A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N¹-acetylspermidine (N¹AcSpd), N⁸-acetylspermidine (N⁸AcSpd), N¹-acetylspermine, N¹,N⁸-diacetylspermidine, and N¹,N¹²-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N¹AcSpd and N⁸AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with ¹³C₂-N¹AcSpd and ¹³C₂-N⁸AcSpd which have the ¹³C₂-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N¹-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N¹-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-¹⁵N₃]-N¹-acetylspermine and [1,4,8-¹⁵N₃]spermidine (¹⁵N₃-Spd), respectively; for SMO, [1,4,8,12-¹⁵N₄]spermine and ¹⁵N₃-Spd, respectively; and for SSAT, ¹⁵N₃-Spd and [1,4,8-¹⁵N₃]-N¹-acetylspermidine, respectively.     

Effect of water content on the solid-state stability in two isomorphic clathrates of cephalosporin: cefazolin sodium pentahydrate (alpha form) and FK041 hydrate

This study clearly demonstrates that clathrated water molecules can contribute to both chemical stabilization and destabilization of clathrates. The solid-state stabilities for two isomorphic clathrates of cephalosporin, cefazolin sodium and FK041, were investigated in terms of the effects of water content. The isomorphic ranges of water content were estimated to be 3.5-5 mol/mol for alpha-form cefazolin sodium and 2-4 mol/mol for FK041 hydrate. Upon the isomorphic dehydration, alpha-form cefazolin sodium was destabilized as the water content decreased below 4.25 mol/mol owing to the disruption of hydrogen bonding network in lattice channels. In this case, the hydration of clathrated water up to 4.25 mol/mol contributed to the physical and chemical stability of the crystals. On the contrary, the isomorphic hydration in FK041 hydrate contributed to the chemical destabilization owing to the high water activity. The difference in water activity between alpha-form cefazolin sodium and FK041 hydrate could be attributed to the size of water channels.     

Studies on chemical carcinogens and mutagens. XXVII alkylating property of mutagenic N-trimethylsilylmethyl-N-nitrosourea,a silicon-analogue of N-neopentyl-N-nitrosourea,in aqueous media

The kinetics for hydrolysis and the chemoselectivity toward nucleophiles of mutagenic N-trimethylsilylmethyl-N-nitrosourea, a silicon analogue of N-neopentyl-N-nitrosourea, were studied.     

Effects of 2-(E-2-decenoylamino)ethyl 2-(cyclohexylethyl) sulfide on various ulcer models in rats.

The effects of 2-(E-2-decenoylamino)ethyl 2-(cyclohexylethyl) sulfide (compd. III-1a) on various experimental ulcers were investigated. The oral administration of compd. III-1a at doses ranging from 30 to 300 mg/kg inhibited the acute gastric ulcerations induced by ethanol, HCl.aspirin and indomethacin in rats. Compound III-1a significantly inhibited the water immersion stress-induced gastric ulcer at doses of 3 mg/kg, p.o. The anti-ulcer activity of plaunotol as a reference drug was equivalent on an ethanol-induced ulcer to that of compd. III-1a, but weaker on HCl.aspirin, indomethacin and stress-induced ulcers than that of compd. III-1a. On indomethacin-produced gastric antral ulcer, compd. III-1a showed the same significant inhibitory activity as spizofurone did at a dose of 100 mg/kg, p.o. Compound III-1a also inhibited hemorrhagic shock-, diethyldithiocarbamic acid (DDC)-and platelet activating factor (PAF)-induced ulcers dose-dependently. Plaunotol only showed significant inhibitory activity on PAF-induced ulcer in these three ucler models. The consecutive administration of compd. III-1a (100 mg/kg, p.o.) twice a day significantly accelerated the healing of an acetic acid-induced ulcer and that of plaunotol (200 mg/kg, p.o.) showed the same activity. Moreover, orally administered compd. III-1a at a dose of 100 mg/kg decreased the gastric acid secretion in pylorus-ligated rats. The results in the present study suggest that compd. III-1a has the dual action on ulcer formation.     

Lignified materials as a potential medicinal resource. IV. Dehydrogenation polymers of some phenylpropenoids and their capacity to stimulate polymorphonuclear cell iodination.

Based on our recent finding regarding diverse biological activities of natural lignified materials, synthetic dehydrogenation homo- and copolymers were prepared using 3 p-hydroxylated cinnamic acids and coniferyl alcohol in order to explore the role of lignin skeleton in the activities displayed by natural lignins. The synthetic polymers stimulated polymorphonuclear cell iodination as potently as the natural lignified materials.     

Formation of O6,7-dimethylguanine residues in calf thymus deoxyribonucleic acid treated with carcinogenic N-methyl-N-nitrosourea in vitro

Treatment of calf thymus deoxyribonucleic acid (DNA) in vitro with a methylating carcinogen, N-methyl-N-nitrosourea (MNU), in phosphate buffer (pH 7.2) resulted in formation of O6,7-dimethylguanine residues in DNA besides the well-known methylated DNA adducts, 7-methylguanine, O6-methylguanine and 3-methyladenine. The product ratio (%) of O6,7-dimethylguanine versus 7-methylguanine was 0.32 after one MNU treatment. The significance of formation of O6,7-dimethylguanine residues in DNA is discussed briefly in relation to the carcinogenicity of MNU.     

Studies on lens-aldose-reductase inhibitor in medicinal plants. II. Active constituents of Monochasma savatierii Franch. et Maxim

The 70% acetone extract of Monochasma savatierii FRANCH. et MAXIM. showed very strong inhibition of rabbit lens aldose reductase (AR). From the active fraction, five iridoid glucosides along with the two phenolic glycosides, acteoside and dehydroacteoside, have been isolated. Among them, acteoside showed the highest activity, being about 2.5 times more potent than baicalein, a known natural inhibitor of AR (IC50 = 9.8 x 10(-7) M). Demethylmussaenoside and 7-O-acetyl-8-epi-loganic acid, which are iridoid glucosides, had weak inhibitory activity.     

Some cytotoxicological aspects of ethyl and fluoroethyl alkanesulfonates in Escherichia coli: role of fluorine substitution

Several fluoroethyl derivatives of alkanesulfonates and N-nitrosourea were tested for cytotoxicity and mutagenicity in E. coli K12 AB1157. Cytotoxicity was potentiated by fluorine substitution in the alkyl moiety of the ethylating agents. Mutagenicity was strongly suppressed by fluorine substitution in the alkanesulfonates, but not in the N-nitrosourea. The capacity to induce the SOS repair network was suppressed, as was mutagenicity, in alkanesulfonates, but not in N-nitrosourea. The potentiating effect of fluorine on the cytotoxicity of alkanesulfonates seems to be due to an as yet unknown killing mechanism. An appreciable suppressive effect on the mutagenicity and the SOS induction is worth notice for the biological role of fluorine substitution in alkylating agents.