Prooxidant Activities of Chlorophylls and Pheophytins on the Photooxidation of Edible Oils

The effect of chlorophyll (Chl) and pheophytin (Phy) on the photooxidation of triglycerides was examined. Prooxidant behavior similar to that noted in previously reported experiments using methyl linoleate as a substrate was observed. The prooxidant activity of Phy was found to be higher than that of Chl. Moreover, Chl b accelerated photooxidation to a greater degree than did Chl a, and Phy b was more active than Phy a.

From the compositional variation of Chl and Phy during photooxidation, it was found that Phy was stable during oxidation of oils. These facts suggest that the Phy content must be noted when considering the oxidative stability of edible oils.     

Specific tritium labeling of glucosyl- and galactosylceramides at the 6-position of the carbohydrate moiety using CrO3-graphite

A new procedure for introducing tritium into the carbohydrate portions of glucosyl- and galactosylceramides was developed using a new catalyst, CrO3-graphite, which specifically oxidizes the primary alcohol group to the aldehyde. About 10% of the glycolipid was converted to the aldehyde and the aldehyde produced was then reduced back to the original form with KB3H4. After methanolysis, more than 96.7% of the radioactivities of [3H]glucosyl- and [3H]galactosylceramides were found to be located in the carbohydrate portions, and the specific activities of the [3H]galactosyl- and [3H]glucosylceramides were 2.08 to 4.30 X 10(4) cpm/nmol, which could be increased greatly by purifying the aldehydes and reducing them with KB3H4. In addition, beta-galactosidase activity was successfully determined with [3H]galactosylceramide as the enzyme substrate; the Km was 18.73 mM and the Vmax was 11.63 nmol/mg/h, indicating that no significant structural modification occurs during the oxidation.     

Inter- and intrasyngenic variations of the hemoglobin components in Paramecium caudatum.

The ciliated protozoan Paramecium contains hemoglobin in heterogeneous monomeric forms. In particular, Paramecium caudatum is characterized by the presence of a major component called Hb10 and a basic component named bHb. We found that in P. caudatum both of these hemoglobin components show some variation according to stock. The types and distributions of these hemoglobin components were examined on 16 stocks in five different syngens and one stock in an unidentified syngen using high performance liquid chromatography. The results indicate that in a variety of stocks the major component, Hb10, was divided into three types, A, B or A + B, and that the basic hemoglobin component was composed of a combination of two or three variants out of four possible, i.e. bHb 1, bHb 2, bHb 3 and bHb 4. Neither the Hb10 types nor the bHb variants, however, could be used to distinguish syngen in P. caudatum, since all of the Hb10 types and bHb variants were widely distributed over syngens and identical profiles appeared to some stocks in different syngens.     

Synthesis of the sugar moiety of TIME-EA4, a glycopeptide isolated from silkworm diapause eggs

We describe the efficient synthesis of the tetrasaccharide, 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,4-di-O-acetyl-3-O-allyl-beta-D-mannopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl azide, which is the protected form of the sugar unit of TIME-EA4 that is isolated from the diapausing eggs of the silkworm, Bombyx mori. The beta-linked D-mannoside of the tetrasaccharide was obtained using the conventional oxidation-reduction method for inversion of the configuration at the C-2 hydroxyl group of beta-D-glucoside. The reduction was effected with NaBH(4) in a methanolic solution in a ratio of 98:2 in favor of the beta-D-mannoside that was obtained in 87% yield.     

Enzymatic polymerization of coniferyl alcohol in the presence of cyclodextrins

The dehydrogenative polymerization of coniferyl alcohol by horseradish peroxidase was performed in 0.10 M phosphate buffer at 27 degrees C. Dehydrogenative polymer (DHP) from coniferyl alcohol was characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. The ratio of 8-O-4':8-5':8-8' linkages was determined by the 1H NMR spectrum of DHP acetate which had good solubility. In "end-wise like" polymerization (the slow addition of hydrogen peroxide), addition of alpha-cyclodextrin to the medium led to DHP with increased 8-O-4' content and a decrease in 8-5' linkages. Under higher pH conditions, DHP with higher 8-O-4' and 8-5' content was obtained in the presence of alpha-cyclodextrin. In the end-wise polymerization (the slow additions of coniferyl alcohol and hydrogen peroxide), using alpha-cyclodextrin also gave DHP with a 8-O-4' richer structure than that prepared in no additive system. The analysis of thioacidolysis products from DHP supported the results of the alpha-cyclodextrin effects on the 8-O-4'-rich structure of DHP. The 8-O-4' structure in DHP prepared in the presence of alpha-cyclodextrin had racemic form as shown by ozonation.     

The Photoxidative Alteration of Chlorophylls in Methyl Linoleate and Prooxidant Activity of Their Decomposition Products

Methyl linoleate containing chlorophylls and/or pheophytins was exposed to light in the presence of oxygen. The photooxidative reaction of both chlorophylls a and b was first-order, and the reaction rate for chlorophyll a was higher than that for chlorophyll b. On the other hand, pheophytins a and b hardly decomposed even after irradiation for 24 hr, and retained a green or a brownish-green color. In qualitative analysis of the photooxidation products of chlorophylls a and b, no pheophytins or pheophorbides were detected, while green and polar red pigments were observed on a thin layer chromatogram near the spot of chlorophyll and the origin, respectively. These photooxidation compounds also had prooxidant effects as well as did chlorophyll.     

Characterization of calcium ion sensitive region for β-mannanase from Streptomyces thermolilacinus

Despite the widespread industrial applications of β-mannanase, the relations between the enzymatic properties and metal ions remain poorly understood. To elucidate the effects of metal ions on β-mannanase, thermal stability and hydrolysis activity were characterized. The stman and tfman genes encoding β-mannanase (EC.3.2.1.78) from Streptomyces thermolilacinus NBRC14274 and Thermobifida fusca NBRC14071 were cloned and expressed in Escherichia coli. The thermal stability of each enzyme shifted to the 7-9°C high temperature in the presence of Ca(2+) compared with that in the absence of Ca(2+). These results show that the thermal stability of StMan and TfMan was enhanced by the presence of Ca(2+). StMan, but not TfMan, required Ca(2+) for the hydrolysis activity. To identify the Ca(2+) sensitive region of StMan, we prepared eight chimeric enzymes. Based on the results of the relationship between Ca(2+) and hydrolysis activity, the region of amino-acid residues 244-349 of StMan was responsible for a Ca(2+) sensitive site.     

Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ([1-14C]N-acetylmannosamine) and a radioactive precursor of ceramide ([3,3-3H2]serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.