Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air-water interface

The interfacial behavior differences of two glutathione peroxidase isoforms have been investigated. The first isoform is the phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12) (GPx-4) isolated from rat testes and the second one is the cytosolic glutathione peroxidase (EC 1.11.1.9) (GPx-1) from bovine erythrocytes. Injected in the subphase buffer of a Langmuir trough, GPx-4 was able to adsorb quickly at the air-water interface whereas the GPx-1 was not. Then, the protein interaction with phospholipid monolayers was explored. Indeed, a monolayer of phospholipids containing a different number of polyunsaturated fatty acyl chains was prepared at the air-water interface. Under each kind of monolayer, the protein solution was injected and its adsorption was visualized by the measurement of successive pressure-area isotherms. We have, then, determined the molecular area increase due to the protein adsorption. It was found that the GPx-4 is adsorbed in each kind of monolayer tested whereas no molecular area increase was detected with the GPx-1. This indicates that the GPx-4 has a higher affinity for the interface, recovered or not by lipids, than the GPx-1. Moreover, the GPx-4 presents a different affinity for the phospholipid monolayers depending on the number of polyunsaturated fatty acyl chains.     

Development of an obidoxime chloride reference material: a metrological approach to the determination of chromatographic purity

A metrological approach to determination of the chromatographic purity of obidoxime chloride and the corresponding obidoxime chloride reference material (RM) with a certified chromatographic purity value have been developed. This value was defined as the ratio of the sum of peak areas of obidoxime chloride isomers to the total peak area of detected substances including impurities (%) under specified HPLC–UV conditions. The RM homogeneity and stability were studied using HPLC with UV detection and evaluated as satisfactory. The certified value calculated from the results of an interlaboratory trial was equal to 99.9% with the expanded uncertainty of 0.6% at the level of confidence 0.95 and the coverage factor 2. The RM certified value, like other results of chromatographic purity determination traceable to the reference measurement procedure, is not traceable directly to the SI mole. However, the results are comparable in metrologically traceable environments, i.e. when relevant measuring laboratory instruments are calibrated with traceability chains to the corresponding SI units. Therefore, the RM can be used as a measurement standard (calibrator) for analytical instruments and as a control sample for quality control of HPLC obidoxime chloride assay results.     

Determination of the carotenoid phytoene in blood by high-pressure liquid chromatography

A sensitive and specific high-pressure liquid chromatographic assay was developed for the determination of phytoene in blood with an overall recovery of 86 ± 6.0% and a limit of detection of 50–100 ng per ml of blood. This method provides for rapid and simple quantitation of phytoene using 1 ml or less of blood.

The assay was used in the determination of phytoene blood levels in the dog following intravenous and oral administration of 10-mg/kg doses.     

Reaction mechanism of the benzoquinone/hydroquinone couple at platinum electrodes in aqueous solutions: Retardation and enhancement of electrode kinetics by single chemisorbed layers

The electrochemical kinetics of the benzoquinone (Q)/hydroquinone (H₂Q) redox couple at platinum electrodes in aqueous solutions has been found to be extremely sensitive to the nature of species adsorbed on the electrode surface at monolayer coverages. Experimental measurements were based on thin-layer cyclic voltammetry; the use of thin-layer electrodes was dictated by the need to minimize surface contamination. Bulky neutral or anionic aromatic adsorbates led to the familiar U-shaped rate-vs.-pH curves; the rate minimum occurred near pH 4. Kinetic effects due to oriental changes of chemisorbed species were noted only when the rate was low. Adsorbed 1 atoms led to comparatively rapid reactivity (rate constant k° > 10^?3 cm s^?1) and virtual independence of pH. Profound retardation resulted from pretreatment ofthe surface with CN^? and SCN^?; total irreversibility (k° < 10^?6 cm s^?1) was observed at pH 4, with a further decrease in rate at pH 7. In contrast, when the surface contained n layer of chemisorbed phenyltriethylammonium cations, the electrode rate increased with increasing pH. The results indicate that different reaction pathways predominate when different absorbates are present.     

Interaction of nitrogen oxides with sublimed layers of (meso-tetraphenylporphyrinato)cobalt(II); IR evidence of oxo-transfer from (nitro)porphyrinatocobalt(III) to free nitric oxide

Interaction of a low-pressure NO2 with sublimed layers of (meso-tetraphenylporphyrinato)cobalt(II) (Co(TPP)) leads to formation of 5-coordinate nitro complex Co(III)(TPP)(NO2). Upon exposure of these layers to pyridine vapors, the fast reaction with formation of 6-coordinate nitro-pyridine porphyrins (Py)Co(III)(TPP)(NO2) occurs. By means of IR spectroscopy and use of nitrogen oxide isotopomers, it is shown that an oxo-transfer reaction occurs from 5-coordinate species to free nitric oxide (NO) while the 6-coordinate complex is rather inert. It is also demonstrated that the stepwise addition of low-pressure NO2 to nitrosyl complex Co(TPP)(NO) leads to formation of the nitro complex most likely by an exchange reaction.     

Synthesis of substituted 5-oxo-1-thiocarbamoyl-3-pyrazoline-4-alkanoic acid derivatives

Cyclization of the 4-methyl-3-thiosemicarbazone of diethyl acetylsuccinate (1a) by the action of ammonium hydroxide followed by acidification afforded ethyl 3-methyl-1-methylthiocarbamoyl-5-oxo-3-pyrazoline-4-acetate (11a). Pmr spectral analyses using shift reagent, Eu(fod)₃, in deuteriochloroform indicated the presence also of approximately 15% of a second tautomer, ethyl 5-hydroxy-3-methyl-1-(methylthiocarbamoyl)pyrazole-4-acetate (11a'). 3-Methyl-1-methyl-thiocarbamoyl-5-oxo-pyrazoline-4-acetamide (11b) was prepared by extending the reaction time of 1a with ammonium hydroxide. Alkaline hydrolysis of 11a provided the corresponding acid 3-methyl-1-methylthiocarbamoyl-5-oxo-3-pyrazoline-4-acetie acid (11c). Regeneration of 11a was achieved by the reaction of ethyl 3-methyl-5-oxo-3-pyrazoline-4-acetate (IV) with methyl isothiocyanate. The latter reaction provided confirmation of structure for 11a. The preparation of other pyrazolin-5-ones by cyclization of thiosemicarbazones of ethyl formylsuccinate and ethyl acetylglutarate also is presented. All spectra are in accord with the proposed structures.     

Azinotriazines. I. Synthesis and spectral data of pyrido[3,2-e] -as-triazines

A series of pyrido[3,2-e]-as-triazines (VI) was prepared via acid-catalyzed cyclization of suitable 2-substituted 3-aminopyridines obtained by reduction of the corresponding 3-nitro-pyridines. Cyclization of 3-amino-2-hydrazinopyridines (III) with triethyl orthoformate and hydrochloric acid, or cyclodehydration of 2-(2-acetylhydrazino)-3-aminopyridines (IV) with alcoholic hydrogen chloride gave 1,2-dihydropyrido[3,2-e]-as-triazine hydrochloride (V). Mild dehydrogenation with alkaline potassium ferricyanide yielded heteroaromatic pyrido[3,2-e]-as-triazines (VI), for which ultraviolet and nmr spectral data are reported.     

DIFFERENTIAL EXPRESSION OF PYRIMIDINE DIMER-BINDING PROTEINS IN NORMAL AND UV LIGHT-TREATED VERTEBRATE CELLS

Abstract— The expression of UV damage-specific DNA-binding proteins was examined in various phylogenetically distant species with differing DNA repair phenotypes. Two distinct constitutive DNA-binding activities, one specific for cyclobutane pyrimidine dimers and the other for non-cyclobutane dimer photoproducts, were detected. The expression of these binding activity was found to be variable throughout the animal kingdom: cold-blooded vertebrastes show a constitutive cyclobutance dimer-binding activity excusively, and primates reveal only non-cyclobutane expression (rather than the constitutive presence)of these UV damage-specific DNA-binding activities after UV traeatment correlate with the cell's capacity for DNA repair. In addition, cyclobutane pyrimidine dimer-binding activities could be detected only in cells with eestablished photoreactivating activity