Impedance spectroscopy and conductometric biosensing for probing catalase reaction with cyanide as ligand and inhibitor

In this work, a new biosensor was prepared through immobilization of bovine liver catalase in a photoreticulated poly (vinyl alcohol) membrane at the surface of a conductometric transducer. This biosensor was used to study the kinetics of catalase–H₂0₂ reaction and its inhibition by cyanide. Immobilized catalase exhibited a Michaelis–Menten behaviour at low H₂0₂ concentrations (< 100 mM) with apparent constant K_M^app = 84 ± 3 mM and maximal initial velocity V_M^app = 13.4 μS min^? 1. Inhibition by cyanide was found to be non-competitive and inhibition binding constant K_i was 13.9 ± 0.3 μM. The decrease of the biosensor response by increasing cyanide concentration was linear up to 50 μM, with a cyanide detection limit of 6 μM. In parallel, electrochemical characteristics of the catalase/PVA biomembrane and its interaction with cyanide were studied by cyclic voltammetry and impedance spectroscopy. Addition of the biomembrane onto the gold electrodes induced a significant increase of the interfacial polarization resistance R_P. On the contrary, cyanide binding resulted in a decrease of Rp proportional to KCN concentration in the 4 to 50 μM range. Inhibition coefficient I₅₀ calculated by this powerful label-free and substrate-free technique (24.3 μM) was in good agreement with that determined from the substrate-dependent conductometric biosensor (24.9 μM).     

Polymeric particulates to improve oral bioavailability of peptide drugs

Oral administration remains the most convenient way of delivering drugs. Recent advances in biotechnology have produced highly potent new molecules such as peptides, proteins and nucleic acids. Due to their sensitivity to chemical and enzymatic hydrolysis as well as a poor cellular uptake, their oral bioavailability remains very low. Despite sophisticated new delivery systems, the development of a satisfactory oral formulation remains a challenge. Among the possible strategies to improve the absorption of drugs, micro- and nanoparticles represent an exciting approach to enhance the uptake and transport of orally administered molecules. Increasing attention has been paid to their potential use as carriers for peptide drugs for oral administration. This article reviews the most common manufacturing methods for polymeric particles and the physiology of particle absorption from the gastrointestinal (GI) tract. In a second part, the use of polymeric particulate systems to improve the oral absorption of insulin is discussed.     

Advantages of hydride generation interface for selenium speciation in waters by high performance liquid chromatography-inductively coupled plasma mass spectrometry coupling

This paper focuses on the analytical performance improvement of the coupled technique HPLC-ICPMS using on-line collision/reaction cell technology for selenium elemental and speciation analyses at the ng (Se) l(-1) level in aquatic environment. Collision/reaction cell operating parameters were optimised, resulting in selected conditions of 5.5 ml min(-1) H(2) and 0.5 ml min(-1) He mixture. The detection limits obtained were around 5 ng (Se) l(-1) for total analysis, and between 7 and 15 ng (Se) l(-1) depending on the species for speciation analysis. The capability of UV irradiation-hydride generation interfacing to increase detector sensitivity was also evaluated for speciation analysis. The detection limits obtained were in the range 2-8 ng (Se) l(-1) depending on the species. Moreover, such interface allowed to prevent bromine introduction to the ICPMS which is particularly convenient for selenium trace analysis in natural waters as (80)Se is preserved free from BrH interferences. The developed method was validated using certified water with low selenium content (TM Rain 95, NWRI, Canada) and applied to the analysis of different waters.     

Control of the evolution of iron peroxide intermediate in superoxide reductase from Desulfoarculus baarsii. Involvement of lysine 48 in protonation

Superoxide reductase is a nonheme iron metalloenzyme that detoxifies superoxide anion radicals O(2)(?-) in some microorganisms. Its catalytic mechanism was previously proposed to involve a single ferric iron (hydro)peroxo intermediate, which is protonated to form the reaction product H(2)O(2). Here, we show by pulse radiolysis that the mutation of the well-conserved lysine 48 into isoleucine in the SOR from Desulfoarculus baarsii dramatically affects its reaction with O(2)(?-). Although the first reaction intermediate and its decay are not affected by the mutation, H(2)O(2) is no longer the reaction product. In addition, in contrast to the wild-type SOR, the lysine mutant catalyzes a two-electron oxidation of an olefin into epoxide in the presence of H(2)O(2), suggesting the formation of iron-oxo intermediate species in this mutant. In agreement with the recent X-ray structures of the peroxide intermediates trapped in a SOR crystal, these data support the involvement of lysine 48 in the specific protonation of the proximal oxygen of the peroxide intermediate to generate H(2)O(2), thus avoiding formation of iron-oxo species, as is observed in cytochrome P450. In addition, we proposed that the first reaction intermediate observed by pulse radiolysis is a ferrous-iron superoxo species, in agreement with TD-DFT calculations of the absorption spectrum of this intermediate. A new reaction scheme for the catalytical mechanism of SOR with O(2)(?-) is presented in which ferrous iron-superoxo and ferric hydroperoxide species are reaction intermediates, and the lysine 48 plays a key role in the control of the evolution of iron peroxide intermediate to form H(2)O(2).     

Synthesis of an iminosugar based peptidomimetic analogue

The synthesis of an 1-deoxymannojirimycin based analogue of a known HIV-protease inhibitor is described. The strategies employed for introduction of the pharmacophore groups onto the azasugar scaffold were based on regioselective reactions of the hydroxyl groups of the natural product and of d-fructopyranoside derivatives.     

Carbohydrate-based DNA ligands: sugar-oligoamides as a tool to study carbohydrate-nucleic acid interactions

Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-DNA interactions. The general design of the ligands 1-3 has been done as to favor the bound conformation of Distamycin-type gamma-linked covalent dimers which is a hairpin conformation. Indeed, NMR analysis of the sugar-oligoamides in the free state has indicated the presence of a percentage of a hairpin conformation in aqueous solution. The DNA binding activity of compounds 1-3 was confirmed by calf thymus DNA (ct-DNA) NMR titration. Interestingly, the binding of the different sugar-oligoamides seems to be modulated by the sugar configuration. Semiquantitative structural information about the DNA ligand complexes has been derived from NMR data. A competition experiment with Netropsin suggested that the sugar-oligoamide 3 bind to DNA in the minor groove. The NMR titrations of 1-3 with poly(dA-dT) and poly(dG-dC) suggested preferential binding to the ATAT sequence. TR-NOE NMR experiments for the sugar-oligoamide 3-ct-DNA complex both in D(2)O and H(2)O have confirmed the complex formation and given information on the conformation of the ligand in the bound state. The data confirmed that the sugar-oligoamide ligand is a hairpin in the bound state. Even more relevant to our goal, structural information on the conformation around the N-glycosidic linkage has been accessed. Thus, the sugar asymmetric centers pointing to the NH-amide and N-methyl rims of the molecule have been characterized.     

Mechanistic diversity covering 15 orders of magnitude in rates: cyanide exchange on [M(CN)(4)](2-) (M = Ni, Pd, and Pt)

Kinetic studies of cyanide exchange on [M(CN)(4)](2-) square-planar complexes (M = Pt, Pd, and Ni) were performed as a function of pH by (13)C NMR. The [Pt(CN)(4)](2-) complex has a purely second-order rate law, with CN(-) as acting as the nucleophile, with the following kinetic parameters: (k(2)(Pt,CN))(298) = 11 +/- 1 s(-1) mol(-1) kg, DeltaH(2) (Pt,CN) = 25.1 +/- 1 kJ mol(-1), DeltaS(2) (Pt,CN) = -142 +/- 4 J mol(-1) K(-1), and DeltaV(2) (Pt,CN) = -27 +/- 2 cm(3) mol(-1). The Pd(II) metal center has the same behavior down to pH 6. The kinetic parameters are as follows: (k(2)(Pd,CN))(298) = 82 +/- 2 s(-1) mol(-1) kg, DeltaH(2) (Pd,CN) = 23.5 +/- 1 kJ mol(-1), DeltaS(2) (Pd,CN) = -129 +/- 5 J mol(-1) K(-1), and DeltaV(2) (Pd,CN) = -22 +/- 2 cm(3) mol(-1). At low pH, the tetracyanopalladate is protonated (pK(a)(Pd(4,H)) = 3.0 +/- 0.3) to form [Pd(CN)(3)HCN](-). The rate law of the cyanide exchange on the protonated complex is also purely second order, with (k(2)(PdH,CN))(298) = (4.5 +/- 1.3) x 10(3) s(-1) mol(-1) kg. [Ni(CN)(4)](2-) is involved in various equilibrium reactions, such as the formation of [Ni(CN)(5)](3-), [Ni(CN)(3)HCN](-), and [Ni(CN)(2)(HCN)(2)] complexes. Our (13)C NMR measurements have allowed us to determine that the rate constant leading to the formation of [Ni(CN)(5)](3-) is k(2)(Ni(4),CN) = (2.3 +/- 0.1) x 10(6) s(-1) mol(-1) kg when the following activation parameters are used: DeltaH(2)() (Ni,CN) = 21.6 +/- 1 kJ mol(-1), DeltaS(2) (Ni,CN) = -51 +/- 7 J mol(-1) K(-1), and DeltaV(2) (Ni,CN) = -19 +/- 2 cm(3) mol(-1). The rate constant of the back reaction is k(-2)(Ni(4),CN) = 14 x 10(6) s(-1). The rate law pertaining to [Ni(CN)(2)(HCN)(2)] was found to be second order at pH 3.8, and the value of the rate constant is (k(2)(Ni(4,2H),CN))(298) = (63 +/- 15) x10(6) s(-1) mol(-1) kg when DeltaH(2) (Ni(4,2H),CN) = 47.3 +/- 1 kJ mol(-1), DeltaS(2) (Ni(4,2H),CN) = 63 +/- 3 J mol(-1) K(-1), and DeltaV(2) (Ni(4,2H),CN) = - 6 +/- 1 cm(3) mol(-1). The cyanide-exchange rate constant on [M(CN)(4)](2-) for Pt, Pd, and Ni increases in a 1:7:200 000 ratio. This trend is modified at low pH, and the palladium becomes 400 times more reactive than the platinum because of the formation of [Pd(CN)(3)HCN](-). For all cyanide exchanges on tetracyano complexes (A mechanism) and on their protonated forms (I/I(a) mechanisms), we have always observed a pure second-order rate law: first order for the complex and first order for CN(-). The nucleophilic attack by HCN or solvation by H(2)O is at least nine or six orders of magnitude slower, respectively than is nucleophilic attack by CN(-) for Pt(II), Pd(II), and Ni(II), respectively.     

Improving imprinted shape cavities of molecularly imprinted sol–gel host matrix with minimal relaxation for sensing of creatinine

Journal of Sol-Gel Science and Technology - Molecularly imprinted polymer (MIP) is of great attention in biomimetic recognition systems due to its selective molecular recognition towards any guest...     

On the Weak Invariance Principle for Non-Adapted Sequences under Projective Criteria

In this paper we study the central limit theorem and its weak invariance principle for sums of non-adapted stationary sequences, under different normalizations. Our conditions involve the conditional expectation of the variables with respect to a given σ-algebra, as done in Gordin (Dokl. Akad. Nauk SSSR 188, 739–741, 1969) and Heyde (Z. Wahrsch. verw. Gebiete 30, 315–320, 1974). These conditions are well adapted to a large variety of examples, including linear processes with dependent innovations or regular functions of linear processes.