Dissociation Rates Of Complexes Research Articles

Copper dynamics in vineyard topsoils as affected by the supply of aerated compost tea: Insights from a batch experiment

Aerated compost teas (ACTs) are rich in soluble humic substances (SHS) that have high affinity for metals, notably Cu. Using a batch experiment, we measured the extent to which two ACTs altered Cu dynamics in vineyard topsoils one day and 21 days after their addition. Soils were extracted with 0.01 M KCl, and total Cu concentration, free Cu ion fraction and size distribution of Cu ligands were measured in the extraction solution to assess the impact of ACT on the mobility of Cu. Diffusive gradient in thin film (DGT) measurements were carried out to assess the effect of ACT on Cu bioavailability, and the dissociation rate of Cu-SHS complexes was measured. The results revealed that ACT increased the mobility of Cu from a factor 1.2 to 5.8 depending on the soil, the ACT and the incubation time. Cu mobilization was associated with an increase in absorbance at 254 nm and a decrease in the free Cu ion fraction in the KCl extract. Associated with the strong agreement between the size distribution of SHS and that of Cu ligands in the KCl extract of soils treated with ACT, these results showed that Cu was mobilized through complexation by the SHS present in ACTs. A fraction of the SHS supplied with ACTs sorbed onto the soil constituents, notably in calcareous soils where this fraction reached 86% for ACT B. Between 15% and 50% of the SHS remaining in solution degraded between day one and day 21 under the presumed action of microflora. This explains why the Cu mobilization efficiency of ACTs was on average lower in calcareous soils than in non-calcareous soils, and decreased with time. Lastly, ACT increased the bioavailability of Cu in soils from a factor 1.3 to 4.2, due to the relatively high dissociation rate of Cu-SHS complexes.

How exogenous ligand enhances the efficiency of cadmium phytoextraction from soils?

Many experiments showed that exogenous ligands could enhance cadmium (Cd) phytoextraction efficiency in soils. Previous studies suggested that the dissociation and the apoplastic uptake of Cd complex could not fully explain the increase of root Cd uptake. Two hypotheses are evaluated to explain enhanced Cd uptake in the presence of ligand: i) enhanced apoplastic uptake of complex due to reduced apoplastic resistance and ii) complex internalization by membrane transporters. Resultsshow that the ligand affinity for Cd is a key characteristic determining the potential mechanism for enhanced Cd uptake. When low molecular weight organic acids are applied, the complex dissociation could generally be fast (> 10−3.3 s−1) and result in the increased Cd uptake. When hydrophilic aminopolycarboxylic acids (APCAs) are applied in experiments without water or temperature stresses to the plant, the root water uptake flux could very likely be high (> 10−7.8 dm s−1), and the strong apoplastic complex uptake could enhance the root Cd uptake. When lipophilic APCAs are applied, the strong internalization of the complex by membrane transporters could result in the increased Cd uptake if the maximum internalization rate is high (> 10−12 mol dm−2 s−1). However, the complex internalization by membrane transporters must be experimentally confirmed.

Simple Physical Model for the Estimation of Irreversible Dissociation Rates for Bimolecular Complexes.

In this article, we propose a simple method of estimating dissociation rates of bimolecular van der Waals complexes ("wells"), rooted in rigid body dynamics, requiring as input parameters only the bimolecular binding energy, together with the intermolecular equilibrium distance and moments of inertia of the complex. The classical equations of motion are solved for the intermolecular and rotational degrees of freedom in a coordinate system considering only the relative motion of the two molecules, thus bypassing the question of whether the energy of the complex is statistically distributed. Well-escaping trajectories are modeled from these equations, and the escape rate as a function of relative velocity and angular momentum is fitted to an empirical function, which is then integrated over a probability distribution of said quantities. By necessity, this approach makes crude assumptions on the shape of the potential well and neglects the impact of energy quantization, and, more crucially, the coupling between the degrees of freedom included in the equations of motion with those that are not. We quantify the error caused by the first assumption by comparing our model potential with a quantum chemical potential energy surface (PES) and show that while the model does make several compromises and may not be accurate for all classes of bimolecular complexes, it is able to produce physically consistent dissociation rate coefficients within typical atmospheric chemistry confidence intervals for triplet state alkoxyl radical complexes, for which the detailed balance approach has been shown to fail.

The interfacial “push and pull” competitive complexation and enhanced separation of praseodymium and neodymium

The separation of praseodymium (Pr) and neodymium (Nd) is particularly challenging within the lanthanide series. A new strategy was developed for enhanced separation of Pr and Nd, using interfacial “push and pull” complexation induced by a kinetic effect. We found that the specific addition order of diethylenetriaminepentaacetic acid (DTPA) had an obvious influence on the separation behaviors of Pr/Nd. Notably, the separation coefficient was up to 3.25 with the addition of DTPA when the extraction process just started. This suggested that different complexation rates decided differences of extraction kinetics between Pr and Nd. It revealed differences in complexation rates of adjacent RE(III) with DTPA and dissociation rates of RE-DTPA complexes by stopped-flow spectrophotometry. The results indicated that the complexation rate constant of Nd3+ was higher than Pr3+. On contrary, the dissociation rate constant of Nd-DTPA was smaller than Pr-DTPA. Molecular dynamics (MD) simulations showed that the interfacial “push and pull” complexation effect led to differences in interfacial molecule arrangements and diffusion mass transfer rates near the interface. And thus, the separation of Pr/Nd can be enhanced. This research provides an efficient strategy for the purification of metals that are difficult to separate.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4β,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, withkoffrates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3β-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14αC-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Kinetic aspects of iron(III)-chelation therapy with deferasirox (DFX) revealed by the solvolytic dissociation rate of the Fe(III)–DFX complex estimated with capillary electrophoretic reactor

Capillary electrophoresis was used to estimate the solvolytic dissociation rate (kd) of metal complexes of deferasirox (DFX, H3L), a drug used to treat iron overload. Inert CoIIIL23− did not dissociate. The estimated kd value for FeIIIL23− was (2.7 ± 0.3) × 10−4 s−1 (298 K, pH 7.4). The kd values of other complexes (AlIIIL23−, NiIIL24−, and MnIIL−) were in the range 10−3–10−4 s−1. In contrast, ZnIIL− and CuIIL− were too labile to allow kd estimation. The fact that the half-life of FeIIIL23− (43.3 min) is shorter than the blood half-life of DFX (8–16 h) implies that the blood concentration of DFX should be high enough to prevent dissociation of FeIIIL23−. The possibility of a safer iron-chelation therapy that avoids excretion of other essential metal ions such as ZnII is discussed, highlighting the importance of selectivity in terms of kinetic stability.

Inhibition of E. coli RecQ Helicase Activity by Structurally Distinct DNA Lesions: Structure—Function Relationships

DNA helicase unwinding activity can be inhibited by small molecules and by covalently bound DNA lesions. Little is known about the relationships between the structural features of DNA lesions and their impact on unwinding rates and processivities. Employing E.coli RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants kobs = kU + kD, and processivities P = (kU/(kU + kD) were determined (kU, unwinding rate constant; kD, helicase-DNA dissociation rate constant). The highest kobs values were observed in the case of intercalated benzo[a]pyrene (BP)-derived adenine adducts, while kobs values of guanine adducts with minor groove or base-displaced intercalated adduct conformations were ~10-20 times smaller. Full unwinding was observed in each case with the processivity P = 1.0 (100% unwinding). The kobs values of the non-bulky lesions T(6-4)T, CPD cyclobutane thymine dimers, and a guanine oxidation product, spiroiminodihydantoin (Sp), are up to 20 times greater than some of the bulky adduct values; their unwinding efficiencies are strongly inhibited with processivities P = 0.11 (CPD), 0.062 (T(6-4)T), and 0.63 (Sp). These latter observations can be accounted for by correlated decreases in unwinding rate constants and enhancements in the helicase DNA complex dissociation rate constants.

Pharmacodynamic model of slow reversible binding and its applications in pharmacokinetic/pharmacodynamic modeling: review and tutorial.

Therapeutic responses of most drugs are initiated by the rate and degree of binding to their receptors or targets. The law of mass action describes the rate of drug-receptor complex association (kon) and dissociation (koff) where the ratio koff/kon is the equilibrium dissociation constant (Kd). Drugs with slow reversible binding (SRB) often demonstrate delayed onset and prolonged pharmacodynamic effects. This report reviews evidence for drugs with SRB features, describes previous pharmacokinetic/pharmacodynamic (PK/PD) modeling efforts of several such drugs, provides a tutorial on the mathematics and properties of SRB models, demonstrates applications of SRB models to additional compounds, and compares PK/PD fittings of SRB with other mechanistic models. We identified and summarized 52 drugs with in vitro-confirmed SRB from a PubMed literature search. Simulations with a SRB model and observed PK/PD profiles showed delayed and prolonged responses and that increasing doses/kon or decreasing koff led to greater expected maximum effects and a longer duration of effects. Recession slopes for return of responses to baseline after single doses were nearly linear with an inflection point that approaches a limiting value at larger doses. The SRB model newly captured literature data for the antihypertensive effects of candesartan and antiallergic effects of noberastine. Their PD profiles could also be fitted with indirect response and biophase models with minimal differences. The applicability of SRB models is probably commonplace, but underappreciated, owing to the need for in vitro confirmation of binding kinetics and the similarity of PK/PD profiles to models with other mechanistic determinants.

A novel assay based on pre-equilibrium titration curves for the determination of enzyme inhibitor binding kinetics

Selection of pharmacological agents based on potency measurements performed at equilibrium fail to incorporate the kinetic aspects of the drug–target interaction. Here we describe a method for screening or characterization of enzyme inhibitors that allows the concomitant determination of the equilibrium inhibition constant in unison with rates of complex formation and dissociation. The assay is distinct from conventional enzymatic assays and is based on the analysis of inhibition curves recorded prior to full equilibration of the system. The methodology is illustrated using bicyclic peptide inhibitors of the serine protease plasma kallikrein.

Charge generation efficiency of electrically doped organic semiconductors

Organic semiconductors (OSCs) have been a significant focus of research for electronic devices over the years exclusively due to their superior mechanical and optical properties as opposed to conventional inorganic semiconductors (ISCs) such as silicon or germanium. These unique materials have smoothened the path for developing extremely light-weight and ultrathin electronic devices with built-in flexibility and transparency for a considerably low cost. However, the commercial application of these organic materials is limited by their inferior electrical conductivity and charge carrier mobility compared with inorganic counterparts. This review article presents an overview of the works published on how to control and adjust the electrical conductivity of OSCs, focusing on electrical doping. The main point of this review is related to the principles and fundamental mechanisms of charge generation efficiency (CGE) in OSCs, which are significantly different from those of ISCs. The reported CGE of OSCs, defined as the ratio of the generated charge carriers to the dopants, is found to be in the range of a few percent and is significantly small compared with the ISCs. The origin of this lies in the lower dissociation rate of the charge transfer complexes (CTCs) formed between the tightly bound ionized dopants and generated charges. The CTCs induce localized electron-hole pairs, and the dissociation of such CTCs into free charge carriers requires large activation energies to overcome the strong Coulombic forces exerted on the generated charges by the dopant ions. Therefore, high doping concentrations are required to generate a large number of free charge carriers in doped OSC films. In this review, the CGE of OSCs is discussed in detail from the point of view of CTC formation and charge separation efficiencies.

Prediction of Absolute Binding and Unbinding Kinetics on Timescales of a Minute from Sub-Microsecond MD Simulations

The prediction of absolute rates of protein-ligand complex association and dissociation that have an impact on pharmacological efficacy is problematic, as the intrinsic timescales of seconds to hours are far outside the scope of current molecular dynamics (MD) techniques. We here present a novel approach to coarse-grain fully atomistic dynamics based on principles from nonequilibrium statistical mechanics to gain access to such timescales: using dissipation-corrected targeted MD simulations, we enforce ligand unbinding and use the resulting bias force to calculate free energies and friction profiles along resulting unbinding paths.

Mechanobiology of Stator Remodeling in the Bacterial Flagellar Motor

Motility is critical for the survival and dispersal of bacteria, and it plays an important role during infection. How bacteria regulate motility is thus of great interest to microbiologists, ecologists, and infectious disease researchers alike. Regulation of motility via chemotaxis and gene regulation is well studied. However, recent work has added a new dimension to this problem. The flagellar motor of bacteria autonomously assembles and disassembles torque-generating stator complexes in response to changes in the external viscous load. in the bacterium Escherichia coli, up to 11 stator complexes drive the motor at high load while all the stator complexes are released at low load. We study this process by artificially manipulating the motor load using electrorotation, where a high frequency rotating electric field applies an external torque on the flagellar motor. Using this technique, we can increase or decrease the motor load at will and measure the resulting stator assembly dynamics. From detailed measurements of stator remodeling in both clockwise and counterclockwise rotating motors, we have found that the motor's response has a conserved torque dependence. increased torque decreases the dissociation rate of stator complexes from the motor, thereby increasing the steady state stoichiometry. We built an equilibrium model that captures the observed dynamics and provides insight into the underlying molecular interactions. Torque-dependent stator remodeling takes place within tens of seconds, making it a highly responsive autonomous control mechanism.

Application of the SPR Biosensor for the Analysis of Protein–Protein Interactions in Aqueous Environment and Bilayer Lipid Membrane As Exemplified by P450scc (CYP11A1)

Protein–protein interactions (PPIs) are crucial for the successful realization of many metabolic and signaling pathways. Of particular interest are PPIs of membrane proteins, which form stable and/or transient complexes for the signal transduction, ion transport, and electron transfer within electron transfer chains. The Surface Plasmon Resonance (SPR) allows analyzing the thermodynamic and kinetic parameters (kon and koff) and equilibrium constants (Kd) of PPI, as well as the assessment of the effects of low molecular weight compounds. The aim of the present study was the adaptation of the SPR protocols for PPIs involving mitochondrial cytochrome P450 CYP11A1, mitochondrial cytochrome b5 (CYB5B), and adrenodoxin (Adx). We found that the Adx–CYP11A1 and CYB5B–CYP11A interactions depend on the method of protein immobilization and on the microenvironment. For example, the koff values for complexes Adx–CYP11A1 and CYB5B–CYP11A1 in the aqueous environment were similar (1.5 ± 0.2) × 10–3 s–1, while the values of kon for these complexes differed from each other by almost one order of magnitude ((6.5 ± 0.5) × 104 and (0.30 ± 0.03) × 104 M–1 s–1, respectively). This is in good agreement with the known high affinity of CYP11A1 to its cognate redox partner Adx. In the lipid environment, the rate of complex dissociation was higher than that in the aqueous environment with koff value equal to (9.1 ± 0.3) × 10–3 s–1. For the CYP11A1–CYB5B complex, the parameters of interaction in the lipid phase were not determined due to unspecific binding of the proteins used as analytes to the lipids immobilized on the L1 chip. We show for the first time that SPR method could be used for the detection and quantitative analysis of PPI involving mitochondrial cytochromes (CYP) with their redox partners in both aqueous and lipid environment. Experimental protocols developed and validated in our previous work and in this one can serve as valuable tools for studies of interactions of CYP proteins and could also be applied for other membrane proteins.

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors.

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.

Rigorous Physicochemical Framework for Metal Ion Binding by Aqueous Nanoparticulate Humic Substances: Implications for Speciation Modeling by the NICA-Donnan and WHAM Codes.

Latest knowledge on the reactivity of charged nanoparticulate complexants toward aqueous metal ions is discussed in mechanistic detail. We present a rigorous generic description of electrostatic and chemical contributions to metal ion binding by nanoparticulate complexants, and their dependence on particle size, particle type (i.e., reactive sites distributed within the particle body or confined to the surface), ionic strength of the aqueous medium, and the nature of the metal ion. For the example case of soft environmental particles such as fulvic and humic acids, practical strategies are delineated for determining intraparticulate metal ion speciation, and for evaluating intrinsic chemical binding affinities and heterogeneity. The results are compared with those obtained by popular codes for equilibrium speciation modeling (namely NICA-Donnan and WHAM). Physicochemical analysis of the discrepancies generated by these codes reveals the a priori hypotheses adopted therein and the inappropriateness of some of their key parameters. The significance of the characteristic time scales governing the formation and dissociation rates of metal-nanoparticle complexes in defining the relaxation properties and the complete equilibration of the metal-nanoparticulate complex dispersion is described. The dynamic features of nanoparticulate complexes are also discussed in the context of predictions of the labilities and bioavailabilities of the metal species.

In situ measurements of micronutrient dynamics in open seawater show that complex dissociation rates may limit diatom growth

In this first in situ study of the dynamic availability of phytoplankton micronutrients, a SeaExplorer glider was combined with Diffusive Gradients in Thin Films and deployed in the Mediterranean Sea. On the basis of their labile metal complex pools, we discovered that Fe and Co can be potentially limiting and Cu co-limiting to diatom growth, contrary to the generally accepted view that phosphorus (phosphate) is the growth limiting element in the Mediterranean Sea. For flagellates and picoplankton, phosphorus remains the main element limiting growth. Our in situ measurements showed that organic complexes of Fe and Cu (>98% of total dissolved concentration), dissociate slower than inorganic complexes of Co, Cd and Ni (>99% of total dissolved concentration being free ions and inorganic complexes). This strengthens the potential growth limiting effect of Fe and Cu versus phosphate, which is present as a free ion and, thus, directly available for plankton.

Dissociation rate kinetics of europium-DTPA complexes

Solvent extraction-based processes for separating lanthanides from actinides depend strongly on the kinetics of ligand complexation and de-complexation with the metal ions. The slower metal-ligand de-complexation process is usually the rate-limiting step, requiring these kinetics to be accurately known. In this study the kinetics of europium(III)-diethylenetriaminepentaacetic (DTPA) acid complex dissociation rates were directly measured using the change in europium(III) luminescence. These dissociation rates were independently determined by minimizing the complex formation rates by using high acidity, and keeping the concentration of europium(III) low to avoid double metal complexes.

Abstract 1008: Peroxynitrite mediates the resistance of tumor cells to cytotoxic T cells by altering the MHC1-peptide repertoire on tumor cells

Abstract Therapeutic advances were able to significantly enhance antigen-specific T cell responses in cancer therapy. However, despite the obvious success, our ability to translate the generation of specific T-cell response into a clinical benefit remains limited. Recently, factors of tumor microenvironment such as hypoxia and oxidative agents were shown to dampen the efficiency of T-cell responses. Previously, we demonstrated that macrophages and myeloid-derived suppressor cells (MDSCs) produce a significant amount of highly oxidative agent, peroxynitrite (PNT), inside the tumor and revealed that PNT inhibited binding of processed peptides to tumor cell MHC class I (MHC I). As a result, tumor cells become resistant to antigen-specific cytotoxic T cells (CTLs). In other studies, it was shown that high levels of PNT (measured by nitrotyrosine level) in tumor tissues are associated with worse clinical outcome. We hypothesized that PNT affects peptide repertoire presented by tumor cell MHC I molecules and by this impairs the immune recognition of tumor cells by CTLs. To test this hypothesis we compared MHC I peptides isolated from PNT-treated and nontreated tumor cells. Quantitative LC-MS/MS analysis revealed that PNT treatment affected the peptide repertoire of MHC I bound peptides. A number of peptides were substantially underrepresented in PNT-treated cells (“nonstable” peptide subset), whereas others remained at the same level after PNT treatment (“stable” peptide subset). We generated CTLs specific to some peptides from “stable” or “nonstable” groups. PNT treatment significantly decreased the cytotoxic activity of T cells specific to “non-stable” but not to “stable” peptides, confirming our initial hypothesis. To further characterize “stable” and “nonstable” peptides we investigated their affinity to MHC I as well as the stability of the formed peptide-MHC I (pMHC) complexes. We found that peptides from both groups demonstrated equally high affinity to MHC I. However, we observed significantly higher dissociation rate of pMHC complexes formed by peptides from “nonstable” group than by “stable” peptides. PNT treatment disrupted proteasome function in tumor cells, suggesting that PNT may impair antigen-processing machinery and “nonstable” peptides quickly dissociating from MHC I were not recovered by malfunctioning proteasome. Consecutively, this could lead to the poorer peptide presentation on the tumor cell surface hindering tumor cell recognition and killing by CTLs. Our findings demonstrate a novel PNT-mediated mechanism of tumor cell resistance to specific CTLs and suggest potential therapeutic avenues for its neutralization. Citation Format: Evgenii N. Tcyganov, Dmitry I. Gabrilovich. Peroxynitrite mediates the resistance of tumor cells to cytotoxic T cells by altering the MHC1-peptide repertoire on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1008.

Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking.

Insulin stimulates glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane. The exocyst complex tethers GLUT4-containing vesicles to the plasma membrane, a process that requires the binding of the G protein (heterotrimeric guanine nucleotide-binding protein) RalA to the exocyst complex. We report that upon activation of RalA, the protein kinase TBK1 phosphorylated the exocyst subunit Exo84. Knockdown of TBK1 blocked insulin-stimulated glucose uptake and GLUT4 translocation; knockout of TBK1 in adipocytes blocked insulin-stimulated glucose uptake; and ectopic overexpression of a kinase-inactive mutant of TBK1 reduced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The phosphorylation of Exo84 by TBK1 reduced its affinity for RalA and enabled its release from the exocyst. Overexpression of a kinase-inactive mutant of TBK1 blocked the dissociation of the TBK1/RalA/exocyst complex, and treatment of 3T3-L1 adipocytes with specific inhibitors of TBK1 reduced the rate of complex dissociation. Introduction of phosphorylation-mimicking or nonphosphorylatable mutant forms of Exo84 blocked insulin-stimulated GLUT4 translocation. Thus, these data indicate that TBK1 controls GLUT4 vesicle engagement and disengagement from the exocyst, suggesting that exocyst components not only constitute a tethering complex for the GLUT4 vesicle but also act as "gatekeepers" controlling vesicle fusion at the plasma membrane.

Nano polyelectrolyte complexes of carboxymethyl dextran and chitosan to improve chitosan-mediated delivery of miR-145

Chitosan (Ch) nanoparticles have emerged as a promising vector for gene delivery, nonetheless slow dissociation rate of the nanoparticles in cytoplasm is a drawback of using Ch. Herein, the Ch-mediated gene delivery was improved using PECs of Ch and carboxymethyl dextran (CMD) to transfer the micro RNA-145 (miR-145). The optimized nano PEC preparation method and effects of Ch molecular weight (Mw) and a CMD to Ch molar ratio (CMD:Ch) on physical characteristics and in vitro efficacy of the nano PECs was determined. The size of the nano PECs depended on the preparation method, Ch Mw, and CMD:Ch ratio. Also, the Ch Mw and CMD:Ch affected stability of the miR-145 PECs in presence of heparin. In vitro tests indicated the different gene transfection efficiency of the nano PECs with various compositions. As a novel vector, these nano PECs have excellent promise for gene delivery with an adequate balance between a complex stability and dissociation rate.