Weakening of the Z-disks of skeletal muscle myofibrils contributes to the tenderization of meat during post-mortem aging. To elucidate the weakening mechanism, we compared Z-disks weakened by post-mortem aging of chicken breast muscle with those of myofibrils treated with a solution containing 0.1 mM CaCl2 and 1 microM calpastatin domain I. In both cases, the Z-disks were weakened with a corresponding liberation of their constituent phospholipids (PLs). The liberation of PLs specific to 0.1 mM calcium ions was minimal at pH 6.5 and maximal at 35 degrees C together with the Z-disk weakening. Binding of calcium ions to PLs in the Z-disks was determined by 45Ca-autoradiography. Acidic PLs were strongly radioactive and neutral PLs were appreciably radioactive. It is very probable that acidic PLs would bind electrostatically to alpha-actinin under physiological conditions, and that this interaction would be broken by the binding of calcium ions at 0.1 mM to PLs, resulting in the partial liberation of PLs from Z-disks. We conclude, therefore, that the liberation of PLs by the binding of 0.1 mM calcium ions was the main cause for Z-disk weakening during the post-mortem aging of chicken.
A randomized crossover study in healthy young Japanese showed no significant effects of a 0.6% energy trans fatty acid (TFA) intake on the serum cholesterol concentrations and parameters of glucose metabolism. The results indicate that TFAs at this dietary level may have no adverse metabolic effects on healthy young Japanese.
Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.
Burn scar contracture that follows the healing of deep dermal burns causes severe deformation and functional impairment. However, its current therapeutic interventions are limited with unsatisfactory outcomes. When we treated deep second-degree burns in rat skin with activin-like kinase 5 (ALK5) inhibitor A-83-01, it reduced wound contraction and enhanced the area of re-epithelialization so that the overall time for wound closing was not altered. In addition, it reduced myofibroblast population in the dermis of burn scar with a diminished deposition of its biomarker proteins such as α-SMA and collagen. Treatment of rat dermal fibroblast with A-83-01 inhibited transforming growth factor-β1 (TGF-β1)-dependent induction of α-SMA and collagen type I. Taken together, these results suggest that topical application of ALK5 inhibitor A-83-01 could be effective in preventing the contraction of burn wound without delaying the wound closure by virtue of its inhibitory activity against the TGF-β-induced increase of myofibroblast population.
We have reported the construction of 1 Mb reduced genome Escherichia coli MGF-01 by a 28-step operation. This time, transcriptome analysis of MGF-01 was performed. Although the transcriptome profiles of the exponential phase in parental strain W3110red were well-conserved in MGF-01, the rspAB operon was highly expressed. A LacZ reporter assay of a series of stepwise deletion strains prepared in the course of MGF-01 construction indicated that rspA was highly expressed after the 5th step. Further analysis indicated that Δ29, one of the deleted regions at the 5th step, relates to an increase in rspA expression, and that transcriptional regulator ydfH, in the Δ29 region, is responsible for the expression of rspA, gel shift assay indicated that YdfH bound directly to the upstream region of rspA. Based on these results, it was concluded that YdfH is a transcriptional repressor of the rspAB operon.
To elucidate the effects of Lilac LAB (Bacillus coagulans lilac-01 and okara [soy pulp] powder) on bowel movements/fecal properties, we conducted a double-blind placebo-controlled randomized trial with healthy Japanese volunteers with a tendency for constipation (n = 297). The subjects ingested 2 g/d placebo (okara powder) or test food (Lilac LAB, 1 × 10(8) CFU) once a day for 2 weeks. In the test group of functionally constipated subjects, the changes in the average scores of self-reported fecal size, sensation of incomplete evacuation, and defecation frequency were significantly improved compared to the placebo group (p < 0.05), and fecal color and odor tended to improve (p = 0.07). In the test food group of all subjects and among the non-functionally constipated subjects, the fecal size tended to improve compared to the placebo group (p = 0.06, p = 0.07, respectively). Lilac LAB was effective in improving bowel movements and fecal properties in functionally constipated persons.
The structure of siccanol, a phytotoxic sesterterpene of fungal origin, was analyzed after chemical conversion by NMR spectroscopy. Siccanol was found to be an epimer of terpestacin that has been isolated from Arthrinium sp., and was thus renamed 11-epiterpestacin. Its stereochemistry was also identical with that of fusaproliferin, a structurally related mycotoxin from Fusarium proliferatum. Therefore, this sesterterpene may also be referred to as 24-deacetyl fusaproliferin. The phytotoxicity of 11-epiterpestacin was almost equal to that of terpestacin, but significantly higher than that of fusaproliferin.
Bacillus megaterium strain NK84-0218 produces a potent antiviral antibiotic, oxetanocin A, which has an oxetanosyl-N-glycoside linkage to an adenine moiety. However, the oxetanocin A productivity of the original strain was unstable and low. In this study, oxetanocin A productivity and resistance was shown to be lost simultaneously when a 51.5-kb plasmid, pOXT1, was cured during cultivation. The deficiency of oxetanocin A productivity and resistance was restored by re-introduction of the pOXT1 plasmid into the cured strain. By a cloning experiment it was shown that a 6.8-kb BglI-D fragment of the pOXT1 plasmid was responsible for oxetanocin A productivity and resistance.
Urolith, which consists of dirty yellow-colored attachments on the toilet bowl, is associated with a variety of odorous chemicals, including ammonia, and causes disadvantages in daily life. Although largely it is derived from microorganisms, little is known about the microbial processes underlying the formation of urolith. In order to gain insight into the types and the activities of microorganisms present in urolith, culturable bacteria were isolated, identified, and physiologically characterized. One of the isolates exhibited higher ability to produce ammonia when it was grown in artificial urine medium. Phylogenetic and physiological analyses indicated that this strain (T-02) belonged to a new group of Staphylococcus species, showing combined phenotypes as between S. lentus and S. xylosus. T-02 exhibited high urease activity and was capable of growing in the urinary condition by forming robust biofilms. The results of this study indicate that T-02 has successfully adapted itself to the environment of urolith.
The new inhibitors of 3alpha-hydroxysteroid dehydrogenase, 0231A 1 and 0231B 2, have a unique benz[c,d]indol-3(1H)-one structure in their molecules. In our advanced studies on indole chemistry, we have developed an efficient synthetic method for benz[c,d]indol-3(1H)-one derivatives. We report here its application to the synthesis of 0231B in 10 steps (8.1% overall yield) from 6-methylindole 8 by introducing an acyl group into the 3-position of the indole nucleus, cyclization of the side chain at the 3-position to the 4-position and subsequent elimination of the phenyl group, and conjugate addition of the substituted phenyl group.
Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83% after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The Km for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other.
The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.
The asymmetric biohydrolysis is described of o-nitrostyrene oxide with high selectivity by whole cells of Aspergillus niger CGMCC 0496. Both the epoxide and diol could be obtained in a high state of optical purity (over 98%). Product inhibition was found when using a high ratio of substrate to cells.
3-Methylaspartase (3-methylaspartate ammonia-lyase, EC 220.127.116.11) from two facultative anaerobes from soil, Citrobacter sp. strain YG-0504 and Morganella morganii strain YG-0601, were purified and crystallized from their crude extracts. Both of the Citrobacter and Morganella enzymes appeared to be a dimer of subunits of M(r) 40,000 and 44,000, respectively. The enzymes had similar enzymological properties: optimum pH for the deamination reaction of (2S,3S)-3-methylaspartic acid, substrate specificity, inhibitor, divalent and monovalent cation requirement, and N-terminal amino acid sequence homology. However, some differences were detected in pH and temperature stability, optimum pH for the amination reaction of mesaconic acid, optimum temperature, specific activity, and stability during electrophoresis. Both enzymes had similar enzymological properties to the known 3-methylaspartase from an obligate anaerobic bacterium, Clostridium tetanomorphum H1, except kinetic constants and substrate specificities.
An intracellular beta-D-xylosidase from Candida utilis IFO 0639 was purified to homogeneity through four chromatographic steps. The molecular mass of the enzyme was estimated to be 92 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.6, and was most active at pH 6.0 and at around 40 degrees C. Ethanol at an optimal concentration (10%, v/v) stimulated the initial enzyme activity by 57%. D-Xylose, the product of the beta-D-xylosidase, has no effect on the enzyme activity at 300 mM. The beta-D-xylosidase was highly specific to the beta-D-xylopyranoside configuration. The enzyme hydrolyzed beta-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 5 by releasing xylose from the non-reducing end. It showed no activity against xylan. The enzyme efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. The fermentation of Muscat juice coupled with the enzyme addition produced a small increase in the concentration of monoterpenols.
We previously isolated Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF). In order to investigate the mechanism of this degradation of BTF, we performed proteomic analysis of cells grown with or without BTF. Three proteins, which resemble dioxygenase pathway enzymes responsible for isopropylbenzene degradation from Rhodococcus erythropolis BD2, were induced by BTF. Genomic PCR and DNA sequence analysis revealed that the Rhodococcus sp. 065240 carries the gene cluster, btf, which is highly homologous to the ipb gene cluster from R. erythropolis BD2. A mutant strain, which could not catalyze BTF defluorination, was isolated from 065240 strain by UV mutagenesis. The mutant strain had one mutation in the btfT gene, which encodes a response regulator of the two component system. The defluorinating ability of the mutant strain was recovered by complementation of btfT. These results suggest that the btf gene cluster is responsible for degradation of BTF.
The radical scavenging mechanisms for the 2-pyrone compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (1), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (4) in several solvent systems were evaluated by the quantitative change in compounds detected at 270 nm and subsequent HPLC analyses. The HPLC profile for each condition suggested that the reaction proceeded by a different mechanism in each solvent system. In organic solvents (CHCl3, iso-propanol, and EtOH), 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo-2H-pyran-3-yl) phenyl]-1-phenyl-2-picrylhydrazine (2) was produced as an adduct of the DPPH radical and 1. On the other hand, the reaction in a buffer solution (an acetate buffer at pH 5.5) gave several degradation products with 1[4-(2,3-dihydro-2,5-dimethyl-3-oxo-fur-2-yl) phenyl]-1-phenyl-2-picrylhydrazine (5), this being structurally elucidated by spectroscopic analyses. The decrease of the DPPH radical in each reaction system suggests that compound 1 could scavenge about 1.5-1.8 equivalents of the radical in organic solvents and about 3.5-3.9 in the buffer solution.
In the course of our work into the use of cane by-products, we have studied the isolation and structural determination of bioactive compounds in sugarcane molasses. In this study, three stereo isomers of syringyl glycerol 3'-O-beta-D-glucopyranoside, three stereo isomers of guaiacyl glycerol 3'-O-beta-D-glucopyranoside, a syringyl glycerol 2'-O-beta-D-glucopyranoside, tachioside and a 2,3-dihydro-3,5-dihydroxy-6-methyl-4-(H)-pyran-4-one (DDMP) were isolated from the 25% methanol eluate by Amberlite XAD-2 column chromatography of sugarcane molasses. The structures of these compounds were determined on the basis of spectroscopic evidence. These isolated compounds were examined for their scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical species, and for their inhibitory activity against mushroom tyrosinase. All of the isolated compounds showed DPPH radical scavenging activity, while DDMP and tachioside showed mushroom tyrosinase inhibitory activity.
An HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) is reported. The activity was evaluated by measuring the decrease of DPPH detected at 517 nm. By using this novel method, we determined the free radical-scavenging activity of several antioxidants: ascorbic acid, alpha-tocopherol, Trolox, and cysteine. The results gave good correlation between the radical-scavenging activity determined by HPLC and by conventional colorimetry. This methodology was applied to determine the free radical-scavenging activity of 8 beverages. The activity of coffee was the highest, followed by red wine, green tea, oolong tea, black tea, rosé wine, white wine, and orange juice. The results well agree with those of previous reports. This method is expected to be useful for a simple and rapid determination of free radical-scavenging activity in colored foods, because coloring substances in foods do not interfere with the measurement.
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.
Guided by their DPPH radical-scavenging activity, nine compounds were isolated from soybean miso. Of these, 8-hydroxydaidzein, 8-hydroxygenistein and syringic acid had as high DPPH radical-scavenging activity as that of alpha-tocopherol. The antiproliferative activity of four of the isolated isoflavones toward three cancer cell lines was examined. 8-Hydroxygenistein showed the highest activity (IC50=5.2 microM) toward human promyelocytic leukemia cells (HL-60).
The effects of dietary 0.2% inositol stereoisomers on the hepatic lipids and myo-inositol (MI) status in rats fed with 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) were investigated. Dietary MI reduced the hepatic lipids in the rats fed with DDT. Dietary D-chiro-inositol (DCI) and L-chiro-inositol (LCI) both had a promoting effect on the increase in hepatic lipids due to DDT feeding. Dietary MI enhanced the hepatic free MI level and the phosphatidylinositol/phosphatidylcholine ratio, but dietary DCI reduced the level and ratio.
We analyzed the kinetics and metabolic pathways of trichloroethylene and 1,1,1-trichloroethane degradation by the ethane-utilizing Mycobacterium sp. TA27. The apparent Vmax and Km of trichloroethylene were 9.8 nmol min(-1) mg of cells(-1) and 61.9 microM, respectively. The apparent Vmax and Km of 1,1,1-trichloroethane were 0.11 nmol min(-1) mg of cells(-1) and 3.1 microM, respectively. 2,2,2-trichloroethanol, trichloroacetic acid, chloral, and dichloroacetic acid were detected as metabolites of trichloroethylene. 2,2,2-trichloroethanol, trichloroacetic acid, and dichloroacetic acid were also detected as metabolites of 1,1,1-trichloroethane. The amounts of 2,2,2-trichloroethanol, trichloroacetic acid, chloral, and dichloroacetic acid derived from the degradation of 3.60 micromol trichloroethylene were 0.16 micromol (4.4%), 0.11 micromol (3.1%), 0.02 micromol (0.6%), and 0.02 micromol (0.6%), respectively. The amounts of 2,2,2-trichloroethanol, trichloroacetic acid and dichloroacetic acid derived from the degradation of 1.73 micromol 1,1,1-trichloroethane were 1.48 micromol (85.5%), 0.22 micromol (12.7%), and 0.02 micromol (1.2%), respectively. More than 90% of theoretical total chloride was released in trichloroethylene degradation. Chloral and 2,2,2-trichloroethanol were transformed into each other, and were finally converted to trichloroacetic acid, and dichloroacetic acid. Trichloroacetic acid and dichloroacetic acid were not degraded by strain TA27.
In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC50 values of 9.1 microM and 23.4 microM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. alpha-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.
The β-xylosidase, which is active against plant complex type N-glycans, was purified to homogeneity from Ginkgo biloba seeds. The N-terminal amino acid sequence, G-S-A-A-G-N-R-, of the Ginkgo β-xylosidase (β-Xyl'ase Gb) was consistent with the deduced internal amino acid sequence of an Arabidopsis β-xylosidase (AtBXL1). β-Xyl'ase Gb hydrolyzed the β1-2 xylosyl residue from Xylβ1-2Manβ1-4GlcNAcβ1-4GlcNAc-PA and Xylβ1-2Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA, but not that from Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-PA.
(1S,2S,5R,6S)-6-(3,4-Methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-1,2-diol ((+)-1-hydroxysamin 1) was synthesized, starting from olefin 8. Stereoselective alpha-hydroxylation was achieved after converting 8 to aldehyde 13. Resulting unstable alpha-hydroxy aldehyde 14 was then transformed to (+)-1-hydroxysamin (1). This is a new efficient synthetic route to 1,2-oxygenated 6-arylfurofuran lignans.
A fungus producing an alpha-glucosidase that synthesizes alpha-1,3- and alpha-1,2-linked glucooligosaccharides by transglucosylation was isolated and identified as Paecilomyces lilacinus. The cell-bound enzyme responsible for the synthesis was extracted by suspension of mycelia with 0.1 M phosphate buffer (pH 8.0), and the extract was purified. The molecular weight and the isoelectric point were estimated to be 54,000 and 9.1, respectively. The enzyme was most active at pH 5.0 and 65 degres C. The enzyme hydrolyzed maltose, nigerose, and kojibiose. The enzyme also hydrolyzed soluble starch and amylose with the rate toward maltose. p-Nitro-phenyl alpha-glucoside and isomaltose were not good substrates. The enzyme had high transglucosylation activity to synthesize oligosaccharides containing alpha-1,3- and alpha-1,2-linkages. At an early stage of the reaction, considerable maltotriose, 4-O-alpha-nigerosyl-D-glucose, and 4-O-alpha-kojibiosyl-D-glucose were synthesized. Afterwards, nigerose and kojibiose were accumulated gradually with glucose as an acceptor.
The tphA1 II and tphA2 II A3 II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1 II and TphA2 II A3 II, indicating that TPADO consists of a reductase (TphA1 II) and a terminal oxygenase component (TphA2 II and TphA3 II). TphA1(II) contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1 II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe2+ was required for it. The Km values for TPA and the Vmax were determined to be 72+/-6 microM and 9.87+/-0.06 U/mg respectively.
4-[2-(3,5-Dimethoxyphenyl)ethenyl]-1,2-benzenediol (7), a stilbene isolated from Sphaerophysa salsula, was synthesized from 3,4-dihydroxybenzaldehyde (1) in five steps in an overall yield of 33%. The spectral data for synthetic 7 are in good agreement with those of the natural product. Hydroxystilbene 7 showed potent antioxidative activity.
(1R,2S)-1-(3'-Chloro-4'-methoxyphenyl)-1,2propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.
A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP_766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.
This study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2-dimethylhydrazine (10 mg/kg body weight) for the initial 10 wk. Dietary supplemental sericin caused a 62% reduction in the incidence of colonic adenoma (P<0.05), but did not affect the incidence of colonic adenocarcinoma. Sericin intake significantly reduced the number of colon adenomas. Consumption of sericin significantly reduced the BrdU labeling index of colonic proliferating cells and the expression of colonic c-myc and c-fos. The levels of colonic 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and inducible nitric oxide synthase protein were significantly suppressed by sericin. The results suggest that dietary sericin suppresses the development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production.
A new lactarane sesquiterpene, 1,2-dehydrolactarolide A (1), together with lactarorufin A (2), 3-O-ethyllactarolide A (3) and 3-O-ethyllactarolide B (4), was isolated from a mushroom of the Russulaceae family, Lactarius vellereus. Additionally, two lactarane sesquiterpenes, lactarorufin A (2) and lactarolide A (5) were isolated from L. subpiperatus. 1,2-Dehydrolactarolide A (1) showed promotional activity (152% at 3.6 x 10(1) microM) toward radicle elongation in lettuce seedlings.
Y-700, 1-[3-cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid, is a newly synthesized inhibitor of xanthine oxidase. This study found that feeding of Y-700 suppressed the development of colonic aberrant crypt foci, precursor lesions of colon cancer, and cell proliferation in 1,2-dimethylhydrazine-treated mice, accompanied by reduced serum urate. These results suggest that Y-700 is a useful agent for the prevention of colon tumorigenesis and that xanthine oxidase plays an important role in the development of colon cancer.
1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-alpha-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.
(3R)-3-[(1R)-1-(tert-Butyldimethylsilyl)oxy-1-(2-methoxy-4,5-methylenedioxyphenyl) methyl]-2-methylene-4-butanolide, which is a key intermediate for the synthesis of 1,2-oxidized furofuran lignan, was stereoselectively synthesized from L-glutamic acid by applying direct methylenation to butanolide.
The two-subunit meta-cleavage enzyme, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked. SDS-PAGE and gel filtration showed that CarB was a alpha2beta2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb. The optimum pH for activity was 8.5 and that of temperature was 35 degrees C. The CarB enzyme had a Km of 14 microM and a kcat/Km of 0.25 microM(-1) s(-1) for 2'-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives. The enzyme was originally isolated as a meta-cleavage enzyme for 2'-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.
When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-alpha-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5-7.0 and was stable at pH 6.0-9.0. The enzyme hydrolyzed the alpha-(1-->2)- L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I <O-alpha-L-fucose-(1-->2)-O-beta-D-galactose-(1-->3)-N-acetyl-O-beta-D- glucosamine-(1-->3)-O-beta-D-galactose-(1-->4)-D-glucose>, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze alpha-(1-->3)-, alpha-(1-->4)- and alpha-(1-->6)-L-fucosidic linkages in LNF-III <O-beta-D-galactose-(1-->4)[O- alpha-L-fucose-(1-->3)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose>, LNF-II <O-beta-D-galactose-(1-->3)[O-alpha-L- fucose-(1-->4)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose> or 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine.
Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.
Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes. A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol.
Effects of the dietary addition of orotic acid to a diet containing casein as a sole protein source on lipid levels in the liver and serum, activities of antioxidant enzymes in the liver, and some enzyme activities in serum, were compared with other diets containing egg protein, soy protein, or wheat gluten, respectively. 1. The contents in the liver of each lipid were increased by the addition of orotic acid as compared with those values without it. The orotic acid added to the casein diet caused accumulation of more liver total lipids, triacylglycerol, 1,2-diacylglycerol, and phospholipids than those fed three other diets. 2. The addition of orotic acid to the casein, but not to the other three diets, lowered the activities of liver superoxide dismutase and increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, the significant increase in serum ornithine carbamoyltransferase activities as the marker of liver lesions may result from the marked accumulation of liver lipids, decreased activities of hepatic superoxide dismutase, and the increased level of hepatic 1,2-diacylglycerol, followed by possibly the increased level of superoxide anion and increased activity of protein kinase C in rats fed the casein diet with orotic acid added.
We investigated the effect of dietary wheat bran (Wb) on colonic tumorigenesis soon after a single administration of 1,2-dimethylhydrazine (DMH). Rats that had been fed on either a fiber-free diet or a 20% Wb diet were injected with 1,2-dimethylhydrazine (20 mg/kg body weight). At 6h, 12h, 1d, 3d, or 7d after the injection, the colorectum was excised for histological analyses. The number of crypt cells more rapidly recovered in the 20% Wb group than in the fiber-free group after its temporary reduction by injection of DMH. At 6h after the DMH treatment, the apoptotic cells were significantly greater in number in the fiber-free group than in the 20% Wb group. In contrast, those in distal colon were significantly fewer in the fiber-free group than in the 20% Wb group at 7d after the treatment. These results suggest that the ingestion of Wb affected the turnover of colonic epithelial cells and would thereby bring about a protective effect against DMH-induced tumorigenesis.
The frequency of appearance of aberrant crypt foci (ACF) in the distal colon was significantly lower in rats fed a high fiber (20% wheat bran) diet than in those fed a fiber-free one at 4 weeks after a single injection of 1,2-dimethylhydrazine (DMH, 20 mg/kg), although crypt/ACF was high in the former relative to the latter. This result suggests that dietary wheat bran effectively serves as a regulator of ACF frequency at early stages after DMH injection.
Using a superoxide (O(2)(-)) generation assay system with differentiated HL-60 cells, 1,2-di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol (DLGG) was identified as an O(2)(-) generation inhibitor from Perilla frutescens var. crispa (a local variety, kida-chirimen shiso). DLGG suppressed the O(2)(-) level in a dose-dependent manner with an IC(50) value of 21 µM, comparable to those of rosmarinic acid (RoA, IC(50) = 29 µM) and caffeic acid (CA, IC(50) = 30 µM). While RoA and CA also dose-dependently inhibited O(2)(-) generation in a xanthine-xanthine oxidase system, DLGG had no effect in the same system. Thus DLGG appeared to decrease the O(2)(-) level in the HL-60 assay system by mechanisms different from those of RoA and CA, which appeared to act as O(2)(-) scavengers.
UDP-N-Acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) is an essential enzyme in the conversion of high mannose type oligosaccharide to the hybrid or complex type. The full length of the rat GnT-I gene was expressed in the filamentous fungus Aspergillus oryzae. A microsomal preparation from a recombinant fungus (strain NG) showed GnT-I activity that transferred N-acetylglucosamine residue to acceptor heptaose, Man(5)GlcNAc(2). The N-linked sugar chain of alpha-amylase secreted by the strain showed a peak of novel retention on high performance liquid chromatography that was same as a reaction product of in vitro GnT-1 assay. The peak of oligosaccharide disappeared on HPLC after beta-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan(5)GlcNAc(2) as a sugar chain of alpha-amylase from strain NG. Chimera of GnT-I with green fluorescent protein (GFP) showed a dotted pattern of fluorescence in the mycelia, suggesting localization at Golgi vesicles. We concluded that GnT-1 was functionally expressed in A. oryzae cells and that N-acetylglucosamine residue was transferred to N-glycan of alpha-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.
Aspergillus saitoi 1,2-alpha-mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T(m) values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 degrees C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.
2-C-Methyl-D-erythritol (A) and 2-C-methyl-L-threitol (B) were respectively synthesized from D-glucose and D-galactose. The 2-C-methyl-1,2,3,4-butanetetrol compound (C) recently isolated from Phlox sublata L was confirmed to be A by comparing the CD and 1H-NMR spectra of its tri-O-benzoate with those of A and B.
Stereoisomers of the tetrahydro-beta-carboline derivative, 1-(2-pyrrolidinethione)-3-yl)-1,2,3,4-tetrahydro-beta-carboline- 3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied. The isomers were identified as (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction. Both (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks.
A variety of 4-aryl- and 4-alkyl-3-(substituted benzylthio)-4H-1,2,4-triazoles were prepared and evaluated for their bleaching activity by the lettuce seedling test. Among the series of tested compounds, 4-(3-fluorophenyl)-3-(4-trifluoromethylbenzylthio)-4H-1,2,4-triazole (39) exhibited the highest bleaching activity, causing complete bleaching symptoms at 10 microM. In the dark condition, compound 39 inhibited the formation of such carotenoids as beta-carotene, violaxanthin, neoxanthin and lutein, resulting in the formation of zeta-carotene, phytoene, phytofluene and beta-zeacarotene, which were not detected in the untreated control. Treatment by compound 39 at 50 microM resulted in the amount of accumulated zeta-carotene being seven-fold higher than that of phytoene, phytofluene and beta-zeacarotene. These results suggest that compound 39 might have interfered with desaturation, especially zeta-carotene desaturation, during carotenoid biosynthesis.
We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.