Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.     

Calreticulin regulates insulin receptor expression and its downstream PI3 Kinase/Akt signalling pathway

Defects in insulin signalling and glucose metabolism are associated with the development of diabetes. Insulin signalling is initiated by the binding of insulin to its receptor and triggering cascades of events including activation of PI3kinase/Akt signalling pathway. Calreticulin (CRT) is a calcium binding chaperone molecule located in the endoplasmic reticulum. Targeted deletion of CRT in mice is embryonic lethal as a result of developmental and metabolic abnormalities. Rescued CRT null mice develop severe hypoglycemia the reason for which is not known. In addition, ventricular cardiomyocytes isolated from CRT null (crt-/-) mice have increased glycogen deposits. Therefore, the aim of this study was to investigate the changes in the glucose uptake and insulin signalling pathway (mainly PI3 kinase/Akt) in the absence of CRT. Here we show a significant increase in the glucose uptake by the crt-/- cells. This increase was accompanied by a significant increase in both insulin receptor beta expression, Insulin receptor substrate-1 phosphorylation, GLUT-1 expression and in insulin stimulated Akt phosphorylation and kinase activity in the crt-/- cells. Intriguingly, the increased expression of insulin receptor beta in the crt-/- was due to decreased levels of p53 protein. The current study is the first evidence for the up-regulation of insulin receptor density and activity in the absence of CRT function.     

Enhanced ubiquitin-proteasome activity in calreticulin deficient cells: a compensatory mechanism for cell survival

Calreticulin is a lectin chaperone essential for intracellular calcium homeostasis. Deletion of calreticulin gene compromises the overall quality control within the endoplasmic reticulum (ER) leading to activation of the unfolded protein response. However, the ER structure of calreticulin deficient cells (crt-/-) is not altered due to accumulation of misfolded proteins. Therefore, the aim of this study was to determine whether the ubiquitin-proteasome pathway is activated in crt-/- cells as a compensatory mechanism for cell survival. Here we show a significant increase in the expression of genes involved in ER associated degradation and activation of the ubiquitin-proteasome system in crt-/- cells. We also demonstrated that the ubiquitination of two proteins processed in ER, connexin 43 and A1AT NHK (alpha1-antitrypsin mutant) are increased in crt-/- cells. Furthermore, we showed that the increased proteasome activity in the crt-/- cells could be rescued upon re-introduction of calreticulin or calsequestrin (a muscle calcium binding protein). We also illustrated that increased cytosolic Ca2+ enhances the proteasome activity. Interestingly, suppression of calnexin function using siRNA further elevated the proteasome activity in crt-/- cells. This is the first report to show that loss of calreticulin function enhances the ubiquitin-proteasome activity which could function as a compensatory mechanism for cell survival.     

Association of Genetic Structure and Diversity in Iranian Wild Germplasms of Mentha longifolia L. Based on Phenotypical,Biochemical, and Molecular Markers

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs.     

From mutations to mechanisms and dysfunction via computation and mining of protein energy landscapes

Background

The protein energy landscape underscores the inherent nature of proteins as dynamic molecules interconverting between structures with varying energies. Reconstructing a protein’s energy landscape holds the key to characterizing a protein’s equilibrium conformational dynamics and its relationship to function. Many pathogenic mutations in protein sequences alter the equilibrium dynamics that regulates molecular interactions and thus protein function. In principle, reconstructing energy landscapes of a protein’s healthy and diseased variants is a central step to understanding how mutations impact dynamics, biological mechanisms, and function.

Results

Recent computational advances are yielding detailed, sample-based representations of protein energy landscapes. In this paper, we propose and describe two novel methods that leverage computed, sample-based representations of landscapes to reconstruct them and extract from them informative local structures that reveal the underlying organization of an energy landscape. Such structures constitute landscape features that, as we demonstrate here, can be utilized to detect alterations of landscapes upon mutation.

Conclusions

The proposed methods detect altered protein energy landscape features in response to sequence mutations. By doing so, the methods allow formulating hypotheses on the impact of mutations on specific biological activities of a protein. This work demonstrates that the availability of energy landscapes of healthy and diseased variants of a protein opens up new avenues to harness the quantitative information embedded in landscapes to summarize mechanisms via which mutations alter protein dynamics to percolate to dysfunction.

     

Reduction of marginal mass required for successful islet transplantation in a diabetic rat model using adipose tissue–derived mesenchymal stromal cells

Background aims

Adipose tissue–derived mesenchymal stromal cells (AT-MSCs), widely known as multipotent progenitors, release several cytokines that support cell survival and repair. There are in vitro and in vivo studies reporting the regenerative role of AT-MSCs possibly mediated by their protective effects on functional islet cells as well as their capacity to differentiate into insulin-producing cells (IPCs).

L-Arginine inhibits vasopressin-stimulated mesangial cell Ca2+

L-Arginine (L-Arg) affects variousparameters that modulate the progression of renal disease. These samefactors [e.g., glomerular filtration rate, changes in mesangialcell (MC) tension, and production of NO] are all controlled atleast in part by changes in MC intracellular Ca²⁺concentration([Ca²⁺]_i). Wetherefore evaluated the effect of L-Arg on MC[Ca²⁺]_i. We found thatL-Arg inhibits the vasopressin-stimulated rise in MC[Ca²⁺]_i both in rat andmurine cell cultures. This effect does not appear to be due tometabolism of L-Arg to either NO or L-ornithine (L-Orn). Blocking the metabolism of L-Arg withN-monomethyl-L-arginine, an NOsynthase inhibitor, or with 20 mM L-valine(L-Val), an inhibitor of Orn formation,does not reverse the inhibition. However, other cationic amino acids,as well guanidine, the functional group ofL-Arg, all inhibit thevasopressin-stimulated rise in[Ca²⁺]_i,consistent with a structural basis for this effect. We conclude that1)L-Arg inhibitsvasopressin-stimulated murine and rat MC [Ca²⁺]_irise, 2) this inhibition is notmediated by metabolism of L-Arg to either NO or L-Orn, and3) the effect ofL-Arg is due to its cationicfunctional group, guanidine.

     

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.     

Energy aware resource allocation of cloud data center: review and open issues

The demand for cloud computing is increasing dramatically due to the high computational requirements of business, social, web and scientific applications. Nowadays, applications and services are hosted on the cloud in order to reduce the costs of hardware, software and maintenance. To satisfy this high demand, the number of large-scale data centers has increased, which consumes a high volume of electrical power, has a negative impact on the environment, and comes with high operational costs. In this paper, we discuss many ongoing or implemented energy aware resource allocation techniques for cloud environments. We also present a comprehensive review on the different energy aware resource allocation and selection algorithms for virtual machines in the cloud. Finally, we come up with further research issues and challenges for future cloud environments.