Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.     

Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.     

Further Study on the Relation of Polyoxin Structure to Chitin Synthetase Inhibition

As a part of studies on the mechanism of action of antibiotics polyoxins, effects of various N-aminoacyl derivatives of polyoxin C and other polyoxins on chitin-UDP acetylglucosaminyl-transferase (EC 2.4.1.16, chitin synthetase) prepared from phytopathogenic fungus Piricularia oryzae were investigated. It was found that they inhibited the enzyme in competition with the substrate UDP-N-acetylglucosamine, Inhibitor constants, Ki, for these polyoxins were determined and the values of binding affinity, ?ΔG_bind of the inhibitors to the enzyme were calculated from the Ki values. In addition, by using these ?ΔG_bind values the values of partial binding affinity, ?Δg for the several atoms and atomic groups or the several moieties contained in the polyoxin J molecule were estimated. From the results obtained, it was concluded that the carbamoylpolyoxamic acid moiety of polyoxins helps to stabilize the polyoxin-enzyme complex through the contributions of its oxygen atom at C?1″, amino group at C?2″, hydroxyl groups at C?3″ and C?4″, aliphatic carbon chain and terminal carbamoyloxy group.

The results obtained by the kinetic investigation using various nucleotides and nucleotide sugars suggested that there was a specific binding site on the enzyme corresponding to the uridine moiety of UDP-N-acetylglucosamine, and that the pyrimidine nucleoside moiety of polyoxins was also bound to this site.     

Studies on the Low Temperature Fermentation

An attempt has been made to isolate the bacteria capable of accumulating amino acids during the growth at low temperature from various natural sources. A psychrophilic strain P 145 forming glutamic acid at 5°C was obtained and identified as a Brevibacterium sp. The bacterium grew in the range of 0° to 37°C and exhibited the optimum growth at 15°C. The bacterium was defined as a facultative psychrophile.

The strain strictly required methionine only at above 28°C; below this temperature it grew normally without the amino acid. When methionine was added thiamine and biotin stimulated the growth of this strain at 28°C.

With the Brevibacterium sp. P 145 isolated from soil, the effect of incubation temperature on the extracellular amino acid accumulation has been examined from cultural and enzymological points of view. The strain was found to accumulate l-glutamic acid up to 5.88 mg/ml and l-alanine 0.38 mg/ml at 5°C, whereas it formed 0.21 mg/ml of l-glutamic acid and 2.54 mg/ml of l-alanine at 28°C.

The accumulation of l-alanine in the medium at 28°C seemed to be related to the thiamine requirement of the strain. In the case of thiamine deficiency, l-alanine was the main product in the culture at 28°C. When the incubation temperature was abruptly shifted from 28° to 5°C or from 5° to 28°C, the amino acid accumulation was also changed to that of the final temperature. l-Alanine dehydrogenase existed even in the cells grown at 5°C but was not active at this low temperature. These results were in accord with the informations obtained from cultural experiments.     

Fatty Acid Hydroxamate Formation by Intact Cells of Micrococcus sp.

When azelaic acid was used as a sole carbon source on the growth of Micrococcus sp. which was isolated from soil, intact cells of the organism catalyzed the enzymic condensation of fatty acids with hydroxylamine. Some of the characteristics of fatty acid hydroxamate formation were investigated.

The enzyme activity was tested with azelaic acid compared to other fatty acids. Azelylhydroxamate formation was activated with the addition of reduced glutathione or 2-mercaptoethanol. The reaction was inhibited by p-chloromercuribenzoate (PCMB), ethylene diamine tetraacetate (EDTA), NaF and benzoate.     

Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.     

High‐resolution prediction of leaf onset date in Japan in the 21st century under the IPCC A1B scenario

Reports indicate that leaf onset (leaf flush) of deciduous trees in cool‐temperate ecosystems is occurring earlier in the spring in response to global warming. In this study, we created two types of phenology models, one driven only by warmth (spring warming [SW] model) and another driven by both warmth and winter chilling (parallel chill [PC] model), to predict such phenomena in the Japanese Islands at high spatial resolution (500 m). We calibrated these models using leaf onset dates derived from satellite data (Terra/MODIS) and in situ temperature data derived from a dense network of ground stations Automated Meteorological Data Acquisition System. We ran the model using future climate predictions created by the Japanese Meteorological Agency's MRI‐AGCM3.1S model. In comparison to the first decade of the 2000s, our results predict that the date of leaf onset in the 2030s will advance by an average of 12 days under the SW model and 7 days under the PC model throughout the study area. The date of onset in the 2090s will advance by 26 days under the SW model and by 15 days under the PC model. The greatest impact will occur on Hokkaido (the northernmost island) and in the central mountains.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

Galectin-9 Ameliorates Clinical Severity of MRL/lpr Lupus-Prone Mice by Inducing Plasma Cell Apoptosis Independently of Tim-3

Galectin-9 ameliorates various murine autoimmune disease models by regulating T cells and macrophages, although it is not known what role it may have in B cells. The present experiment shows that galectin-9 ameliorates a variety of clinical symptoms, such as proteinuria, arthritis, and hematocrit in MRL/lpr lupus-prone mice. As previously reported, galectin-9 reduces the frequency of Th1, Th17, and activated CD8⁺ T cells. Although anti-dsDNA antibody was increased in MRL/lpr lupus-prone mice, galectin-9 suppressed anti-dsDNA antibody production, at least partly, by decreasing the number of plasma cells. Galectin-9 seemed to decrease the number of plasma cells by inducing plasma cell apoptosis, and not by suppressing BAFF production. Although about 20% of CD19^−/low CD138⁺ plasma cells expressed Tim-3 in MRL/lpr lupus-prone mice, Tim-3 may not be directly involved in the galectin-9-induced apoptosis, because anti-Tim-3 blocking antibody did not block galectin-9-induced apoptosis. This is the first report of plasma cell apoptosis being induced by galectin-9. Collectively, it is likely that galectin-9 attenuates the clinical severity of MRL lupus-prone mice by regulating T cell function and inducing plasma cell apoptosis.