Engineering an allosteric binding site for aminoglycosides into TEM1-β-Lactamase

Allosteric regulation of enzyme activity is a remarkable property of many biological catalysts. Up till now, engineering an allosteric regulation into native, unregulated enzymes has been achieved by the creation of hybrid proteins in which a natural receptor, whose conformation is controlled by ligand binding, is inserted into an enzyme structure. Here, we describe a monomeric enzyme, TEM1-β-lactamase, that features an allosteric aminoglycoside binding site created de novo by directed-evolution methods. β-Lactamases are highly efficient enzymes involved in the resistance of bacteria against β-lactam antibiotics, such as penicillin. Aminoglycosides constitute another class of antibiotics that prevent bacterial protein synthesis, and are neither substrates nor ligands of the native β-lactamases. Here we show that the engineered enzyme is regulated by the binding of kanamycin and other aminoglycosides. Kinetic and structural analyses indicate that the activation mechanism involves expulsion of an inhibitor that binds to an additional, fortuitous site on the engineered protein. These analyses also led to the defining of conditions that allowed an aminoglycoside to be detected at low concentration.     

Glass and Photonics — an Overview

Editor's Note: This issue of the International Journal of Applied Glass Science is the second in a planned series which engage internationally recognized authorities to serve as Guest Editors. We are especially pleased that Drs' Kathleen Richardson and Heike Ebendorff-Heidepriem accepted our invitation to organize this issue on Glass and Photonics, arguably one of the most important branches of glass science throughout the world. We extend sincere thanks to them for their care, diligence, and exceptional dedication in completing this task. We also extend our sincere thanks to the authors who committed their time and energy to bring this issue to completion.     

Interaction Between an Anionic and an Amphoteric Surfactant. Part I: Monomer–Micelle Equilibrium

A mixture of anionic and amphoteric surfactants is composed of three components at intermediate pH levels: anionic, cationic (protonated amphoteric), and zwitterionic (unprotonated amphoteric). Knowledge of the composition of each surfactant in both monomer and micellar forms (monomer–micelle equilibrium) is important in applications using this mixture. Hydrogen ion titration of the mixed surfactant solution as a function of surfactant composition is combined with the pseudophase separation model and regular solution theory for the three-surfactant mixture to calculate the concentration of each surfactant in monomer and in micelle forms at different pH levels. The specific systems studied here contain sodium dodecyl sulfate (SDS) and dimethyldodecylamine oxide (DDAO), which are used in a wide range of consumer products. The degree of protonation of monomeric DDAO is not affected by the presence of SDS, indicating an insignificant formation of ion pairs between these monomers. However, the presence of SDS in micelles shifts the micellar pK _a of DDAO protonation significantly and the method used here allows the quantification of partial fugacities of each individual surfactant in micelle form. The composition in the monomer phase at each pH will aid in understanding and predicting solution compositions corresponding to anionic/amphoteric surfactant precipitation boundaries, which is the focus of the subsequent paper in this series.

Purification and Stability of an Antithrombus Enzyme from Bacillus subtilis

An antithrombus enzyme (ATE) was precipitated by (NH4)2SO4 or ethanol from supernatant of Bacillus subtilis culture broth then purified using ion exchange chromatography on CM-sepharose fast flow. The effects of ionic strength and pH value on protein adsorption, the gradient elution at different flow rates arid step elution were examined respectively. The recovery yield of the optimised process was 74.5% with a purification factor 8.1. The ATE molecular weight was estimated as 30ku by SDS-PAGE. The experimental results showed that the enzyme was stable in the range of pH 7 to pH11, and temperature 25℃ to 37℃.     

The Flow and Fracture of Advanced Glasses — an Overview

Editors' note: On appropriate occasions, the International Journal of Applied Glass Science will engage the services of Guest Editors to help focus on topics of special interest to the international glass community. In this inaugural issue of this initiative, Professors Tanguy Rouxel and Richard Brow have accomplished just that in their overview of the subject “Flow and Fracture of Advanced Glasses.” An introduction to this material and brief biographical sketches are given below. Our sincere thanks are extended to them for their help and cooperation throughout this undertaking, and especially the authors who contributed so much of their time, energy, and talents to making this special issue possible.     

A holistic mathematical modelling and simulation for cathodic delamination mechanism – a novel and an efficient approach

This paper addresses a holistic mathematical design using a novel approach for understanding the mechanism of cathodic delamination. The approach employed a set of interdependent parallel processes with each process representing: cation formation, oxygen reduction and cation transport mechanism, respectively. Novel mathematical equations have been developed for each of the processes based on the observations recorded from experimentation. These equations are then solved using efficient time-iterated algorithms. Each process consists of distinct algorithms which communicate with each other using duplex channels carrying signals. Each signal represents a distinct delamination parameter. As a result of interdependency of various processes and their parallel behaviour, it is much easier to analyse the quantitative agreement between various delamination parameters. The developed modelling approach provides an efficient and reliable prediction method for the delamination failure. The results obtained are in good agreement with the previously reported experimental interpretations and numerical results. This model provides a foundation for the future research within the area of coating failure analysis and prediction.     

Phase transitions and dimensional and morphological changes in an “Aluminum Hydroxide-Alumina” system under a shock-wave action

A method is proposed to study the sequence of phase transitions in powdered materials under a shock-wave action. It is shown that the aluminum hydroxide-alumina system has the following sequence of phase transformations under a shock-wave action: bayerite boehmite -Al₂O₃ -Al₂O₃. It is found that there are no transitional high-temperature modifications of alumina. A method is developed for obtaining a submicron alumina powder, which allows obtaining materials with a controlled phase composition, including a thermodynamically stable -modification of Al₂O₃. The specific features of the morphological structure and the phase and structural characteristics of powders after a shock-wave action are considered.Translated from Fizika Goreniya i Vzryva, Vol. 41, No. 1, pp. 110–119, January–February, 2005.     

1,2,3-Triazolophanes—Cyclophanes with an Array of Molecular Structures and Supramolecular Architectures

1,2,3-Triazolophanes are a class of cyclophanes that has attracted enormous attention in recent times. With the advent of the intramolecular copper-mediated alkyne azide cycloaddition (CuAAC) reaction 1,2,3-triazolophanes of various molecular structure and supramolecular architecture have been synthesized. 1,2,3-Triazolophanes find a variety of applications as molecular hosts, ion sensors, peptidomimics, and supramolecular building blocks, among others.     

Structure and Properties of an Amorphous Nickel–Titanium Alloy after Explosive Compaction

Xray phase, differentialthermal, and thermogravimetric analyses are employed to study the action of shockwave loading on the state of Ni₅₀$Ti₅₀ amorphous alloy produced by synthesis using mechanical activation. It is shown that, on the one hand, shockwave loading leads to additional disordering of the amorphous matrix, and, on the other hand, it improves the structure of microcrystals of the crystalline constituent of the Ni₅₀$Ti₅₀ amorphous alloy.     

Synthesis and characterization of an isocyanurate–oxazolidone polymer: Effect of stoichiometry

Isocyanurate–oxazolidone polymers were synthesized by using various reactant stoichiometry of a diglycidyl ether of bisphenol-A (DGEBA) and a polymeric diphenyl methane diisocyanate (pMDI). The reaction was catalyzed by tris-2,4,6-dimethylaminoethylphenol (Ancamine K54). The effects of stoichiometry that the reaction had on the molecular structure and mechanical and thermomechanical properties were evaluated. Two main structures obtained from the reaction of DGEBA with pMDI, namely isocyanurate and oxazolidone, were clearly shown by Fourier transformed infrared spectroscopy (FTIR) analyses. It was found that the amount of DGEBA present determines the amount of oxazolidone formed. Where excess DGEBA was used, structural transformation reaction from isocyanurate to oxazolidone was observed. The amount of pMDI, on the other hand, influenced the amount of isocyanurate structure formed. As the relative amounts of isocyanurate and oxazolidone contents changed with stoichiometry of the reactants, the effects on the crosslink density in the samples were clearly shown by both mechanical and thermomechanical measurements. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 879–888, 2001     

Modelling and constraint optimisation of an aromatic nitration in liquid–liquid medium

A methodology, which determines the operating conditions simultaneously optimising the chemical yield and considering the safety aspect, has been developed for a chemical reaction which is carried out batch-wise. To illustrate the methodology, the aromatic nitration of toluene by mixed acid has been chosen as a typical exothermic and non-selective reaction. This reaction takes place in a two-phase medium and, therefore, involves simultaneously chemical reaction and mass transfer phenomena. A kinetic model recently proposed for the slow and fast liquid–liquid reaction regimes was integrated to the mass balance. Nitration experiments were carried out in order to compare experimental composition profiles with simulated ones. Afterwards, an optimisation procedure has been used to maximise conversion, by manipulating the operating conditions subject to safety constraints. The p-nitrotoluene yield was chosen as the criterion to be maximised. Experimental validation for the optimisation procedure has been carried out. A monofluid heating–cooling system controlled by a predictive controller was used for the temperature control of the reactor. Simulation and experimental results are presented, discussed and compared.     

Preparation of a Nickel Ion Containing Langmuir–Blodgett Multilayer and an Ultra-Thin Nickel Film Deposited on an Interpenetrating Polymer Network Substrate

The nickel ion containing Langmuir–Blodgett (LB) multilayer was prepared by transferring nickel acetate, spread on the surface of a sub-phase of ultra-pure water and stearic acid–chloroform mixture, onto an interpenetrating polymer network (IPN) substrate. The substrate was prepared by dip-pulling a hydrophilic silicon wafer or a glass plate into precursors, followed by solidification at room temperature, in order to create an ultra-thin and uniform surface for metal deposition. The multilayer was then converted into an ultra-thin nickel film after chemical reduction by 0.01 mol/l sodium borohydride. The surface pressure of the monolayer and dipping speed of substrate were determined by measuring the transfer coefficient. Fourier transform infrared spectroscopy (FT-IR) was used to investigate the interactions of the nickel ions with the IPN during the multilayer deposition on substrate, and the metal transformations of nickel ion in the ultra-thin film. The shifted peak location for –C=O verified the interactions between the IPN and the nickel ion and the transformation of the nickel ion by reduction. Further reduction caused the organic phase to dissolve, resulting in most of it being removed from the multilayer. The surface morphologies of the LB multilayer were detected by atomic force microscopy (AFM). Compared with the IPN, which formed a uniform and flat film, within a range of nanometers, the Ni/IPN multilayer and Ni ultra-thin film both showed some surface fluctuations, although these were still within nanometer range. The roughness of the nickel ion containing multilayer and the ultra-thin nickel film was 1.123 nm and 0.773 nm with a maximum height of 12.451 nm and 14.933 nm, respectively, which were larger than that of pure IPN substrate of 0.593 nm with the max height of 6.795 nm.     

Cyanoethylation of cyclopentadiene: Isolation of penta- and hexa(β-cyanoethyl)cyclopentadiene and an unexpected acrylonitrile polymer

Summary The cyanoethylation of cyclopentadiene was examined under a variety of reaction conditions. Approximately 9% of cyclopentadiene was converted to the desired hexa(-cyanoethyl)cyclopentadiene when the amount of acrylonitrile to cyclopentadiene was increased from 2 to 7 equivalents under phase transfer conditions. The low conversion is due to competitive formation of various unreactive side-products. For example, termination leading to less-highly substituted products was confirmed by isolation of crystalline 1,1,2,3,4-penta(-cyanoethyl)cyclopentadiene. Another side product isolated and characterized by ¹³C NMR was an oligomer of acrylonitrile attached to hexa(-cyanoethyl)cyclopentadiene; the anion (at a -alkyl position) of the latter apparently acted as anionic initiator. Attempts to induce thermal polymerization and cyclotrimerization of the pendent cyano groups of hexa(-cyanoethyl)cyclopentadiene were unsuccessful even with added Lewis acid catalysts. Reduction to the hexaamine was only partially successful.     

Synthesis,antiapoptotic biological activity and structure of an oxo–vanadium(IV) complex with an OOO ligand donor set

The oxo–vanadium(IV) complex VO(oda)(H₂O)₂ (1) (oda=oxydiacetate, O(CH₂COO⁻)₂) was obtained by reaction of aerobic aqueous solutions of VO(acac)₂ with oxydiacetic acid, O(CH₂COOH)₂. The antiapoptotic biological activity of 1 was studied in insulin-producing cells. Chemically generated nitric oxide (NO) triggers apoptotic events, such as the appearance of oligonucleosomes in cytosol, and this response was prevented by the presence of 1 in the culture medium. The molecular structure of 1 has been determined by X-ray diffraction analysis.     

Binuclear rhodium(III) and iridium(III) monoselenocarboxylates: Synthesis,spectroscopy and structure of an ortho-metallated iridium complex featuring an IrCCC(μ-Se) chelate ring

Monoselenocarboxylate–bridged binuclear complexes of Rh^III and Ir^III, [(Cp1MCl)₂(μ-SeCOAr)₂] (1) (M = Rh or Ir; Cp1 = pentamethylcyclopentadienyl; Ar = Ph, C₆H₄Me–4), have been isolated either by the reaction between [Cp1₂M₂(μ-Cl)₂Cl₂] with KSeCOAr in acetonitrile or by treatment of [Cp1MCl(solvent)₂][PF₆] with KSeCOAr in acetone. The novel binuclear complexes, [Cp1IrCl(μ-SeCOAr)(κ²-SeCOC₆H₃R–)IrCp1] (2) (R = H or Me-4) with ortho-metallation at one of the iridium centres have been isolated following the use of excess AgPF₆. The single crystal structure of [Cp1IrCl(μ-SeCOC₆H₅)(κ²-SeCOC₆H₄–)IrCp1] (2a) exhibits two phenylcarboselenolate moieties situated in syn fashion with respect to the “Ir₂Se₂” plane, one of which leans towards the metal centre in order to undergo ortho-metallation after iridium–chlorine bond dissociation.     

Crystallization behavior and sintering of cordierite synthesized by an aqueous sol–gel route

The purpose of the research was to investigate crystallization behavior and sintering of cordierite synthesized by a low-price aqueous sol–gel route starting from silicic acid and magnesium and aluminum salts. Viscous sintering of the gel occurred in the temperature range of 800–850 °C, followed by μ-cordierite crystallization at about 900 °C, which proves the homogeneity of the gel. Decreasing of μ-cordierite crystallinity in a wide temperature range prior to commencing of α-cordierite crystallization at about 1200 °C indicates reconstructive type of μ- → α-cordierite transformation. The transformation was fully completed at 1350 °C. The value of the Avrami parameter indicates that μ-cordierite crystallization was controlled by surface or interface nucleation, which implies that viscous sintering occurred in the primary gel particles, which leads to shrinkage, and thereafter nucleation occurred on the surface or interface of the particles. The overall activation energy of μ-cordierite crystallization was 382.0 kJ/mol. The sinterability of the powder obtained by calcination at 1300 °C, where well-crystallized α-cordierite was formed, was better than that of the powder obtained by calcination at 850 °C, where the most intensive shrinkage occurred before the onset of crystallization of μ-cordierite.     

Electrochemical behavior of an inorganic–organic hybrid based on isopolymolybdate anions and ethylenediamine

The one-dimensional inorganic–organic compound, (C₂H₁₀N₂)[Mo₃O₁₀], was synthesized hydrothermally, and characterized by single crystal X-ray diffraction. The compound was used as a bulk-modifier to fabricate a renewable three-dimensional chemically modified carbon paste electrode (Mo-CPE) by direct mixing. The electrochemical properties of (C₂H₁₀N₂)[Mo₃O₁₀]-modified Mo-CPE were investigated with respect to their pH-dependence, stability and electrocatalytic activity. The hybrid material bulk modified Mo-CPE not only displays good electrocatalytic activity toward the reduction of BrO₃ ⁻, IO₃ ⁻, NO₂ ⁻ and H₂O₂, but also exhibits good stability and multiple repeatability by simply polishing on the surface of a wet filter paper, a feature which is important for practical applications.