A Quantitative Analytical Method to Test for Salt Effects on Giant Unilamellar Vesicles

Today, free-standing membranes, i.e. liposomes and vesicles, are used in a multitude of applications, e.g. as drug delivery devices and artificial cell models. Because current laboratory techniques do not allow handling of large sample sizes, systematic and quantitative studies on the impact of different effectors, e.g. electrolytes, are limited. In this work, we evaluated the Hofmeister effects of ten alkali metal halides on giant unilamellar vesicles made of palmitoyloleoylphosphatidylcholine for a large sample size by combining the highly parallel water-in-oil emulsion transfer vesicle preparation method with automatic haemocytometry. We found that this new quantitative screening method is highly reliable and consistent with previously reported results. Thus, this method may provide a significant methodological advance in analysis of effects on free-standing model membranes

The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.This work was supported by grants from the Norwegian Cancer Society (to ØH), Junta de Andalucía, grant CVI-02483 (to JMSR), The Research Council of Norway (grant 185181 to A.M.), the Western Norway Health Authorities (grant 911618 to A.M.) and The Kristian Gerhard Jebsen Foundation (to AM)

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

N,N-bis(cyclohexanol)amine aryl esters inhibit P-glycoprotein as transport substrates

P-Glycoprotein (Pgp) inhibition by three sets of four isomers of N,N-bis(cyclohexanol)amine aryl esters was assessed on rhodamine 123 (R123) efflux in human MDR1-gene transfected mouse T-lymphoma L5178 cells and on Sf9 ATPase activity. The most active compounds inhibited Pgp with IC(50) values much lower than those of either cyclosporin A (CSA) or GF120918. As to R123 efflux inhibition, the role of the bond present in the second aryl moiety appeared important since the triple bond derivatives (3a-d) were the most powerful as compared to the double bond (2a-d) and the single bond (1a-d) counterparts. Concentration-inhibition curves of 2c and 3d exhibited a biphasic behaviour suggesting the existence of two binding sites in the recognition domain of Pgp. Persistence of inhibition by these compounds resulted to be intermediate between that caused by CSA and GF120918. R123 exhibited positive interaction with CSA, 1d, 1c, 2d, 2c and 3c, the concentration-inhibition curves being shifted leftward when R123 concentration was increased, while it exhibited negative interaction with 3d and no effect with GF120918. Sf9 ATPase activity was stimulated in an increasing order of potency by 2c, 3c, 2d, CSA, epirubicin and 3d. In a decreasing order of potency 3d, 2c, GF120918, CSA, 2d and 3c inhibited at sub-nanomolar concentrations epirubicin-stimulated ATPase activity. In conclusion, isomeric geometry and restriction of molecular flexibility of N,N-bis(cyclohexanol)amine aryl esters were crucial for their presentation to and inhibition of Pgp as transport substrates, R123 and epirubicin cooperating with them to this inhibition

Interfacial water structure controls protein conformation

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed

Bone mass preservation with high-dose cholecalciferol and dietary calcium in HIV patients following antiretroviral therapy. Is it possible?

Objective: To evaluate whether treatment with 100,000 IU/month (equivalent to 3200 IU/day) of cholecalciferol and 1 g/day of dietary calcium supplementation in HIV patients following different cART regimens yields normal levels of vitamin D3 and PTH as well as whether changes in bone mineral density are clinically significant. Methods: Consecutive HIV patients following different cART regimens received 100,000 IU/month (equivalent to 3200 IU/day) of cholecalciferol and 1 g/day of dietary calcium supplementation. The participants underwent BMD assessment via dual energy X-ray absorptiometry of the spine and hip at baseline (T0) and after 24 months (T1). Levels of 25(OH) vitamin D3 and parathyroid hormone (PTH) were assessed at T0 and T1. Quantitative variables were assessed with a paired t-test, independent t-test or ANOVA, as appropriate. A chi-squared analysis was used to assess the association between qualitative variables. A p-value <0.05 was considered significant. Patients were divided into three groups depending on the cART regimen. Results: A total of 79 patients were included (40 males, 51% and 39 females 49%), with a mean age of 46.6 (SD ±11.2) years, a baseline CD4 count of 649 cells/µl and a mean 25 hydroxycholecalciferol (25(OH) D3) value of 25+10 ng/ml. After 24 months, the 25(OH) D3 increased to 40+11 ng/ml. The initial BMDs at T0 were estimated as 0.919 (±0.27) and 0.867 (±0.14) g/cm2 at the spine and hip, respectively. After 24 months, the BMD was 0.933 (±0.15) g/cm2 at the spine and 0.857 (±0.14) g/cm2 at the hip. Based on a BMD change exceeding 3%, a worsening was observed in 23% of patients at the spine and 27% at the hip, whereas stability or improvement was demonstrated in 77% of patients at the spine and 73% at the hip. Subgrouping patients based on antiretroviral therapy indicated that, at T1, there was a statistically significant increase in vitamin D3 concentration in all patients, while PTH concentration was not significantly reduced in patients taking tenofovir or efavirenz. BMD stability or improvement was demonstrated in 77% of patients at the spine and 73% at the hip after 24 months. The multivariate analysis confirms a decrease in vitamin D3 and an increase in PTH levels in smokers, as well higher vitamin D3 concentrations in males and lower spine BMDs in menopausal females. Conclusion: The proposed protocol of cholecalciferol and dietary calcium supplementation is safe and valid for correcting vitamin D abnormalities in almost all patients as well as reducing PTH levels in a high percentage of patients; however, it is not sufficient for normalization, particularly in patients exposed to tenofovir or efavirenz. At the spine, no significant BMD change was found in any of the therapy groups. At the hip, our data confirm a modest negative effect on bone mass caused by tenofovir and efavirenz

Despondency among HIV-positive older men and women in Uganda.

Forty people over 60 years of age took part in longitudinal research over the course of a year on the impact of the HIV epidemic in southern Uganda. In this paper we focus mainly on the data from 26 of the 40 who were HIV-positive. While we observed that feelings of depression were frequently experienced by many of the people in our study, the state of 'being depressed' was not constant. Participants regularly expressed economic frustration (because of a lack of money to buy food and other commodities including sugar and soap); medical problems (including those related to HIV) as well as old age, the burden of dependents (including concerns about school fees for grandchildren), feelings of sadness and isolation, and a lack of support from others, as well as stigma, whether real or perceived. However, while worries, sorrow and despondent thoughts were reported in many of the interviews across the study, moods fluctuated moving from happiness and hope, to sadness and despair, from month to month. Concerns regarding the psychological wellbeing amongst older people, including those living with HIV and older carers in Uganda deserve greater attention

Recent Results in the Approximation of Nonlinear Optimal Control Problems

This survey paper presents recent advances for the numerical solution of Hamilton-Jacobi-Bellman equations related to optimal control problems. The Dynamic Programming approach suffers for the "curse of dimensionality" and the solution of the nonlinear partial differential equations characterizing the value function of optimal control problems in high dimension is out of reach. However, a combination of various techniques can circumvent this difficulty and find the solution of optimal control problems up to dimension 10, a range of dimensions which could be enough for many applications. We illustrate here some of these techniques: patchy domain decomposition, fast marching and fast sweeping and an acceleration method based on the coupling between value and policy iteration. Numerical examples will illustrate the main features of those methods. © 2014 Springer-Verlag