Enhancement of In Vivo Antioxidant Ability in the Brain of Rats Fed Tannin

The effect of the oral administration of mimosa tannin (MMT) on the rat intra-hippocampal antioxidant ability was examined. Wistar rats at the age of 6 weeks were reared for 8 weeks with the rodent diet (RD) consisting of 0.1 g/kg of MMT (RD–MMT). The antioxidant ability of rat brain was evaluated from the decay of a brain–blood-barrier permeable stable nitroxide, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM) measured by the microdialysis-electron spin resonance system under a freely moving state. The decay rate of PCAM in the brain of rats fed RD–MMT was significantly larger than that of rats fed control rodent diet, which indicates the increase of the antioxidant ability in the brain of rats fed RD–MMT. In vitro study showed that MMT did not reduce PCAM directly but enhanced the reduction of PCAM by ascorbic acid. These results indicate that MMT is a potent antioxidant in vitro and in vivo.     

Role of mitochondrial Bax, caspases, and MAPKs for ceramide-induced apoptosis in renal proximal tubular cells

It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3–6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.     

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0–5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.     

Calcium-Dependent Interaction Sites of Tropomyosin on Reconstituted Muscle Thin Filaments with Bound Myosin Heads as Studied by Site-Directed Spin-Labeling

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca²⁺ ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca²⁺. These findings suggest that even in the myosin-bound (open) state, Ca²⁺ may regulate actomyosin contractile properties via Tm.     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.     

Red Pigment Formation by the Reaction of Oxidized Ascorbic Acid and Protein in a Food Model System of Low Moisture Content

Ascorbic acid-protein mixtures of low moisture content stored for 2~3 days in aerobic conditions at 60°C produced a red coloration, which was shown to have resulted from an amino-carbonyl reaction of oxidized ascorbic acid (dehydroascorbic acid, DHA) by the facts that DHA-casein or ovalbumin systems yielded a rapid red coloration in low moisture conditions as well as in an ethanol suspension. Zein showed only a weak red coloration with DHA. An apparent decrease in the free amino group, as determined by the TNBS method, was observed to be associated in parallel with the red pigment formation. The electrophoretic pattern of ovalbumin changed significantly upon incubation with DHA, together with the formation of the red pigment. The red pigment extracted from a DHA-casein system showed an absorption spectrum, TLC Rf value, and hydrolyzed products (DHA and scorbamic acid) identical to those of 2,2′-nitrilo di-2(2)-deoxy-l-ascorbic acid mono ammonium salt, produced from DHA and amino acid. Formation of the red pigment was also observed in the reaction of DHA with N_α-acetyl lysine. These results indicate that the ε-amino group of lysine in protein can be attributed to the formation of a red pigment identical with NDA.     

A Root Growth-promoting Factor,Capillarol, from Artemisia capillaris Thunb

A root growth-promoting factor was isolated from Artemisia capillaris Thunb. The chemical structure of this compound was elucidated as methyl 3-(3-methylbut-2-enoyl)-4-hydroxycinnamate (capillarol) on the basis of its spectroscopic analysis. At 5×10^–4m this compound promoted rice root growth to 180% of the control value.     

Analysis of terminal sugar moieties and species-specificities of acrosome reaction-inducing substance in Xenopus (ARISX)

The acrosome reaction of Xenopus sperm is triggered by the acrosome reaction-inducing substance in Xenopus (ARISX), an oviductal pars recta-derived, sugar-rich substance decorated on the entire surface of the vitelline envelope (VE) during ovulation. Here we addressed the functional importance of the sugar moiety in ARISX. Among various lectins examined, soybean agglutinin and Dolichos biflorus agglutinin were shown to abolish the acrosome reaction-inducing activity of ARISX present in pars recta extract or on the VE, indicating the importance of the terminal alpha-N-acetylgalactosamine residue for the function of ARISX. Consistently, the acrosome reaction-inducing activity was not affected by proteinase K digestion, in spite of the simultaneous shift of ARISX to a smaller molecular weight. Indirect immunofluorescence microscopic examinations showed that ARISX was distributed as two types of structures on VE; thick fiber-like materials and thin filamentous materials, and that a new structure appeared on the fertilization envelope instead of the thin filamentous materials. Sperm from several amphibian species were subjected to an in vitro assay during induction of the acrosome reaction with ARISX. The resulting limited population of sperm from a non-Xenopus species underwent acrosome reaction, implying a weak species-specificity of ARISX.