Modeling of Ca2+ flux in pancreatic beta-cells: role of the plasma membrane and intracellular stores

We have developed a detailed mathematical model of ionic flux in beta-cells that includes the most essential channels and pumps in the plasma membrane. This model is coupled to equations describing Ca2+, inositol 1,4,5-trisphosphate (IP3), ATP, and Na+ homeostasis, including the uptake and release of Ca2+ by the endoplasmic reticulum (ER). In our model, metabolically derived ATP activates inward Ca2+ flux by regulation of ATP-sensitive K+ channels and depolarization of the plasma membrane. Results from the simulations support the hypothesis that intracellular Na+ and Ca2+ in the ER can be the main variables driving both fast (2-7 osc/min) and slow intracellular Ca2+ concentration oscillations (0.3-0.9 osc/min) and that the effect of IP3 on Ca2+ leak from the ER contributes to the pattern of slow calcium oscillations. Simulations also show that filling the ER Ca2+ stores leads to faster electrical bursting and Ca2+ oscillations. Specific Ca2+ oscillations in isolated beta-cell lines can also be simulated.     

Theoretical Ab initio study of the effects of methylation on the nature of hydrogen bonding in A:T base pair

We report the results of a theoretical ab initio study of methylation in Watson-Crick A:T base pairs. Equilibrium geometries were obtained without symmetry restrictions by the gradient procedure at DFT level of theory with the standard 6-31G(d) basis set. Each local minima was verified by energy second derivative calculations. Single-point calculations for the DFT geometries have been performed at the MP2/6-31G(d,p), MP2/6-31++G(d,p), and MP2/6-311++G(2d,2p) levels of theory. The geometrical parameters, relative stabilities and counterpoise corrected interaction energies are reported. In addition, using a variation-perturbation energy decomposition scheme, we have found the important contributions to the total interaction energy.     

Association between physique characteristics and hand skeletal aging status

The aim of the present study was to evaluate the association between skeletal aging traits (obtained from evaluation of hand radiographs) and physique characteristics, including indices of obesity, skeletal size, muscular development, and the somatotypes of Heath and Carter (1967) and of Deriabin (1985). To achieve our aims, we used multiple regression analysis, and Pearson's and canonical correlation analyses. The studied population comprised 629 males and 561 females aged 18-90 years, all members of the Chuvasha (Russian Federation). The present study provides evidence for a connection between hand bone aging traits and physique characteristics. The cortical index showed a small but significant correlation with adiposity and the index of overall physique, but bone mineral density (BMD) traits were not correlated with the index of overall physique. Deriabin's set of somatotypes was significantly correlated with BMD, but could explain only 2% of its variation. Significant correlations of the hand osteoarthritis trait were observed with indices of adiposity. Also observed were statistically significant canonical correlations with both sets of somatotypes, which enabled us to predict 2-3% of hand osteoarthritis variation by a combination of somatotype components. Osseometric score evinced statistically significant correlations with most indices of physique as well as with a combination of somatotype components. These findings may prove helpful in epidemiological studies, because they show that body composition is the factor that is statistically significantly associated with skeletal aging.     

The presence of valine at residue 129 in human prion protein accelerates amyloid formation

The polymorphism at residue 129 of the human PRNP gene modulates disease susceptibility and the clinico-pathological phenotypes in human transmissible spongiform encephalopathies. The molecular mechanisms by which the effect of this polymorphism are mediated remain unclear. It has been shown that the folding, dynamics and stability of the physiological, alpha-helix-rich form of recombinant PrP are not affected by codon 129 polymorphism. Consistent with this, we have recently shown that the kinetics of amyloid formation do not differ between protein containing methionine at codon 129 and valine at codon 129 when the reaction is initiated from the alpha-monomeric PrP(C)-like state. In contrast, we have shown that the misfolding pathway leading to the formation of beta-sheet-rich, soluble oligomer was favoured by the presence of methionine, compared with valine, at position 129. In the present work, we examine the effect of this polymorphism on the kinetics of an alternative misfolding pathway, that of amyloid formation using partially folded PrP allelomorphs. We show that the valine 129 allelomorph forms amyloids with a considerably shorter lag phase than the methionine 129 allelomorph both under spontaneous conditions and when seeded with pre-formed amyloid fibres. Taken together, our studies demonstrate that the effect of the codon 129 polymorphism depends on the specific misfolding pathway and on the initial conformation of the protein. The inverse propensities of the two allelomorphs to misfold in vitro through the alternative oligomeric and amyloidogenic pathways could explain some aspects of prion diseases linked to this polymorphism such as age at onset and disease incubation time.     

Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors

Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.     

Influence of viscosity on myocardium mechanical activity: a mathematical model

We have previously proposed and validated a mathematical model of myocardium contraction-relaxation cycle based on current knowledge of regulatory role of Ca2+ and cross-bridge kinetics in cardiac cell. That model did not include viscous elements. Here we propose a modification of the model, in which two viscous elements are added, one in parallel to the contractile element, and one more in parallel to the series elastic element. The modified model allowed us to simulate and explain some subtle experimental data on relaxation velocity in isotonic twitches and on a mismatch between the time course of sarcomere shortening/lengthening and the time course of active force generation in isometric twitches. Model results were compared with experimental data obtained from 28 rat LV papillary muscles contracting and relaxing against various loads. Additional model analysis suggested contribution of viscosity to main inotropic and lusitropic characteristics of myocardium performance.     

Stable benefit of embryonic stem cell therapy in myocardial infarction

Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to beta-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to beta-adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.     

The mechanism of EDHF-mediated responses in subcutaneous small arteries from healthy pregnant women

We studied the importance of endothelium-derived hyperpolarizing factor (EDHF) vs. nitric oxide (NO) and prostacyclin (PGI(2)) in bradykinin (BK)-induced relaxation in isolated small subcutaneous arteries from normal pregnant women. We also explored the contribution of cytochrome P-450 (CYP450) product of arachidonic acid (AA) metabolism, hydrogen peroxide (H(2)O(2)), and gap junctions that have been suggested to be involved in EDHF-mediated responses. Isolated arteries obtained from subcutaneous fat biopsies of normal pregnant women (n = 30) undergoing planned cesarean section were mounted in a wire-myography system. In norepinephrine-constricted vessels, incubation with N(G)-nitro-L-arginine methyl ester (L-NAME) resulted in a significant reduction in relaxation to BK. Simultaneous incubation with L-NAME and indomethacin failed to modify this response further. BK-mediated dilatation in the presence of K(+)-modified solution was decreased to similar level as obtained after incubation with L-NAME. Incubation with L-NAME abolished BK-induced responses in K(+)-modified solution. Sulfaphenazole, a specific inhibitor of CYP450 epoxygenase, and catalase (an enzyme that decomposes H(2)O(2)) did not affect the EDHF-mediated relaxation because concentration-response curves to BK were similar in arteries after incubation with L-NAME vs. L-NAME + sulfaphenazole and L-NAME + catalase. The inhibitor of gap junctions, 18 alpha-glycyrrhetinic acid, significantly reduced BK-mediated relaxation both without and with incubation with L-NAME. We found that both NO and EDHF, but not PGI(2), are involved in the endothelium-dependent dilatation to BK. BK-induced relaxation is almost equally mediated by NO and EDHF. CYP450 epoxygenase metabolites of AA or H(2)O(2) do not account for EDHF-mediated response; however, gap junctions are involved in the EDHF-mediated responses to BK in subcutaneous small arteries in normal pregnancy.