Contribution of Natural Inhibitors to the Understanding of the PI3K/PDK1/PKB Pathway in the Insulin-mediated Intracellular Signaling Cascade

The critical initial steps in insulin action include phosphorylation of adapter proteins and activation of phosphatidylinositol 3-kinase (PI3K). One of important components in this process is a protein called Akt/protein kinase B (PKB). The work of numerous different researchers indicates a role of PKB in regulating insulin-stimulated glucose uptake. The crucial role of lipid second messengers in PKB activation has been dissected through the use of the PI3K-specific inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Receptor-activated PI3K synthesizes the lipid second messenger PtdIns[3,4,5]-trisphosphate, leading to the recruitment of PKB to the membrane. Membrane attachment of PKB is mediated by its pleckstrin homology domain binding to PtdIns[3,4,5]-trisphosphate or PtdIns[3,4]-bisphosphate with high affinity. Activation of PKB alpha is then achieved at the plasma membrane by phosphorylation of Thr308 in the activation-loop of the kinase domain and Ser473 in the carboxy-terminal regulatory region, respectively. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) is responsible for T308 phosphorylation. The usage of specific inhibitors and natural compound has significantly contributed to investigate the molecular mechanism of PI3K/PDK1/PKB signaling pathway, leading to the putative therapeutics benefits of patients. This review focuses on the contribution of natural inhibitor or compound in our understanding of the mechanism by which insulin induces, especially in PI3K/PDK1/PKB signaling.     

Synthesis and Characterization of Mesoporous Silica Functionalized with Calix[]arene Derivatives

This work reports a new method to covalently attach calix[4]arene derivatives onto MCM-41, using a diisocyanate as a linker. The modified mesoporous silicates were characterized by fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA) and elemental analysis. The FTIR spectra and TGA analysis verified that the calix[4]arene derivates are covalently attached to the mesoporous silica. The preservation of the MCM-41 channel system was checked by X-ray diffraction and nitrogen adsorption analysis.     

Determinants of Lipid Domain Size

Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system.     

Characterization of a Low Shrinkage Dental Composite Containing Bismethylene Spiroorthocarbonate Expanding Monomer

In this study, a novel dental composite based on the unsaturated bismethylene spiroorthocarbonate expanding monomer 3,9-dimethylene-1,3,5,7-tetraoxa-spiro[5,5]undecane (BMSOC) and bisphenol-S-bis(3-meth acrylate-2-hydroxypropyl)ether (BisS-GMA) was prepared. CQ (camphorquinone) of 1 wt % and DMAEMA (2-(dimethylamino)ethyl methacrylate) of 2 wt % were used in a photoinitiation system to initiate the copolymerization of the matrix resins. Distilled water contact angle measurements were performed for the wettability measurement. Degree of conversion, volumetric shrinkage, contraction stress and compressive strength were measured using Fourier Transformation Infrared-FTIR spectroscopy, the AccuVol and a universal testing machine, respectively. Within the limitations of this study, it can be concluded that the resin composites modified by bismethylene spiroorthocarbonate and BisS-GMA showed a low volumetric shrinkage at 1.25% and a higher contact angle. The lower contraction stress, higher degree of conversion and compressive strength of the novel dental composites were also observed.     

Fabrication of Calix[]arene Derivative Monolayers to Control Orientation of Antibody Immobilization

Three calix[4]arene (Cal-4) derivatives which separately contain ethylester (1), carboxylic acid (2), and crownether (3) at the lower rim with a common reactive thiol at the upper rim were synthesized and constructed to self-assembled monolayers (SAMs) on Au films. After spectroscopic characterization of the monolayers, surface coverage and orientation of antibody immobilized on the Cal-4 derivative SAMs were studied by surface plasmon resonance (SPR) technique. Experimental results revealed that the antibody could be immobilized on the Cal-4 derivatives spontaneously. The orientation of absorbed antibody on the Cal-4 derivative SAMs is related to the SAM’s dipole moment. The possible orientations of the antibody immobilized on the Cal-4 derivative 1 SAM are lying-on or side-on, while on the Cal-4 derivative 2 and Cal-4 derivative 3 head-on and end-on respectively. These experimental results demonstrate the surface dipole moment of Cal-4 derivative appears to be an important factor to antibody orientation. Cal-4 derivatives are useful in developing site direct protein chips.     

Analogies Between Digital Radio and Chemical Orthogonality as a Method for Enhanced Analysis of Molecular Recognition Events

Acoustic wave biosensors are a real-time, label-free biosensor technology, which have been exploited for the detection of proteins and cells. One of the conventional biosensor approaches involves the immobilization of a monolayer of antibodies onto the surface of the acoustic wave device for the detection of a specific analyte. The method described within includes at least two immobilizations of two different antibodies onto the surfaces of two separate acoustic wave devices for the detection of several analogous analytes. The chemical specificity of the molecular recognition event is achieved by virtue of the extremely high (nM to pM) binding affinity between the antibody and its antigen. In a standard ELISA (Enzyme-Linked ImmunoSorbent Assay) test, there are multiple steps and the end result is a measure of what is bound so tightly that it does not wash away easily. The fact that this “gold standard” is very much not real time, masks the dance that is the molecular recognition event. X-Ray Crystallographer, Ian Wilson, demonstrated more than a decade ago that antibodies undergo conformational change during a binding event[1, 2]. Further, it is known in the arena of immunochemistry that some antibodies exhibit significant cross-reactivity and this is widely termed antibody promiscuity. A third piece of the puzzle that we will exploit in our system of acoustic wave biosensors is the notion of chemical orthogonality. These three biochemical constructs, the dance, antibody promiscuity and chemical orthogonality will be combined in this paper with the notions of in-phase (I) and quadrature (Q) signals from digital radio to manifest an approach to molecular recognition that allows a level of discrimination and analysis unobtainable without the aggregate. As an example we present experimental data on the detection of TNT, RDX, C4, ammonium nitrate and musk oil from a system of antibody-coated acoustic wave sensors.     

Immobilization of Ferrocene-Modified SNAP-Fusion Proteins

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit [7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.     

In Vitro Treatment of Melanoma Brain Metastasis by Simultaneously Targeting the MAPK and PI3K Signaling Pathways

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAF^V600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1_DL2, harboring a BRAF^V600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1_DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1_DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1_DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.     

Synthesis of Analogues of Gingerol and Shogaol,the Active Pungent Principles from the Rhizomes of Zingiber officinale and Evaluation of Their Anti-Platelet Aggregation Effects

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.     

Homogeneous crystal nucleation in droplets as a method for determining the line tension of a crystal-liquid-vapor contact

The line tension of a three-phase contact is believed to play an important role in phase transition and phase equilibria in multiphase nanoscale systems hence the need in developing various methods for its experimental evaluation. In this paper we suggest an indirect experimental method for determining the line tension of a solid-liquid-vapor contact based on experiments on homogeneous crystallization of droplets. The underlying idea explores our recent finding that the line tension can give rise to an important contribution to the free energy of formation of a crystal nucleus in a surface-stimulated mode when one of its facets forms at the droplet surface and thus represents a “crystal-vapor” interface. The proposed method requires experimental data on the rate of homogeneous crystal nucleation as a function of droplet size. However, it can provide a rough estimate of line tension even if the rate is known only for one droplet size. Using the method to examine experimental data on homogeneous crystal nucleation in droplets of 19, 49, and radii at , we evaluated the line tension of ice-(liquid)water-(water)vapor contact to be positive and of the order of consistent with the current expectations.     

Effects of shear flow on a semidilute polymer solution under phase-separating condition

We studied the effects of a step-up shear flow from zero shear rate to the given shear rate, , on formation of shear-induced structures for a semidilute polystyrene (PS)/diethyl malonate (DEM) solution below its cloud point temperature where the solution undergoes phase separation via spinodal decomposition (SD) in quiescent state. We elucidated that the effects of step-up shear can be divided into two regions: below and above a critical shear rate, . At , growing phase-separated domains via SD are found to be deformed under the flow, so that FFT spectra of the shear-microscopy images become elliptical with the wave number q_mx at the maximum intensity parallel to the flow being smaller than the corresponding wave number q_mz parallel to the neutral axis. However, strikingly enough, the aspect ratio q_mz/q_mx of the elliptic spinodal ring observed for this system was much smaller than that observed for binary fluids. The unique feature was proposed to be the elastic effect inherent in this system. When is larger than , however, initially phase-separating structures via SD are strongly deformed and distorted. Interestingly enough, the light scattering pattern was transformed from the isotropic ring pattern into the butterfly pattern. This is interpreted as follows: when , there may not be enough time for the domains composed of elastically deformed swollen-network chains to relax, and consequently the domains are cooperatively disrupted. The disrupted domains tend to squeeze solvent in order to release the elastic free energy stored in the deformed swollen-network chains, resulting in anisotropic domain more extended to neutral axis than flow direction and hence giving rise to the butterfly pattern.     

Relative binding free energy calculations of inhibitors to two mutants (Glu46-- Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches

We present free energy perturbation calculations on the complexesof Glu46— Ala46 (E46A) and Glu46— Gln46 (E46Q) mutantsof ribonuclease T1 (RNaseT1) with inhibitors 2‘-guanosinemonophosphate (GMP) and 2’adenosine monophosphate (AMP)by a thermodynamic perturbation method implemented with moleculardynamics (MD). Using the available crystal structure of theRNaseT1–GMP complex, the structures of E46A-GMP and E46Q-GMPwere model built and equilibrated with MD simulations. The structuresof E46A-AMP and E46Q-AMP were obtained as a final structureof the GMP—AMP perturbation calculation respectively.The calculated difference in the free energy of binding (Gbind)was 0.31 kcal/mol for the E46A system and —1.04 kcal/molfor the E46Q system. The resultant free energies are much smallerthan the experimental and calculated value of 3 kcal/mol forthe native RNase T1, which suggests that both mutants have greaterrelative adenine affinities than native RNaseT1. EspeciallyE46Q is calculated to have a larger affinity for adenine thanguanine, as we suggested previously from the calculation onthe native RNaseT1. Thus, the molecular dynamics/free energyperturbation method may be helpful in protein engineering, directedtoward increasing or changing the substrate specificity of enzymes.     

Partially reversible adsorption of annexin A1 on POPC/POPS bilayers investigated by QCM measurements, SFM, and DMC simulations

The kinetics of annexin A1 binding to solid-supported lipid bilayers consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS; 4:1) has been investigated as a function of the calcium ion concentration in the bulk phase. Quartz crystal microbalance measurements in conjunction with scanning force microscopy, fluorescence microscopy, and computer simulations indicate that at a given Ca2+ concentration annexin A1 adsorbs irreversibly on membrane domains enriched in POPS. By contrast, annexin A1 adsorbs reversibly on the POPC-enriched phase, which is composed of single POPS molecules embedded within a POPC matrix. The overall area occupied by the POPS-enriched phase is controlled by the CaCl2 concentration. Monte Carlo simulations suggest that the area of the POPS-enriched phase increases by a factor of 7 when the Ca2+ concentration is changed from 0.01 to 1 mM.     

Saturated thermodynamic properties for the air-water system at elevated temperatures and pressures

A new thermodynamic model is proposed to calculate the thermodynamic properties for the air-water system in which the dry air was assumed to be a mixture of nitrogen and oxygen with the mole fractions of 0.7812 and 0.2188, respectively. For the vapor phase, fugacity coefficients were calculated with the modified Redlich-Kwong equation of state in which a new interaction parameter of oxygen and water was correlated from the experimental data of oxygen-water system. The dissolved gas followed Henry's law. Henry's constant of nitrogen was calculated with the Helgeson equation of state and that for oxygen was correlated from the experimental data of oxygen-water system. The proposed model was verified by comparing the calculated results with the available experimental data. It is shown that the proposed model is suitable for predicting saturated thermodynamic properties for the air-water system up to 300°C and . Furthermore, the prediction results of the proposed model are better than those calculated with the model of Rabinovich and Beketov (Moist Gases, Thermodynamic Properties. Begell: House, 1995), and the application range is wider than that of the model of Hyland and Wexler (ASHRAE Trans. 89(2A) (1983a, b) 500-519, 520-535) which are among the best of today's models.     

二元水溶液渗透蒸发膜扩散过程的修正自由体积模型

在自由体积扩散理论的基础上,提出了渗透蒸发过程中二元水溶液在聚合物膜中扩散的修正自由体积模型。模型中定义了2个共存因子,用以描述非理想物系在渗透蒸发膜扩散过程中的共存效应。结合4种水溶液在聚酰亚胺(PMDA-ODA)膜中的吸附实验数据所计算的膜相浓度,用本模型拟合了上述物系的渗透蒸发实验数据,获得较一致的结果。     

Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy

Cell membranes are typically very complex, consisting of a multitude of different lipids and proteins. Supported lipid bilayers are widely used as model systems to study biological membranes. Atomic force microscopy and force spectroscopy techniques are nanoscale methods that are successfully used to study supported lipid bilayers. These methods, especially force spectroscopy, require the reliable preparation of supported lipid bilayers with extended coverage. The unreliability and a lack of a complete understanding of the vesicle fusion process though have held back progress in this promising field. We document here robust protocols for the formation of fluid phase DOPC and gel phase DPPC bilayers on mica. Insights into the most crucial experimental parameters and a comparison between DOPC and DPPC preparation are presented. Finally, we demonstrate force spectroscopy measurements on DOPC surfaces and measure rupture forces and bilayer depths that agree well with X-ray diffraction data. We also believe our approach to decomposing the force-distance curves into depth sub-components provides a more reliable method for characterising the depth of fluid phase lipid bilayers, particularly in comparison with typical image analysis approaches.     

Introduction of a New Coupling Agent for Selective Coupling of Hydroxy and Amino Group‐Containing Polymers

A new bifunctional coupling agent with an oxazinone and a lactamate group is synthesized. It is shown by means of model reactions that in the presence of aliphatic hydroxy compounds the reaction of the coupling agent is highly selective. At 220 °C, a conversion of the lactamate group of about 90% is observed under elimination of lactam. In a second reaction the oxazinone group is converted with an aliphatic amino compound. This high selectivity is utilized in the synthesis of segmented polyester/polyamide block copolymers by sequential conversion of the coupling agent with hydroxy‐terminated polyesters [poly(butylene terephthalate) (PBT) and polycaprolactone (PCL)] and amino‐terminated polyamide 12 (PA12) under the conditions of reactive extrusion. In the case of the PA12/PBT block copolymer, ductile behavior is observed, whereas the PA12/PCL block copolymer shows elastic properties. Both polymers are phase separated on the nanometer‐scale, as evidenced by AFM and SEM. The thermal behavior of the polymers is affected by the influence of the individual blocks.