Purification and characterization of chitinase A of Streptomyces cyaneus SP-27: an enzyme participates in protoplast formation from Schizophyllum commune mycelia

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.     

Role of acetyl-L-carnitine in the brain: revealed by Bioradiography

To elucidate the role of acetyl-l-carnitine in the brain, we used a novel method, ‘Bioradiography,’ in which the dynamic process could be followed in living slices by use of positron-emitter labeled compounds and imaging plates. We studied the incorporation of 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) into rat brain slices incubated in oxygenated Krebs-Ringer solution. Under the glucose-free condition, [¹⁸F]FDG uptake rate decreased with time and plateaued within 350 min in the cerebral cortex and cerebellum, and the addition of 1 or 5 mM acetyl-l-carnitine did not alter the [¹⁸F]FDG uptake rate. When a glutaminase inhibitor, 0.5 mM 6-diazo-5-oxo-l-norleucine (DON), was added under the normal glucose condition, [¹⁸F]FDG uptake rate decreased. Acetyl-l-carnitine (1 mM), which decreased [¹⁸F]FDG uptake rate, reversed this DON-induced decrease in [¹⁸F]FDG uptake rate in the cerebral cortex. These results suggest that acetyl-l-carnitine can be used for the production of releasable glutamate rather than as an energy source in the brain.     

Continuous treatment with organic nitrate affects hepatic cytochrome P450

We previously reported that cytochrome P450 (P450) is a key enzyme of organic nitrate biotransformation and that P450 levels of the heart and its vessels markedly decreased at the development of nitrate tolerance. Escape from tolerance of organic nitrate by induction of cytochrome P450. Most organic nitrates, including nitroglycerin (NTG), are metabolized in the liver, where nitric oxide (NO) is concomitantly produced from the organic nitrates. Therefore, organic nitrate administration may also affect hepatic P450 levels, since the liver is the major organ of P450-related metabolism. Male Wistar rats were intravenously administrated NTG or isosorbide dinitrate (ISDN) for 24-96 h. Hepatic P450 was drastically decreased after 48 h or 72 h of continuous NTG or ISDN infusion, when nitrate tolerance was observed, but it recovered 48 h after cessation of the drug administration. hemeoxygenase-1 (HO-1) was induced within 24 h of continuous NTG infusion, but it returned to normal levels 48 h after cessation of the NTG. The administration of sodium nitroprusside, an agent to which the animals showed no tolerance, did not induce HO-1 or P450 depletion as judged by SDS-PAGE in combination with Western-blotting. These results suggest that P450-dependent drug metabolism may be drastically affected after continuous organic nitrate administration.     

53BP2S, interacting with insulin receptor substrates, modulates insulin signaling

It is well known that insulin receptor substrates (IRS) act as a mediator for signal transduction of insulin, insulin-like growth factors, and several cytokines. To identify proteins that interact with IRS and modulate IRS-mediated signals, we performed yeast two-hybrid screening with IRS-1 as bait. Out of 109 cDNA-positive clones identified from a human placental cDNA library, two clones encoded 53BP2, p53-binding protein 2 (53BP2S), a short form splicing variant of the apoptosis-stimulating protein of p53 that possesses Src homology region 3 domain, and ankyrin repeats domain, and had been reported to interact with p53, Bcl-2, and NF-kappaB. Interaction of 53BP2S with IRS-1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays in COS-7 cells and 3T3-L1 adipocytes. The Src homology region 3 domain and ankyrin repeats domain of 53BP2S were responsible for its interaction with IRS-1, whereas the phosphotyrosine binding domain and a central domain (amino acid residues 750-861) of IRS-1 were required for its interaction with 53BP2S. In CHO-C400 cells, expression of 53BP2S reduced insulin-stimulated IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation. In addition, the amount of the phosphatidylinositol 3-kinase regulatory p85 subunit associated with tyrosine-phosphorylated proteins, and activation of Akt was enhanced by 53BP2S expression. Although 53BP2S also enhanced Akt activation in 3T3-L1 adipocytes, insulin-induced glucose transporter 4 translocation was markedly inhibited in accordance with reduction of insulin-induced AS160 phosphorylation. Together these data demonstrate that 53BP2S interacts and modulates the insulin signals mediated by IRSs.     

Synthesis of a burkholdine analogue containing β-hydroxytyrosine

Synthesis of a β-OHTyr-containing Bk analogue, a cyclic octalipopeptide with antifungal activities, is described. Since β-OHTyr-containing peptides generally are unstable in strong acidic conditions, synthesis of β-HOTyr-containing peptides by SPPS have rarely been reported. To overcome this problem, we found that using distilled TFA removed the protecting groups of side chains of β-OHTyr-containing Bk analogue, which was prepared by Fmoc-SPPS.

Abbreviations: β-OHTyr: β-hydroxytyrosine; β-OHAsn: β-hydroxyasparagine; Bk: burkholdine; FAA: fatty acyl amino acid; β-MeOTyr: β-methoxytyrosine; SPPS: solid phase peptide synthesis; MIC: minimun inhibitory concentration; DMF: dimethyl formamide; DIPEA: diisopropylethylamine; DIPC: diisopropylcarbodiimide; HOBt: 1-hydroxybenzotriazole; Fmoc: 9-fluorenylmethyloxycarbonyl; HFIP: 1,1,1,3,3,3-hexafluoropropan-2-ol; TFA: trifluoroacetic acid; LAP: N-lauryl ?3-amino-4-carbamolypropanoic acid; HPLC: high performance liquid chromatography; ESI-TOFMS: electrospray ionization-time of flight mass spectrometry; Bn: benzyl; Boc: t-butyloxycatbonyl; 2-CTC: 2-chlorotritylchloride.     

Influence of eye colors of Caucasians and Asians on suppression of melatonin secretion by light

This experiment tested effects of human eye pigmentation depending on the ethnicity on suppression of nocturnal melatonin secretion by light. Ten healthy Caucasian males with blue, green, or light brown irises (light-eyed Caucasians) and 11 Asian males with dark brown irises (dark-eyed Asians) volunteered to participate in the study. The mean ages of the light-eyed Caucasians and dark-eyed Asians were 26.4 +/- 3.2 and 25.3 +/- 5.7 years, respectively. The subjects were exposed to light (1,000 lux) for 2 h at night. The starting time of exposure was set to 2 h before the time of peak salivary melatonin concentration of each subject, which was determined in a preliminary experiment. Salivary melatonin concentration and pupil size were measured before exposure to light and during exposure to light. The percentage of suppression of melatonin secretion by light was calculated. The percentage of suppression of melatonin secretion 2 h after the start of light exposure was significantly larger in light-eyed Caucasians (88.9 +/- 4.2%) than in dark-eyed Asians (73.4 +/- 20.0%) (P < 0.01). No significant difference was found between pupil sizes in light-eyed Caucasians and dark-eyed Asians. These results suggest that sensitivity of melatonin to light suppression is influenced by eye pigmentation and/or ethnicity.     

S-allyl cysteine prevents CCl(4)-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50-200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

Effect of AOB, a fermented-grain food supplement, on oxidative stress in type 2 diabetic rats

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic complications. Antioxidant Biofactor (AOB) is a mixture of commercially available fermented grain foods and has strong antioxidant activity. This study investigated the effect of AOB supplementation of standard rat food on markers of oxidative stress and inflammation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with type 2 diabetes. Blood glucose, hemoglobin A1c, plasma free fatty acid, triacylglycerol and plasminogen activator inhibitor-1 (PAI-1) were significantly higher in OLETF rats than in non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats at 29 weeks. AOB (6.5% of diet) was given to rats during 29-33 weeks of diabetic phase in OLETF rats. OLETF rats with AOB supplementation showed decreased blood glucose, hemoglobin A1c, triacyglycerol, low density lipoprotein, cholesterol and PAI-1. Mitochondrial ROS production was significantly increased in heart, aorta, liver and renal artery of OLETF rats. Uncoupling protein 2 (UCP2) is known to regulate ROS production. We found aortic UCP2 protein expression increased in OLETF rats, and AOB returned UCP2 expression to normal. Aortic endothelial NO synthase (eNOS) was also increased in OLETF rats more than in LETO rats at 33 weeks. In contrast, phosphorylated vasodilator-stimulated phosphoprotein, an index of the NO-cGMP pathway, was significantly diminished. AOB increased eNOS proteins in LETO and OLETF rats. In conclusion, AOB significantly improved the NO-cGMP pathway via normalizing ROS generation in OLETF rats. The data suggest that dietary supplementation with AOB contributes to nutritional strategies for the prevention and treatment of type 2 diabetes mellitus.