Activation of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine with poly(salicylic acid 5-aminodisulfide)

5-Aminosalicylic acid (5-ASA) inhibited by a mixed mechanism the peroxidase catalyzed oxidation of tetramethylbenzidine (TMB) in 0.015 M phosphate-citrate buffer (pH 6.4) supplemented with 5% DMSO and 5% DMF. Poly(salicylic acid 5-aminodisulfide) (poly(SAADS)) in 0.01 M phosphate buffer (pH 6.2-7.4) supplemented with 5% DMSO and 5% DMF effectively activated the peroxidase-catalyzed oxidation of TMB. The activation was quantitatively characterized by coefficients (M^–1) determined at different pH values: increased linearly with increase in pH up to the maximal value of 2.44·10⁵ M^–1 at pH 7.0. The activating effect of poly(SAADS) on the peroxidase-catalyzed oxidation of TMB is explained by the activator properties of polyelectrolyte, with its anionic form interacting with peroxidase sites responsible for the acid-base catalysis.     

Initiation and inhibition of free-radical processes in biochemical peroxide systems: A review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase-and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Peroxidase-catalyzed co-oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (K _i, μmol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (α, M^?1), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).     

Activation of peroxidase-catalyzed oxidation of chromogenic substrates by tetrazole and its 5-substituted derivatives

Peroxidase-catalyzed oxidation of 2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid) (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB) is activated by tetrazole and its 5-substituted derivatives—5-amino-(AmT), 5-methyl-(MeT), 5-phenyl-(PhT), and 5-CF₃-(CF₃-T) tetrazoles. In phosphate-citrate or phosphate buffer (pH 6.4 or 7.2; 20°C), the activating effect of tetrazoles on TMB and ABTS oxidation decreased in the series AmT > MeT > T > PhT > CF₃-T and T > AmT > MeT > PhT, respectively. The coefficient (degree) of activation (), expressed in M^–1, determined for both substrates and all activators, depended on substrate type, buffer nature, and pH (it increased as pH increased from 6.4 to 7.2). For TMB oxidation, good correlation between log and the Hammet constants _meta for m-substituents in the benzene series NH₂, CH₃, C₆H₅, and CF₃ was found. It is suggested that AmT, MeT, and T can be used as activators of peroxidase-catalyzed oxidation of TMB and ABTS in enzyme immunoassay and designing peroxidase-based biosensors.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 148–157.Original Russian Text Copyright © 2005 by Karasyova, Gaponik, Metelitza.     

Inactivation of Glucose-6-Phosphate Dehydrogenase in Solution by Low- and High-Frequency Ultrasound

We compared the kinetics of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) inactivation in 0.1 M phosphate buffer (pH 7.4) at 36–50° under conditions of exposure to low-frequency (LF, 27 kHz, 60 W/cm²) or high-frequency (HF, 880 kHz, 1.0 W/cm²) ultrasound (USD). The inactivation of G6PDH was characterized by effective first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(usd), ultrasonic inactivation. Dilution of the enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k _in > k _in ^*. The rate constants increased with temperature. The Arrhenius plots of the temperature dependences of k _in and k _in(usd) had an break point at 44°C. The activation energy ( _act) of the total inactivation of G6PDH was higher than _act for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: _act was higher in the case of inactivation with low-frequency ultrasound (LF-USD) than high-frequency ultrasound (HF-USD). The rate of the ultrasonic inactivation of this enzyme substantially decreased in the presence of low concentrations of HO^. radical scavengers (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in solutions exposed to USD.     

红腹锦鸡肺的组织结构与微血管构筑

为了了解红腹锦鸡(Chroysolophus pictus)肺的微细结构和微血管构筑特征,为呼吸生物学研究提供形态学依据,用组织学方法和微血管铸型技术在光镜和扫描电镜下观察研究了红腹锦鸡肺的组织结构与微血管构筑情况。结果表明,红腹锦鸡肺主要由各级支气管构成,从三级支气管上呈辅射状分出许多呼吸毛细管(微气管),并相互吻合成网状,呼吸毛细管外面包围有丰富的毛细血管;红腹锦鸡肺毛细血管垂直围绕在各微气管外,并相互吻合成密集的立体微血管网;毛细血管管径4.5~7.0μm,微气管直径11~50μm。并对肺微血管构筑情况与呼吸效率的关系作了探讨。     

The Effect of Tetrazole and Its Amino Derivatives on the Kinetics of Peroxidase Oxidation of Chromogenic Substrates

The peroxidase-catalyzed oxidation of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (PDA), and 3,3,5,5-tetramethylbenzidine (TMB) was found to be activated by tetrazole and 5-aminotetrazole (AT) and weakly inhibited by 1,5-diaminotetrazole. The activating action of tetrazole and AT on the PDA and TMB oxidation was clearly discompetitive and that on ABTS was non-competitive. The coefficients (degrees) of activation were determined for three substrates and two activators; they depended on the substrate type and the buffer nature and increased along with the pH growth from 6.4 to 7.2. For AT and tetrazole, the maximal values were 4140 and 800 M^–1, respectively, upon the PDA oxidation and 3570 and 540 M^–1, respectively, upon the TMB oxidation. Lower values (145 and 58 M^–1 for tetrazole and AT, respectively) were characteristic of the peroxidase oxidation of ABTS. The activation of peroxidase oxidation of the substrates by tetrazole and AT at pH 5.4 was explained by the nucleophilic nature of the activators interacting with the amino acid residues in the peroxidase active site according to the mechanism of acid–base catalysis.     

Olive Phenolics as c-Met Inhibitors: (-)-Oleocanthal Attenuates Cell Proliferation,Invasiveness, and Tumor Growth in Breast Cancer Models

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.     

Flavonoids: Efficient protectors of glucose-6-phosphate dehydrogenase from ultrasonic cavitation-induced inactivation

Seven structurally diverse flavonoids have been shown to decrease glucose-6-phosphate dehydrogenase (G6PDH) inactivation in 0.1 M phosphate buffer (pH 7.4), induced by exposure to a high temperature (44°C), or by a low-frequency ultrasound (27 kHz, 60 Wt/cm²). The activity of the compounds was assessed by their ability to change effective first-order rate constants characterizing the total (thermal and ultrasonic), thermal, and ultrasonic inactivation of 2.5 nM G6PDH (k _in, k*_in, and k _in(us), respectively). The value dependences of these constants on flavonoid concentrations (0.01–50 μM) were obtained. Rank order of potency exhibited by the compounds in protecting G6PDH appeared as follows: hesperidin > morin > silibin > naringin = quercetin > kampferol ? astragalin. The data obtained confirm the crucial role of free radicals formed in the field of ultrasonic cavitation (HO^· and O ₂ ^·? in G6PDH inactivation in solutions.