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X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis., Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.

We have conducted a study to compare various preparation methods, including a certain number of new methods, to find which ones are best suited to the morphological evaluation of nerve fibers in the pyramidal tract of the human medulla oblongata. Our main concern was to find fixation and staining methods that would minimize errors, especially regarding the tissue shrinkage ratio and the ease of staining. From the two fixation methods we examined, the most satisfactory was the secondary chromic acid fixation (which gave the best overall results when followed by nitrocellulose embedding), as it gives the lowest shrinkage ratio with the narrowest range (10 +/- 0%). Among the ten staining methods we tested, we found that the most suitable for morphological evaluation were the discriminative staining methods (Luxol fast blue-Periodic acid-Schiff-Hematoxylin stain, Masson-Goldner-Goto method and modified Hematoxylin-Eosin stain) and the silver impregnation methods (Luxol fast blue-Silver impregnation and Luxol fast blue-Silver impregnation-Periodic acid-Schiff-Hematoxylin).

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS 1632 , ISB27, ISBj8, and IS 1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster ( nifDKENXSBZHQW-fixBCX ) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB . ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.

Although phytoremediation is a promising method for the removal of cadmium (Cd) from contaminated soil, plants remove Cd from soil slowly. To increase Cd uptake by plants, the effects of the application of chloride ions (Cl−) and a decrease in soil pH on the amount of Cd taken up by plants were investigated in a pot experiment using soil collected from paddy fields contaminated with Cd from mining wastewater. Three treatments were applied (all quantities were expressed on a 350 g soil basis): control (0.6 g (NH4)2SO4 as N fertilizer), Cl− application (0.5 g NH4Cl as N fertilizer and 0.15 g CaCl2) and low pH (0.8 g powdered sulfur plus NH4Cl and CaCl2). Sunflower (Helianthus annuus), kenaf (Hibiscus cannabinus) and sorghum (Sorghum vulgare) plants were grown in the treated soil for 33 days in a growth chamber under a 26°C/24°C day/night (16 h/8 h) regime. At harvest, the plants were separated into roots, stems and leaves, and the Cd concentration was determined. The application of Cl− approximatel...

To clarify the genetic basis to the diversity of denitrifying ability in soybean bradyrhizobia, we compared the end-products of denitrification (N2, N2O and NO2 � ) with the existence of denitrifying genes (napA, nirK, norCB and nosZ) of sixty phylogenetically diverse strains of Bradyrhizobium japonicum and B. elkanii. The results indicate that the existence of denitrifying genes directly determines phenotype (end-products) in most strains of B. japonicum. The denitrifying capability and gene set were reflected by phylogenetic position based on repeated sequences (RS)-fingerprints and 16S rRNA gene sequences. However, the denitrifying genes in HRS (highly repeated sequence-possessing) strains of B. japonicum, which were identified based on RS-fingerprints as having heavy hybridization, resulted in an inconsistent correlation probably because of genomic rearrangements. The evolutionary and ecological implications of the denitrifying genes and capability in soybean bradyrhizobia are discussed.

To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new 15 N-labeled N 2 detection methodology, which is free from interference from atmospheric N 2 contamination. 30 N 2 ( 15 N 15 N) and 29 N 2 ( 15 N 14 N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N 2 gas having natural abundance of 15 N (0.366 atom%) as a carrier gas. The detection limit was 0.04% 30 N 2 , and the linearity extended at least to 40% 30 N 2 . When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with 15 NO 3 − , denitrification product ( 30 N 2 ) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.

Using a preparation method composed of secondary chromic acid fixation, nitrocellulose embedding and Luxol fast blue-PAS-hematoxylin stain (a discriminative staining method) for the purpose of axonal discrimination, we examined 43 human spinal cords (31 males and 12 females) at the C5 level to find the differences due to gender and aging in axons of the lateral corticospinal tract. These results can be of great help in understanding the motor functions in relation to gender differences and the aging process.

Indirect enzyme-linked immunosorbent assays (ELISAs) have been applied to detect immunoglobulin Y antibodies to different serotypes of Salmonella in the yolks of chicken eggs with heat-extracted antigens of Salmonella enterica serotypes Agona (SA), Cerro (SC), Enteritidis (SE), Montevideo (SM), and Putten (SP). The egg yolk samples examined were classified as positive if their ELISA absorbance values exceeded the value for eggs from specific-pathogen-free flocks by more than two standard deviations. Of 30 egg yolk samples from three flocks vaccinated with a killed SE vaccine, 29 were antibody positive by the ELISA assay for the SE antigen. Four to 29 of the 29 yolk samples showed positive results for the other serovars, although the absorbance values for SE were higher than those obtained for the other serotypes in each of the yolk samples. All 30 yolks from three flocks that were not administered any SE vaccines were found to be antibody negative for SE, and two samples were determined to be positive for SC. Thirty-nine or 40 eggs were obtained from each of four layer flocks in a commercial egg production farm where the laying houses were naturally contaminated with SA, SC, SM, SP, Salmonella serovar Infantis (SI), and untypeable strains. The ELISA absorbance values for SM in the egg yolks obtained from the two flocks molted through feed withdrawal when the birds restarted laying were significantly (P < 0.05) higher than those observed in the yolks obtained before the molt. In egg yolks from the two other flocks that were molted through a wheat bran diet, there was no significant difference between the absorbance values before and after the molt. The observations in the present study provide further evidence to suggest that a molt initiated through the administration of a wheat bran diet can reduce the risk for Salmonella problems in a commercial egg-producing setting.

N 2 O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N 2 O uptake and conversion of 15 N-N 2 O into 15 N-N 2 . Free-living cells of USDA110 showed N 2 O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N 2 O reductase activity. When detached soybean nodules formed with USDA110 were fed with 15 N-N 2 O, they rapidly emitted 15 N-N 2 outside the nodules at a ratio of 98.5% of 15 N-N 2 O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N 2 O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N 2 O (0.34 ppm). These results indicate that the conversion of N 2 O to N 2 depends exclusively on the respiratory N 2 O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N 2 O.

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex.