Immobilization of Baeyer-Villiger monooxygenase from acetone grown Fusarium sp.

OBJECTIVE: A novel biocatalyst for Baeyer-Villiger oxidations is necessary for pharmaceutical and chemical industries, so this study aims to find a Baeyer-Villiger monooxygenase (BVMO) and to improve its stability by immobilization. RESULTS: Acetone, the simplest ketone, was selected as the only carbon source for the screening of microorganisms with a BVMO. A eukaryote, Fusarium sp. NBRC 109816, with a BVMO (FBVMO), was isolated from a soil sample. FBVMO was overexpressed in E. coli and successfully immobilized by the organic-inorganic nanocrystal formation method. The immobilization improved the thermostability of FBVMO. Substrate specificity investigation revealed that both free and immobilized FBVMO were found to show catalytic activities not only for Baeyer-Villiger oxidation of ketones to esters but also for oxidation of sulfides to sulfoxides. Furthermore, a preparative scale reaction using immobilized FBVMO was successfully conducted. CONCLUSIONS: FBVMO was discovered from an environmental sample, overexpressed in E. coli, and immobilized by the organic-inorganic nanocrystal formation method. The immobilization successfully improved its thermostability.

Rice endosperm protein slows progression of fatty liver and diabetic nephropathy in Zucker diabetic fatty rats.

We previously reported that rice endosperm protein (REP) has renoprotective effects in Goto-Kakizaki rats, a non-obese diabetic model. However, whether these effects occur in obese diabetes remains unclear. This study aimed to clarify the effects of REP on obese diabetes, especially on fatty liver and diabetic nephropathy, using the obese diabetic model Zucker diabetic fatty (ZDF) rats. In total, 7-week-old male ZDF rats were fed diets containing 20 % REP or casein (C) for 8 weeks. Changes in fasting blood glucose levels and urinary markers were monitored during the experimental period. Hepatic lipids and metabolites were measured and renal glomeruli were observed morphologically. HbA1c levels were significantly lower in rats fed REP, compared with C (P<0·05). Compared with C in the liver, REP prevented lipid accumulation (total lipid, TAG and total cholesterol, P<0·01). Liver metabolome analysis indicated that levels of metabolites associated with glycolysis, the pentose phosphate pathway and carnitine metabolism were significantly greater in the REP group than in the C group (P<0·05), suggesting activation of both glucose catabolism and fatty acid oxidation. The metabolite increases promoted by REP may contribute to suppression of liver lipid accumulation. Urinary excretion of albumin and N-acetyl-ß-d-glucosaminidase was significantly reduced in rats fed REP for 8 weeks (P<0·01). In addition, there was a distinct suppression of mesangial matrix expansion and glomerular hypertrophy in response to REP (P<0·01). Thus, REP had preventive effects on obese diabetes, fatty liver and diabetic nephropathy.

Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes.

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception.

Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl ß-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl ß-D-glucopyranoside arabinosylation to produce HexVic in planta.

First syntheses of (-)-tauranin and antibiotic (-)-BE-40644 based on lipase-catalyzed optical resolution of albicanol.

First syntheses of sesquiterpene quinones (-)-tauranin and (-)-BE-40644 which exhibited strong cytotoxicity against several cancer cell lines, were achieved from (8aS)-albicanol obtained by enzymatic optical resolution. By comparison of the sign of specific rotation between synthetic (12bS)-BE-40644 and natural (-)-BE-40644, the absolute configurations of natural (-)-BE-40644 were determined to be 4aS, 6aS, 12aR, 12bS.

Structural Revision of a Naphthodipyranodione from Gentian Root and its Degradation Pathway from Gentiopicroside.

Based on NOE experiments, the structure of naphthodipyranodione from Gentianaceae plants was revised to 1,2-dihydro-4H,6H,8H-naphto[1,2-d:4,5- c'd']dipyrano-4,8-dione. Naphthodipyranodione was assumed to be formed by the degradation of gentiopicroside by enzymatic hydrolysis at low water -concentration. The degradation pathway was a unique domino-reaction triggered by enzymatic hydrolysis. Naphthodipyranodione may become an index compound for the drying and/or fermenting procedure of Gentian root.

Randomized, double-blind, placebo-controlled, parallel-group study of the effect of Lactobacillus paracasei K71 intake on salivary release of secretory immunoglobulin A.

Lactobacillus paracasei K71 was shown to be effective in alleviating the severity of atopic dermatitis in a randomized controlled trial, and a preliminary open-label trial suggested that strain K71 intake enhanced secretory immunoglobulin A (sIgA) release in the saliva. This study investigated the effect of K71 on sIgA release in a randomized, double-blind, placebo-controlled, parallel-group trial. The trial included 62 Japanese subjects aged 20-64 years with relatively low rates of salivary sIgA release. Subjects (n=31 in each group) were randomly given a tablet containing 100 mg (approximately 2 × 1011 bacteria) of K71 or a placebo tablet daily for 12 weeks. After eliminating data for eight subjects (four in each group) who met the exclusion criteria for efficacy analysis, data for 54 subjects were analyzed. The change in the rate of salivary sIgA release 8 weeks after initiation of the study compared with baseline was significantly higher in the K71 tablet group (105.5 ± 119.0 µg/min) than in the placebo group (52.7 ± 62.6 µg/min; p=0.047). There were no adverse events associated with intake of tablets containing K71. The safety of intake of L. paracasei K71 was also confirmed in an independent open-label trial with 20 healthy subjects who consumed excessive amounts of K71-containing food. L. paracasei K71 intake may therefore have some benefits in promoting mucosal immune function.

A Randomized, Double-Blind, Crossover Pilot Trial of Rice Endosperm Protein Supplementation in Maintenance Hemodialysis Patients.

In maintenance hemodialysis (MHD) patients, low protein intake is associated with protein-energy wasting, a risk factor that affects outcome. However, increased protein intake may lead to hyperphosphatemia and hyperkalemia, which are also mortality risk factors. Here, we evaluated the safety and effects of purified rice endosperm protein (REP), which contains less phosphorus and potassium than soy and casein proteins, as a supplemental protein source for MHD patients. This randomized, double-blind, placebo-controlled, crossover pilot study of REP supplementation (5 g/day × 4 weeks) was carried out in 50 Japanese adult MHD patients (1 dropped out); the primary outcome was the change in the urea kinetic-based normalized protein catabolic rate (nPCR), an indicator of protein intake in MHD patients. Intention-to-treat analyses of 24 patients in the REP-first group and 25 in the placebo-first group showed that REP supplementation increased nPCR significantly by 0.07 g/kg/day (95% confidence interval, 0.03-0.11), whereas changes in serum phosphorus and potassium concentrations were not different from the placebo. REP supplementation did not show a significant effect on other nutritional or metabolic parameters and no specific complications. In conclusion, purified REP with efficient bioavailability may be safe and useful for dietary supplementation in MHD patients.

Anxiolytic effect of Gardeniae Fructus-extract containing active ingredient from Kamishoyosan (KSS), a Japanese traditional Kampo medicine.

Kamishoyosan (KSS), a Kampo formula used to treat menopausal psychotic syndromes in women, consists of ten crude herbal drugs. The anxiolytic effect of KSS was investigated by the social interaction (SI) test using mice, and whether the effect of KSS was due to the stimulating and/or sedating effects was examined by the open field locomotion test. Furthermore, the present study examined the effect of individual crude drugs in KSS by the SI test to clarify its active components. Oral administration of KSS increased the total SI time in a dose-dependent manner (50--200 mg/kg), but this effect was not observed over a dose of 300 mg/kg. On the other hand, there were no significant changes observed for the open field locomotion test. These results suggest that the appearance of KSS-induced SI behavior is due to an anxiolytic effect. The unaltered results of the open field test indicated that KSS was neither a stimulant nor sedative. To identify the essential herbs in KSS, the effects of "the component herbs in KSS" and "KSS minus one component herb" using the SI test were examined. An increase in the SI time was observed for hot water extracts of Menthae herba and Gardeniae Fructus, the same as for the KSS treatment. On the other hand the effect of KSS on the SI time was reduced to the control level for KSS minus Gardeniae Fructus, KSS minus Paeoniae Radix, KSS minus Glycyrrhizae Radix and KSS minus Hoelen. Oral administration of Gardeniae Fructus-extract or its common constituent, geniposide increased the SI time in a dose-dependent manner. These results indicate that Gardeniae Fructus and geniposide play a role in the anxiolytic effect of KSS.

Rice protein hydrolysates stimulate GLP-1 secretion, reduce GLP-1 degradation, and lower the glycemic response in rats.

Rice has historically been consumed in Asia as a major source of carbohydrates, however, little is known regarding the functional roles of rice proteins as dietary factors. In the present study, we investigated whether peptides derived from rice proteins could stimulate GLP-1 secretion, which results in reducing glycemia via the incretin effect in normal rats. Hydrolysates were prepared from the protein fraction of rice endosperm or rice bran, and the effects of these hydrolysates on GLP-1 secretion were examined in a murine enteroendocrine cell line GLUTag. Plasma was collected after oral administration of the rice protein hydrolysates, under anesthesia, or during glucose tolerance tests in rats. In anesthetized rats, plasma dipeptidyl peptidase-IV (DPP-IV) activity was measured after ileal administration of the rice protein hydrolysates. GLP-1 secretion from GLUTag cells was potently stimulated by the rice protein hydrolysates, especially by the peptic digest of rice endosperm protein (REPH) and that of rice bran protein (RBPH). Oral administration of REPH or RBPH elevated plasma GLP-1 concentrations, which resulted in the reduction of glycemia under the intraperitoneal glucose tolerance test. In addition, the plasma DPP-IV activity was attenuated after ileal administration of REPH or RBPH, which resulted in a higher ratio of intact (active) GLP-1 to total GLP-1 in the plasma. These results demonstrate that rice proteins exert potent stimulatory effects on GLP-1 secretion, which could contribute to the reduction of postprandial glycemia. The inhibitory effect of these peptides on the plasma DPP-IV activity may potentiate the incretin effect of GLP-1.

Topical nipradilol: effects on optic nerve head circulation in humans and periocular distribution in monkeys.

PURPOSE: To investigate the effects of topical nipradilol on blood velocity in the optic nerve head (ONH) in normal humans and the ocular and periocular distribution of topically instilled nipradilol in monkeys. METHODS: In normal humans, 0.25% nipradilol was instilled in one eye and vehicle in the other twice daily for 7 days, and blood velocity in the ONH was measured by the laser speckle method. In monkeys, after a single instillation of 1% [(14)C]nipradilol in one eye, distribution of radioactivity was evaluated by whole-head autoradiography. RESULTS: Twice-daily 7-day instillation of nipradilol temporarily but significantly increased human ONH blood velocity, in the ipsilateral eye only (P = 0.005), independent of a reduction in the intraocular pressure. In monkeys, equivalent nipradilol concentration in the periocular tissue around the optic nerve insertion was higher on the ipsilateral side than on the contralateral side (140 +/- 25 ng/g and 42 +/- 10 ng/g, P = 0.022, n = 5). Radioactivity was higher in the periocular tissue behind the equator than around the optic nerve insertion on the ipsilateral side (P = 0.004), but not on the contralateral side. The equivalent nipradilol concentration in the ipsilateral posterior retina-choroid was 636 +/- 92 ng/g, which was significantly higher than that on the contralateral control side (521 +/- 92 ng/g, P < 0.001). CONCLUSIONS: The ipsilateral increase in ONH blood velocity induced by topical nipradilol in humans was attributed to drug that penetrated locally. Whole-head autoradiographic study suggests that topically instilled nipradilol can rapidly reach the posterior periocular tissue at pharmacologic concentrations.

Cloning of the pepX gene of Lactobacillus helveticus IF03809 encoding salt-tolerant X-prolyl dipeptidyl aminopeptidase and characterization of the enzyme.

X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactobacillus helveticus IF03809 expressed nearly full activity under high salt conditions, such as 2 M NaCl. We cloned and sequenced the pepX gene for X-PDAP. The calculated M, of deduced X-PDAP (803 amino acids) was 90,847 and the protein was distantly related (35 to 44% identity) to known X-PDAPs of Lactobacillus sp. including L. helveticus CNRZ32 (40% identity). Native and recombinant X-PDAP were purified to homogeneity from both L. helveticus IF03809 and Escherichia coli DH5alpha harboring the pepX gene on a plasmid, respectively. The native enzyme appeared to be a dimer of 220 kDa, as estimated by gel filtration column chromatography. It hydrolyzed an X-prolyl-linkage, but not prolyl- or X-prolyl-X-peptide bonds, and tolerated up to 2 M NaCl as well as some other chlorides of monovalent cations. Determination of the flanking sequences revealed two divergent genes. The upstream region of the pepX gene encodes oppA gene for a putative oligopeptide permease, while the downstream region encodes tnp gene specifying a possible transposase of the IS3 family. The oppA gene shares a 176 bp-promoter region with pepX in the intergenic region, implying a relationship between this oligopeptide transport system and X-PDAP.

Beneficial effect of a diet containing heat-killed Lactobacillus paracasei K71 on adult type atopic dermatitis.

The purpose of this study was to investigate the clinical effect of a supplementary diet containing heat-killed lactic acid bacterium Lactobacillus paracasei K71 (LAB diet) on adult patients with atopic dermatitis (AD). A randomized, double-blind, placebo-controlled study was conducted in 34 adult type AD subjects who were treated with conventional topical corticosteroid and tacrolimus. LAB diet or placebo was added over 12 weeks. The primary end-point was the clinical severity of AD which was evaluated by a severity scoring system proposed by the guideline of the Japanese Dermatological Association. The effect was also secondarily evaluated by itch scores of visual analog scales (VAS), quality-of-life (QOL) impairment scores of Skindex 16 and consumption amounts of topical therapeutics. Data on these four assessment variables were collected at baseline and at week 4, 8 and 12. Within the study population, the skin severity scores were significantly decreased from baseline at week 8 (P<0.05) and at week 12 (P<0.01) in the LAB diet group but not in the placebo group. Influence of LAB diet on itch scores or QOL impairment scores was not evident. The consumption of topical therapeutics in the placebo group was 1.9-times greater in total amount compared with the corresponding value in the LAB diet group during the intervention period, although there was no significant difference. No LAB diet- or placebo-related adverse events were observed. We concluded that the LAB diet may have some benefits as a complementary therapy for adult AD patients who are managed with the conventional treatment.

Synthesis of ß-methoxyacrylate natural products based on box-Pd(II)-catalyzed intermolecular methoxycarbonylation of alkynoles.

Bis(oxazoline)-palladium(II) catalyzed carbonylation of homopropargyl alcohols afforded acyclic methoxyacrylate 2 and 6-membered lactone 3a-k in good combined yield. In the case of propargyl alcohols, 5-membered lactones 3p, 3q, 16 were obtained in moderate yields. The one-pot synthesis of kawa lactones 3a, 3r, 3s and formal synthesis of dihydroxycystothiazole A and dihydroxycystothiazole C are presented. To elucidate the stereochemistry of (+)-annularin G and (-)-annularin H, the first asymmetric syntheses of these natural products were achieved.

Antidiabetic effect of orally administered conophylline-containing plant extract on streptozotocin-treated and Goto-Kakizaki rats.

Conophylline, a vinca alkaloid from Ervatamia microphylla, is known to induce the differentiation of pancreatic precursor cells to insulin-producing cells. In the present research we examined the antidiabetic effects of this alkaloid in vivo by oral administration. Crude conophylline preparations were prepared from the leaves of Tabernaemontana divaricata collected in Okinawa Prefecture, Japan. Conophylline was orally absorbed and showed an increase in its plasma level in both normal and streptozotocin-induced diabetic Sprague-Dawley rats. The plasma conophylline concentration reached its maximum from 1.5 to 3h after a single oral administration and gradually decreased in 24h. The alkaloid decreased the blood glucose level and increased the plasma insulin level in streptozotocin-induced diabetic rats after repetitive administration for 15 days. Fasting blood glucose levels in rats treated orally with conophylline at 0.11 and 0.46 mg/kg/day were 411+/-47 and 381+/-65 mg/dl, respectively; whereas the glucose level of the control rats was 435+/-46 mg/dl. Conophylline also decreased the fasting blood glucose level in Goto-Kakizaki rats in a dose-dependent manner after repetitive administration for 42 days. These results suggest that the extract from conophylline-containing leaves may be useful as a functional food for the treatment of type 2 diabetes mellitus.

Concise syntheses of coronarin A, coronarin E, austrochaparol and pacovatinin A.

Total syntheses of (+)-coronarin A (1), (+)-coronarin E (2), (+)-austrochaparol (3) and (+)-pacovatinin A (4) were achieved from the synthetic (+)-albicanyl acetate (6). Dess-Martin oxidation of (+)-albicanol (5) derived from the chemoenzymatic product (6) gave an aldehyde (7), which was subjected to Julia one-pot olefination using beta-furylmethyl-heteroaromatic sulfones (8 or 9 ) gave (+)-trans coronarin E (2) and (+)-cis coronarin E (12) with high cis-selectivity. The synthesis of (+)-coronarin A (1) from (+)-trans coronarin E (2) was achiev-ed, while (+)-cis coronarin E (12) was converted to the natural products (+)-(5S,9S,10S)-15,16-epoxy-8(17),13(16),14-labdatriene (13) and (+)-austrochaparol (3). By the asymmetric synthesis of (+)-3, the absolute structure of (+)-3 was determined to be 5S, 7R, 9R, 10S configurations. Homologation of (+)-albicanol (5) followed by allylic oxidation gave (7 alpha)-hydroxy nitrile (17), which was finally converted to the natural (+)-pacovatinin A (4) in 8 steps from (+)-albicanol (5).

Control of chemoselectivity of microbial reaction with resin adsorbent: enhancement of Baeyer-Villiger oxidation over reduction.

Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer-Villiger (BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded, and BV oxidation was scarcely found in the absence of the polymer.

Chemoenzymatic synthesis of sacranosides a and B.

Direct beta-glucosidation between (-)-myrtenol and nerol and D-glucose (3) using the immobilized beta-glucosidase from almonds with the synthetic prepolymer ENTP-4000 gave myrtenyl O-beta-D-glucoside (4) and neryl O-beta-D-glucoside (10), respectively. The coupling of the myrtenyl or neryl O-beta-D-glucopyranoside congeners (7 or 13) and 2,3,4-tri-O-benzoyl-beta-L-arabinopyranosyl bromide (8) afforded the coupled products (9 or 14), respectively. Deprotection of the coupled products (9 or 14) afforded the synthetic myrtenyl 6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside (Sacranoside A, 1) or neryl 6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside (Sacranoside B, 2), respectively.

Chemoenzymatic synthesis of naturally occurring benzyl 6-O-glycosyl-beta-D-glucopyranosides.

Direct beta-glucosidation between benzyl alcohol and D-glucose (5) using the immobilized beta-glucosidase from almonds with the synthetic prepolymer ENTP-4000 gave a benzyl beta-D-glucoside (1) in 53% yield. The coupling of the benzyl beta-D-glucopyranoside congener (8) derived from 1 with phenyl 2,3,4-tri-O-acetyl-1-thio-beta-D-xylopyranoside (9), ethyl 2,3,4-tri-O-acetyl-1-thio-alpha-L-rhamnopyranoside (13), and 2,3,4-tri-O-acetyl-alpha-L-arabinopyranosyl bromide (15) afforded 10, 14, and 16, respectively, as coupled products. Deprotection of 10, 14, and 16 provided the synthetic benzyl beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranoside (2), benzyl alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (3), and benzyl alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside (4), respectively.