Increased bioavailability of diosmetin-7-glucoside-γ-cyclodextrin inclusion complex compared with diosmin in Sprague-Dawley rats.

Diosmin (DSN) is found mainly in citrus fruits, and has potent antioxidant effects. This study aimed to evaluate pharmacokinetics of diosmetin-7-glucoside-γ-cyclodextrin (DIOSG-CD) inclusion complex. The area under the curve values from AUC0-24 of DIOSG-CD, prepared by reacting DSN and naringinase with γ-CD, were approximately 800-fold higher than those of DSN following their administration in Sprague-Dawley rats.

Mutagenic, Acute, and Subchronic Toxicity Studies of the Hesperetin-7-Glucoside-ß-Cyclodextrin Inclusion Complex.

Hesperetin glucosides such as hesperidin and hesperetin-7-glucoside are abundantly present in citrus fruits and have various pharmacological properties. However, the potential toxicity of hesperetin glucosides remains unclear. An initial assessment of the safety of hesperetin-7-glucoside-ß-cyclodextrin inclusion complex (HPTGCD) as a functional food ingredient was undertaken to assess toxicity and mutagenic potential. A bacterial reverse mutation assay (Ames test) using Salmonella typhimurium (strains TA98, TA1535, TA100, and TA1537) and Escherichia coli (strain WP2 uvrA) with HPTGCD (up to 5000 µg/plate) in the absence and presence of metabolic activation was negative. In a single oral (gavage) toxicity study in male and female rats, HPTGCD at dose up to 2000 mg/kg did not produce mortality nor clinical signs of toxicity or change in body weight. In a subchronic oral (dietary admix) toxicity study in rats receiving 0, 1.5, 3, and 5% HPTGCD for 13 weeks, no adverse effects were noted and the no-observed-adverse-effect level (NOAEL) was 5% in the diet (equivalent to 3267.7 mg/kg/day for males and to 3652.4 mg/kg/day for females). These results provide initial evidence of the safety of HPTGCD.

Bioavailability comparison between a compound comprising hesperetin-7-glucoside with ß-cyclodextrin and a mixture of hesperidin and dextrin in healthy adult human males.

The pharmacokinetics of compounds comprising hesperetin-7-glucoside with ß-cyclodextrin and physically mixed hesperidin/dextrin was compared in 8 healthy adult male subjects in a nonrandomized, double-blind, cross-over, controlled study. For 0-24 h, the area under the curve of the total plasma hesperetin concentration after hesperetin-7-glucoside with ß-cyclodextrin consumption was >100-fold higher than that after hesperidin/dextrin consumption.

Distribution of hydrocarbon-degrading bacteria in the soil environment and their contribution to bioremediation.

A real-time PCR quantification method for indigenous hydrocarbon-degrading bacteria (HDB) carrying the alkB gene in the soil environment was developed to investigate their distribution in soil. The detection limit of indigenous HDB by the method was 1 × 10(6) cells/g-soil. The indigenous HDB were widely distributed throughout the soil environment and ranged from 3.7 × 10(7) to 5.0 × 10(8) cells/g-soil, and the ratio to total bacteria was 0.1-4.3 %. The dynamics of total bacteria, indigenous HDB, and Rhodococcus erythropolis NDKK6 (carrying alkB R2) during bioremediation were analyzed. During bioremediation with an inorganic nutrient treatment, the numbers of these bacteria were slightly increased. The numbers of HDB (both indigenous bacteria and strain NDKK6) were gradually decreased from the middle stage of bioremediation. Meanwhile, the numbers of these bacteria were highly increased and were maintained during bioremediation with an organic nutrient. The organic treatment led to activation of not only the soil bacteria but also the HDB, so an efficient bioremediation was carried out.

Analysis of nitrification in agricultural soil and improvement of nitrogen circulation with autotrophic ammonia-oxidizing bacteria.

Accumulations of inorganic nitrogen (NH4⁺, NO2⁻, and NO3⁻) were analyzed to evaluate the nitrogen circulation activity in 76 agricultural soils. Accumulation of NH4⁺ was observed, and the reaction of NH4⁺→ NO2⁻ appeared to be slower than that of NO2⁻ → NO3⁻ in agricultural soil. Two autotrophic and five heterotrophic ammonia-oxidizing bacteria (AOB) were isolated and identified from the soils, and the ammonia-oxidizing activities of the autotrophic AOB were 1.0 × 10³-1.0 × 106 times higher than those of heterotrophic AOB. The relationship between AOB number, soil bacterial number, and ammonia-oxidizing activity was investigated with 30 agricultural soils. The ratio of autotrophic AOB number was 0.00032-0.26% of the total soil bacterial number. The soil samples rich in autotrophic AOB (>1.0 × 104 cells/g soil) had a high nitrogen circulation activity, and additionally, the nitrogen circulation in the agricultural soil was improved by controlling the autotrophic AOBs.

Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots.

Peptide uptake by plant roots from degraded soybean-meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed-phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean-meal products for 24 h. Carboxyfluorescein-labeled root hair-promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts.

Further stabilization of Leu¹55 mutant thermolysins by mutation of an autodegradation site.

The autodegradation-resistant mutant thermolysins (TLNs), L155A (Leu(155) to Ala) and L155S (Leu(155) to Ser), were previously constructed by site-directed mutagenesis to enhance thermostability. These mutations suppressed autodegradation at position 154-155, resulting in increased thermostability. However, a new autodegradation site became apparent in these mutant TLNs, at position 155-156. In this study, further stabilization of the mutant TLNs to suppress this new autodegradation was attempted by the substitution of Ile(156) to Asp and Val (L155A-I156N, L155A-I156V, L155S-I156N, and L155S-I156V). SDS-PAGE analysis showed that the autodegradation at 155-156 of all double-mutant TLNs was suppressed. Thermostability at 80 °C was enhanced in all double-mutant TLNs (half-life at 80 °C: WT, 18.3 min; L155A, 25.0 min; L155S, 24.0 min; L155A-I156N, 60.8 min; L155A-I156V, 62.4 min; L155S-I156N, 93.3 min; and L155S-I156V, 40.0 min), and k (cat)/K (m) values were: WT, 220; L155A, 240; L155S, 123; L155A-I156N, 62; L155A-I156V, 760; L155S-I156N, 240; and L155S-I156V, 520 min(-1) mM(-1).

Expression in Escherichia coli, refolding, and purification of the recombinant mature form of human matrix metalloproteinase 7 (MMP-7).

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).

Analysis of relationship between microbial and methanogenic biomass in methane fermentation.

To analyze the relationship between biomass of microorganisms and methane production, the total biomass of bacteria and archaea (BA) during methane fermentation was analyzed by the environmental DNA analysis method. In the case of using methanogenic sludge as a seed which is generally used for methane fermentation, the total BA biomass reached to 1.5 x 10(8) to 3.6 x 10(8) cells/ml when methane was produced. On the other hand, soil suspension was used as a seed; methane was not produced for 14-day cultivation. However, the total BA biomass reached to above 1.5 x 10(8) cells/ml. The methanogen biomass was counted by using a fluorescence microscope (coenzyme F420), and the methanogen biomass and the ratio of methanogens in the total of BA were analyzed during methane fermentation. At the methane-producing phase, the methanogen biomass reached to 1.3 x 10(8) cells/ml, and the ratio of methanogens was above 70% of the total BA. When the ratio of methanogens in a seed was changed, the methane-producing phase was moved. However, the relationship between methanogens and other microorganisms at the methane-producing phase was almost similar.

Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay.

Thirty-six bacteria that degraded long-chain hydrocarbons were isolated from natural environments using long-chain hydrocarbons (waste car engine oil, base oil or the c-alkane fraction of base oil) as the sole carbon and energy source. A phylogenetic tree of the isolates constructed using their 16S rDNA sequences revealed that the isolates were divided into six genera plus one family (Acinetobacter, Rhodococcus, Gordonia, Pseudomonas, Ralstonia, Bacillus and Alcaligenaceae, respectively). Furthermore, most of the isolates (27 of 36) were classified into the genera Acinetobacter, Rhodococcus or Gordonia. The hydrocarbon-degradation similarity in each strain was confirmed by the 2,6-dichlorophenol indophenol (2,6-DCPIP) assay. Isolates belonging to the genus Acinetobacter degraded long-chain normal alkanes (n-alkanes) but did not degrade short-chain n-alkanes or cyclic alkanes (c-alkanes), while isolates belonging to the genera Rhodococcus and Gordonia degraded both long-chain n-alkanes and c-alkanes.

Effect on epidermal cell of soybean protein-degraded products and structural determination of the root hair promoting peptide.

Peptide(s) produced from degraded soybean protein by an alkaline protease from Bacillus circulans HA12 (degraded soybean-meal products; DSP) increased the number of both the root hair cells (trichoblasts) and hairless cells (atrichoblasts) of Brassica rapa by about 4.4 times and 1.9 times, respectively. To identify the root hair-promoting peptide(s) in DSP, the origin protein of the root hair-promoting peptide(s) was identified as Kunitz trypsin inhibitor (KTI). The root hair-promoting peptide in the degraded products of KTI was purified and produced a signal of 1,198.2 Da with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A search of the amino acid sequence of KTI located the peptide GGIRAAPTGNER, which had a molecular weight identical to 1,198.2 Da. The peptide GGIRAAPTGNER was chemically synthesized, and the synthetic peptide possessed root hair-promoting activity. Thus, it is concluded that this peptide in DSP is the foreign bioactive peptide promoting the differentiation of root hairs.

Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment.

To construct an efficient lipid-containing wastewater treatment system, microorganisms that degrade lipids efficiently were isolated from various environmental sources. Strain DW2-1 showed the highest rate of degradation of 1% (w/v) salad oil among the isolated strains. Strain DW2-1 was identified as Burkholderia sp. and designated Burkholderia sp. DW2-1. The rate of degradation of salad oil, olive oil, sesame oil, and beef tallow by strain DW2-1 were 96.7%, 92.3%, 90.1% and 77.4%, respectively, during a 48-h cultivation. Strain DW2-1 grew well in a synthetic wastewater medium (>1 x 10(10) colony forming unit [CFU]/ml) between 20 degrees C and 38 degrees C, and its rate of degradation of salad oil was above 90% after a 48-h cultivation. The lipase and biosurfactant (BSF) activities of strain DW2-1 after a 48-h cultivation were 1720 U/l and 480 U/ml, respectively. In continuous cultures for lipid-containing wastewater treatment, DW2-1 was stably maintained and degraded more than 90% of salad oil during a 7-d cultivation.

Isolation and characterization of a bluegill-degrading microorganism, and analysis of the root hair-promoting effect of the degraded products.

Bluegill-degrading bacteria were isolated from various environmental sources. Brevibacillus sp. BGM1 degraded bluegill efficiently at 50 degrees C, and its culture supernatant showed the highest peptide and amino acid concentrations as trichloroacetic acid (TCA) soluble fraction (ASF) (10.7 mg/ml) of all supernatants obtained with bluegill as a substrate. Strain BGM1 secreted a protease(s) into the medium, and the concentration of peptides and amino acids gradually increased. The fertile effect of the degraded bluegill products (DGP) on Brassica rapa was also investigated. The root hair density of B. rapa grown with DGP at a concentration of 30 mug peptides and amino acids/ml was about 1.7 times higher than when grown with the same concentration of undegraded bluegill. DGP was shown to increase root hair numbers and adventitious root formation. The results of this study suggest that a specific peptide(s) for promotion of root hair is produced from the order Perciformes with a protease(s) from BGM1.

Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site.

The relationship between the autodegradation and thermostability of thermolysin (TLN) was studied. Four autodegradation sites in TLN were identified in the presence of Ca(2+). One of the sites was identified as Gly(154)-Leu(155), and Leu(155) was substituted with various amino acids, X = Ala, Ser, Phe, and Gly, by site-directed mutagenesis. The thermostability at 80 degrees C increased with the amino acid substitutions in the order of Ala>Phe>Ser>Gly>Leu (WT TLN). An additional autodegradation fragment that was not observed with WT TLN appeared for all mutant TLNs examined. The autodegradation site shifted from the Gly(154)-Leu(155) bond to the X(155)-Ile(156) one with the mutation at Leu(155). Furthermore, the Ile(164)-Asp(165) bond was recognized newly as an autodegradation site in the mutant TLNs for the production of AF3'.