to the clock is well studied, the molecular mechanisms by which circadian clocks respond to temperature remain poorly understood. We found that temperature phase shifts Drosophila circadian clocks through degradation of the pacemaker protein TIM. This degradation is mechanistically distinct from photic CRY-dependent TIM degradation. Thermal TIM degradation is triggered by cytosolic calcium increase and CALMODULIN binding to TIM and is mediated by the atypical calpain protease SOL. This thermal input pathway and CRY-dependent light input thus converge on TIM, providing a molecular mechanism for the integration of circadian light and temperature inputs. Mammals use body temperature cycles to keep peripheral clocks synchronized with their brain pacemaker. Interestingly, downregulating the mammalian SOL homolog SOLH blocks thermal mPER2 degradation and phase shifts. Thus, we propose that circadian thermosensation in insects and mammals share common principles.

Mouse hippocampal GABAB1 but not GABAB2 subunit-containing receptor complex levels are paralleling retrieval in the multiple-T-maze.

GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM) task, a robust paradigm for evaluation of spatial learning. Trained or untrained (yoked control) C57BL/6J male mice (n = 10/group) were subjected to the MTM task and sacrificed 6 h following their performance. Hippocampi were taken, membrane proteins extracted and run on blue native PAGE followed by immunoblotting with specific antibodies against GABAB1, GABAB1a, and GABAB2. Immunoprecipitation with subsequent mass spectrometric identification of co-precipitates was carried out to show if GABAB1 and GABAB2 as well as other interacting proteins co-precipitate. An antibody shift assay (ASA) and a proximity ligation assay (PLA) were also used to see if the two GABAB subunits are present in the receptor complex. Single bands were observed on Western blots, each representing GABAB1, GABAB1a, or GABAB2 at an apparent molecular weight of approximately 100 kDa. Subsequently, densitometric analysis revealed that levels of GABAB1 and GABAB1a but not GABAB2- containing receptor complexes were significantly higher in trained than untrained groups. Immunoprecipitation followed by mass spectrometric studies confirmed the presence of GABAB1, GABAB2, calcium calmodulin kinases I and II, GluA1 and GluA2 as constituents of the complex. ASA and PLA also showed the presence of the two subunits of GABAB receptor within the complex. It is shown that increased levels of GABAB1 subunit-containing complexes are paralleling performance in a land maze.

Role of calcium on the initiation of sperm motility in the European eel

Sperm from European eel males treated with hCGrec was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCl+EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca2+]i fluorescence (and in [Na+]i in some cases) were evaluated by flow cytometry.After sperm motility was activated in a medium containing Ca2+ (seawater, SW) the intracellular fluorescence emitted by Ca2+ increased 4–6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCl+EDTA) resulted in a percentage of motility similar to seawater, the [Ca2+]i levels did not increase at all. This result strongly suggests that increasing [Ca2+]i is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca2+]i concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm.

Long-Term Dietary Alpha-Linolenic Acid Supplement Alleviates Cognitive Impairment Correlate with Activating Hippocampal CREB Signaling in Natural Aging Rats.

Alpha-linolenic acid (ALA) is a major precursor of the essential n-3 polyunsaturated fatty acid (PUFA), whose deficiency alters the structure and function of membranes and induces cerebral dysfunctions. The major purpose of this study was to investigate the protective effect of prolonged ALA intake on cognitive function during natural aging. Female Sprague-Dawley rats aged 6months were chronically treated with ALA and/or lard per day for 12months. Regular diet-treated rats, both young and old (4 and 18months old, respectively) served as controls. Rats fed on regular diet during aging showed memory deficits in Morris water maze, which were further exacerbated by lard intake. However, supplementation with ALA for 12months dose-dependently improved the performance in spatial working memory tasks. Memory performance correlated well with the activation of cAMP response element-binding protein (CREB) and increases in both levels of brain-derived neurotrophic factor (BDNF) and its specific receptor tyrosine kinase B (TrkB) phosphorylation in the hippocampus. Further study identified that hippocampal extracellular signal-related kinase (ERK) and Akt rather than calcium calmodulin kinase IV (CaMKIV) and protein kinase A (PKA), the upstream signalings of CREB, were also activated by ALA supplement. Moreover, memory improvement was accompanied with alterations of hippocampal synaptic structure and number, suggestive of enhancement in synaptic plasticity. Together, these results suggest that long-term dietary intake of ALA enhances CREB/BDNF/TrkB pathway through the activation of ERK and Akt signalings in hippocampus, which contributes to its ameliorative effects on cognitive deficits in natural aging.

Editorial: Plant responses to bacterial quorum sensing molecules.

Editorial: Plant responses to bacterial quorum sensing molecules.

Prenatal cocaine exposure impairs cognitive function of progeny via insulin growth factor II epigenetic regulation

Studies have showed that prenatal cocaine exposure (PCOC) can impair cognitive function and social behavior of the offspring; however, the mechanism underlying such effect is poorly understood. Insulin-like growth factor II (Igf-II), an imprinted gene, has a critical role in memory consolidation and enhancement. We hypothesized that epigenetic regulation of hippocampal Igf-II may attribute to the cognitive deficits of PCOC offspring. We used Morris water maze and open-field task to test the cognitive function in PCOC offspring. The epigenetic alteration involved in hippocampal Igf-II expression deficit in PCOC offspring was studied by determining Igf-II methylation status, DNA methyltransferases (DNMT) expressions and l-methionine level. Moreover, IGF-II rescue experiments were performed and the downstream signalings were investigated in PCOC offspring. In behavioral tests, we observed impaired spatial learning and memory and increased anxiety in PCOC offspring; moreover, hippocampal IGF-II mRNA and protein expressions were significantly decreased. Hippocampal methylation of cytosine-phospho-guanine (CpG) dinucleotides in differentially methylated region (DMR) 2 of Igf-II was elevated in PCOC offspring, which may be driven by the upregulation of l-methionine and DNA methyltransferase (DNMT) 1. Importantly, intra-hippocampal injection of recombinant IGF-II reactivated the repressed calcium calmodulin kinase II α (CaMKIIα) and reversed cognitive deficits in PCOC offspring. Collectively, our findings suggest that cocaine exposure during pregnancy impairs cognitive function of offspring through epigenetic modification of Igf-II gene. Enhancing IGF-II signaling may represent a novel therapeutical strategy for cocaine-induced cognitive impairment.

Streptozotocin induces endoplasmic reticulum stress and apoptosis via disruption of calcium homeostasis in mouse pancreas

Calcium homeostasis refers to the regulation of calcium ion concentration in the body. This concentration is tightly controlled by a stabilizing system consisting of calcium channels and calcium buffering proteins. Calcium homeostasis is crucial for cell survival. Various forms of cell death (e.g., necrosis and apoptosis) also share calcium signaling pathways and molecular effectors. Calcium acts not only as a ubiquitous second messenger involved in apoptosis along with various cell death inducers but also a regulator for the synthesis of enzymes/hormones such as insulin. We hypothesized that streptozotocin disrupts calcium homeostasis and the altered intracellular calcium levels may induce cell death. After streptozotocin administration, blood glucose level was increased while insulin levels decreased. The expression of insulin response markers also decreased relative to the vehicle group. L-type voltage-gated calcium channel expression and sarcoplasmic reticulum Ca2+ ATPase were increased by streptozotocin. Calcium buffering protein calbindin-D9k and calmodulin family members were also increased. The expression of genes involved in transporting calcium ions to the endoplasmic reticulum (ER) was decrease while the expression of those affecting the removal of calcium from the ER was increased. Depletion of calcium from the ER leads to ER-stress and can induce apoptosis. In the streptozotocin-treatment group, apoptosis markers were increased. Taken together, these results imply that the disruption of calcium homeostasis by streptozotocin induces ER-stress and leads to the apoptosis of pancreatic cells. Additionally, findings from this study suggest that imbalances in calcium homeostasis could promote pancreatic beta cell death and result in type I diabetes.

Activation of calcium/calmodulin-dependent kinase II following bovine rotavirus enterotoxin NSP4 expression.

The rotavirus nonstructural protein 4 (NSP4) is responsible for the increase in cytoplasmic calcium concentration through a phospholipase C-dependent and phospholipase C-independent pathways in infected cells. It is shown that increasing of intracellular calcium concentration in rotavirus infected cells is associated with the activation of some members of protein kinases family such as calcium/calmodulin-dependent kinase II, which plays a crucial role in replication and pathogenesis of the virus. The aim of this study was to expression bovine rotavirus NSP4 gene in HEK293 cell and evaluation of its biological effect related to activation of calcium/calmodulin-dependent kinase II in cell culture. MA104 cells was used as a sensitive cell for propagation of virus and defined as a positive control. The NSP4 gene was amplified and inserted into an expression vector, and introduced as a recombinant plasmid into HEK293T cells. Western blot analysis was performed as a confirmation test for both expression of NSP4 protein and activation of calcium/calmodulin-dependent kinase II. Expression of NSP4 and activated form of calcium/calmodulin-dependent kinase II were demonstrated by western blotting. It was shown that the expression of biologically active full- length NSP4 protein in HEK293T cells may be associated with some biological properties such as calcium calmodulin kinase II activation, which was indicator of rotaviruses replication and pathogenesis.