Bioscience Biotechnology and Biochemistry

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.

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.

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.

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 4.3.1.2) 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.

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.

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).

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.

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.

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.