Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Overexpressed Ca-v beta 3 inhibits N-type (Ca(v)2.2) calcium channel currents through a hyperpolarizing shift of "ultra-slow" and "closed-state" inactivation

It has been shown that P auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a hovel inhibitory effect of beta3 Subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/ cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at physiological holding potentials (HPs) of -60 and -80 mV At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca,.2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced Current suppression was reversed at a HP of - 120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba-2divided by (40 mM) as a charge carrier also largely diminished the effect of P3 at -80 mV Therefore, experimental conditions (HP, divalent cation concentration, and P3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel P3 effect. Steady-state inactivation curves revealed that N-type channels exhibited closed-state inactivation without P3, and that P3 caused an similar to40 mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of P3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV Similar ultra-slow inactivation was observed for N-type channels Without P3. Thus, P3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by Causing a hyperpolarizing shift of the inactivation without affecting ultra-slow and closed-state inactivation properties.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product fort-nation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation. (c) 2006 Elsevier Inc. All rights reserved.

Determination of chromophore charge states in the low pH color transition of the fluorescent protein Rtms5(H146S) via time-dependent DFT

The red fluorescent protein Rtms5H146S displays a transition from blue (absorbance λmax 590 nm) to yellow (absorbance λmax not, vert, similar453 nm) upon titration to low pH. The pKa of the reaction depends on the concentration of halide, offering promise for new expressible halide sensors. The protonation state involved in the low pH form of the chromophore remains, however, ambiguous. We report calculated excitation energies of different protonation states of an RFP chromophore model. These suggest that the relevant titration site is the phenoxy moiety of the chromophore, and the relevant base and conjugate acid are anionic and neutral chromophore species, respectively.

A numerical study of steady state evaporative conditions applied to mine tailings

The suction profile of a desiccating soil is dependent on the water table depth, the soil-water retention characteristics, and the climatic conditions. In this paper, an unsaturated flow model, which simulates both liquid and vapour flow, was used to investigate the effects of varying the water table depth and the evaporation rate on the evaporative fluxes from a desiccating tailings deposit under steady-state conditions. Results obtained showed that at a critical evaporation rate, beyond which evaporation is no longer dictated by climatic conditions, the matric suction profiles remain basically unchanged. The critical evaporation rate varies inversely with the water table depth. It is associated with the maximum evaporative flux that might be extracted from a soil at steady-state conditions. The time required to establish steady-state conditions is directly proportional to the water table depth, and it acquires a maximum value at the critical evaporation rate. A detailed investigation of the movement of the drying front demonstrated the significance of attaining a matric suction of about 3000 kPa on the contribution to flow in the vapour phase.

The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.

Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts:the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

Spin dependent photoconductivity in silicon-on-sapphire

Resonant and non resonant spin dependent photoconductivity is observed in(100) silicon films grown on sapphire by CVD and MBE techniques. The CVD films are either in their as-grown state or have undergone single or double solid phase epitaxial regrowth. For all samples, a resonant decrease in photoconductivity is observed at a field of about 0.34 T for a microwave frequency of about 9.7 GHz and at about 3.3 mT when the frequency is about 92 MHz. For all samples the maximum fractional change in photoconductivity is approximately 10-4 independent of magnetic field strength.

Fully probabilistic control for stochastic nonlinear control systems with input dependent noise

Robust controllers for nonlinear stochastic systems with functional uncertainties can be consistently designed using probabilistic control methods. In this paper a generalised probabilistic controller design for the minimisation of the Kullback-Leibler divergence between the actual joint probability density function (pdf) of the closed loop control system, and an ideal joint pdf is presented emphasising how the uncertainty can be systematically incorporated in the absence of reliable systems models. To achieve this objective all probabilistic models of the system are estimated from process data using mixture density networks (MDNs) where all the parameters of the estimated pdfs are taken to be state and control input dependent. Based on this dependency of the density parameters on the input values, explicit formulations to the construction of optimal generalised probabilistic controllers are obtained through the techniques of dynamic programming and adaptive critic methods. Using the proposed generalised probabilistic controller, the conditional joint pdfs can be made to follow the ideal ones. A simulation example is used to demonstrate the implementation of the algorithm and encouraging results are obtained.

The development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ:evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)-RARα, RARβ, and RARγ-is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

A Limit Theorem for Multi-Type Subcritical Age-Dependent Branching Processes with two Types of Immigration

Марусия Н. Славчова-Божкова - В настоящата работа се обобщава една гранична теорема за докритичен многомерен разклоняващ се процес, зависещ от възрастта на частиците с два типа имиграция. Целта е да се обобщи аналогичен резултат в едномерния случай като се прилагат “coupling” метода, теория на възстановяването и регенериращи процеси.

Comparison between Numerical and Simulation Methods for Age-dependent Branching Models with Immigration

2000 Mathematics Subject Classification: primary: 60J80, 60J85, secondary: 62M09, 92D40

Intracellular redox state modulates the T cell surface proteome – relevance to ageing

During ageing an altered redox balance has been observed in both intracellular and extracellular compartments, primarily due to glutathione depletion and metabolic stress. Maintaining redox homeostasis is important for controlling proliferation and apoptosis in response to specific stimuli for a variety of cells. For T cells, the ability to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols. Here we describe the effects of depleting intracellular glutathione concentration for T cell exofacial expression of thioredoxin 1 and IL-2 production, and have determined the distribution of Trx1 with ageing. Using buthionine sulfoximine to deplete intracellular glutathione in Jurkat T cells we show using Western blotting that cell surface thioredoxin-1 is lowered and that the response to the lectin phytohaemagglutinin measured by ELISA as IL-2 production is also decreased. Using flow cytometry we show that the distribution of Trx1 on primary CD4+ T cells is age-dependent, with lower surface Trx1 expression and greater variability of surface expression observed with age. Together these data suggest that a relationship exists between the intracellular redox compartment and exofacial surface. Redox imbalance may be important for impaired T cell function during ageing.

Market reform and fiscal laxity in Communist and post-Communist Hungary: A path-dependent approach

Purpose – The purpose of this paper is to present a conceptual framework in order to analyse and understand the twin developments of successful microeconomic reform on the one hand and failed macroeconomic stabilisation attempts on the other hand in Hungary. The case study also attempts to explore the reasons why Hungarian policymakers were willing to initiate reforms in the micro sphere, but were reluctant to initiate major changes in public finances both before and after the regime change of 1989/1990. Design/methodology/approach – The paper applies a path-dependent approach by carefully analysing Hungary's Communist and post-Communist economic development. The study restricts itself to a positive analysis but normative statements can also be drawn accordingly. Findings – The study demonstrates that the recent deteriorating economic performance of Hungary is not a recent phenomenon. By providing a path-dependent explanation, it argues that both Communist and post-Communist governments used the general budget as a buffer to compensate the losers of economic reforms, especially microeconomic restructuring. The gradualist success of the country – which dates back to at least 1968 – in the field of liberalisation, marketisation and privatisation was accompanied by a constant overspending in the general government. Practical implications – Hungary has been one of the worst-hit countries of the 2008/2009 financial crisis, not just in Central and Eastern Europe but in the whole world. The capacity and opportunity for strengthening international investors' confidence is, however, not without doubts. The current deterioration is deeply rooted in failed past macroeconomic management. The dissolution of fiscal laxity and state paternalism in a broader context requires, therefore, an all-encompassing reform of the general government, which may trigger serious challenges to the political regime as well. Originality/value – The study aims to show that a relatively high ratio of redistribution, a high and persistent public deficit and an accelerated indebtedness are not recent phenomena in Hungary. In fact, these trends characterised the country well before the transformation of 1989/1990, and have continued in the post-socialist years, too. To explain such a phenomenon, the study argues that in the last couple of decades the hardening of the budget constraint of firms have come at the cost of maintaining the soft budget constraint of the state.

The effect of the advanced placement examination for credit policy by the Florida State universities on the acceleration of a student's postsecondary education

The purpose of this research study was to determine if the Advanced Placement program as it is recognized by the universities in the Florida State University System (SUS) truly serves as an acceleration mechanism for those students who enter an SUS institution with passing AP scores. Despite mandates which attempt to control uniformity of policy, each public university in Florida determines which courses will be exempted and the number of credits they will grant for passing Advanced Placement courses.^ This is a descriptive study in which the AP policies of each of the SUS institutions were compared. Additionally, the college attendance and graduation data on members of a cohort of 593 Broward County high school graduates of the class of June, 1992 were compared. Approximately 28% of the cohort members entered university with passing Advanced Placement scores.^ The rate of early and on time graduation was significantly dependent on the Advanced Placement standing of the students in the cohort. Given the financial and human cost involved, it is recommended that all state universities bring their Advanced Placement policies into line with each other and implement a uniform Advanced Placement policy. It is also recommended that a follow-up study be conducted with a new cohort bound under the current 120 credit limitation for graduation. ^