Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

Synthesis of daunosamine-containing disaccharides

Treatment of 2,3,6-trideoxy-1,4-di-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexopyranose (1) with benzyl 2,3-dideoxy-d-glycero-pentopyranoside and p-toluenesulfonic acid gave a mixture of benzyl 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3- (trifluoroacetamido)-l-lyxo-hexopyranoside (49%) and benzyl 2,3-dideoxy-4-O-[2,3,6-trideoxy-4-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-α-l-lyxo-hexopyranosyl]-d-glycero-pentopyranoside (4, 20 %). The structure of the disaccharide 4 was confirmed by a detailed, mass-spectrometric analysis in three modes, namely, negative- and positive-ion, chemical ionization, and electron impact. Similar treatment of the bis(p-nitrobenzoate) 1 with ethyl 2,3-dideoxy-d-glycero-pentopyranoside gave the ethyl glycoside and the desired disaccharide, showing that the transglycosylation is not restricted to benzyl glycosides. Removal of the p-nitrobenzoyl and the benzyl groups from 4 gave the disaccharide 2,3-dideoxy-4-O-(2,3,6-trideoxy-3-trifluoroacetamido-α-l-lyxo-hexopyranosyl)-d-glycero-pentopyranose.     

Synthesis of 7-(tetrahydropyran-2-yl)- and 7-(4-acetoxytetrahydropyran-2-yl)-ε-rhodomycinone and 7-(tetrahydropyran-2-yl)-ε-pyrromycinone

The ¹³C.n.m.r spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of α(1→3)- and α-(1→6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in α-(1→6)-linkages and the latter in α-(1→3)-linkages. The structural conclusions arrived at by ¹³C-n.m.r. spectroscopy are consistent with data from methylation analysis and ¹H-n.m.r. spectroscopy.     

The reduction of aldehydes by broad bean and maize alcohol dehydrogenases and a study of the substrate binding to the enzyme protein

Alcohol dehydrogenase was prepared from 2-day germinating maize and 3-day germinating broad-bean seeds by ammonium sulphate fractionation of sodium phosphate extracts, chromatography onDEAE cellulose and Sephadex G-200. The activity of the broad beanADH amounted to182 800 units per mg protein, that of maizeADH 79 000 units per mg protein. Besides oxidation of a series of alcohols at pH optimum in the alkaline region and with KM equalling 10-2M, alcohol dehydrogenases isolated from both plants catalyze the reduction of acetaldehyde, n-propanal, n-butanal, isobutanal and crotonal at pH optimum in the neutral region with KM equalling 10-3M. The inhibition studies using fatty acids and chloride ions revealed that the oxidation of alcohols is inhibited competitively by both types of inhibitors, with inhibition constants of 10-2M and 10-1M, respectively. The inhibition in the presence of acetaldehyde is non-competitive since the inhibitors do not compete with acetaldehyde and do not form an enzyme-NADH-inhibitor complex, yet they obviously react with the enzyme-NAD product only, thus giving rise to an enzyme-NAD-inhibitor complex. These differences in the behaviour of inhibitors may be interpreted in the sense that the binding sites of ethanol and acetaldehyde as substrates for broad bean and maize alcohol dehydrogenases are non equivalent. The nonequivalency discussed in the text.     

Reactions of 3-(1-arylhydrazono-L-threo-2,3,4-trihydroxybutyl)-1-methyl-2-Quinoxalinones

The 1-methyl derivatives (3 and 4) of 3-(1-phenyl- (1) and 3-(1-p-bromophenylhydrazono-L-threo-2,3,4-trihydroxybutyl)-2-quinoxalinone (2) were prepared by methylation. Periodate oxidation of 3 gave 1-methyl-3-[1-(phenylhydrazono)glyoxal-1-yl]-2-quinoxalinone (5), which, on reduction with sodium borohydride, gave the corresponding 3-[2-hydroxy-1-(phenylhydrazono)ethyl] derivative (8). Reaction of 5 with hydroxylamine or benzoylhydrazine gave the corresponding 2-oxime (6) and 2-(benzoylhydrazone) (7), respectively. Acetic anhydride causes one molecule of 3 or 4 to undergo elimination of two molecules of water, with simultaneous acetylation and ring closure to afford pyrazoles 9 and 10, respectively. Pyrolysis of the triacetate of 3 led to the elimination of acetic acid from the sugar and the hydrazone residue, to give the 3-[5-(acetoxymethyl)-1-phenylpyrazol-3-yl] derivatives (9). Acetic acid was found to effect the same rearrangement, but without acetylation, of 1, 2, and 3 to give the 3-[5-(hydroxymethyl)] derivatives 11, 12, and 13, respectively. The structure of these pyrazoles was confirmed by a series of reactions, including methylation and acetylation. The n.m.r. and i.r. spectra of the compounds were investigated.     

The use of Opalina sudafricana in a biological test for diagnosing disordered metabolism of tryptophan in human subjects

Injections of urine of patients with bladder cancer linked with bilharziasis, simple urinary bilharziasis, ascariasis or ancylostomiasis, induced cyst formation found in Opalina sudafricana when injected into its host Bufo regularis. It is suggested that the carcinogenic tryptophan metabolites present in the injected urine reach the parasites in the recta of the experimental toads and stimulate them to divide mitotically to form small forms which eventually encyst. This test may be of a diagnostic help in detecting any abnormality in tryptophan metabolism in some human patients.     

Molecular identification and determination of the trichothecene chemotypes of fungi causing crown and root in different growth stages of wheat in north of Iran

In order to determine the crown and root agents and their mycotoxins produced in different growth stages of wheat including seedling, tillering and heading, sampling was done in north of Iran, during 2011–2012. From 160 isolates of Fusarium, eight species were obtained including F. graminearum, F. culmorum, F. equiseti, F. nygamai, F. semitectum, F. solani, F. acuminatum and F. oxysporum. Sampling at different growth stages showed that F. graminearum was the predominant causal agent of crown and root at the heading stage, whereas other species of Fusarium were mostly observed at the seedling and tillering stages. Moreover, identification of pathogenic species was confirmed using species-specific primers pairs. In F. graminearum isolates, presence of Tri13 gene, responsible for nivalenol (NIV) and deoxynivalenol (DON) mycotoxins biosynthesis, was detected using specific PCR primers. Finally, the ability of trichothecene production of five F. graminearum isolates was confirmed with high-performance liquid chromatography.     

Intake and digestibility in sheep fed on alkali-treated straw and concentrates,as complete diets or separate feeds

Twelve mature wethers were used in an experiment of change-over design to determine the effect of complete diet feeding on intake and digestibility of diets based on alkalitreated straw and a concentrate containing rolled barley and extracted soya bean meal. Two diets were used: D₁ with a 50:50, and D₂ with a 75:25 ratio of straw:concentrates on a dry matter basis. The straw and concentrates of each diet were given either separately (S) or as a complete diet (C). Complete diets gave a slightly, but not significantly higher intake (C, 86.5) and S, 82.5 g dry matter /kg W^0.75 per day). The separate method gave a significantly higher digestibility coefficient for dry matter (C, 62.2 and S, 65.5; P < 0.05) and for crude fibre (C, 57.2 and S, 62.5; P < 0.01). Water intake increased by 42.7 ml g^?1 Na derived from NaOH. There were no significant interactions between the method of feeding and the type of diet in intake and digestibility of the diets or in the intake of water.