Development of an Umami Taste Sensitivity Test and Its Clinical Use

There is a close relationship between perception of umami, which has become recognized as the fifth taste, and the human physical condition. We have developed a clinical test for umami taste sensitivity using a filter paper disc with a range of six monosodium glutamate (MSG) concentrations. We recruited 28 patients with taste disorders (45–78 years) and 184 controls with no taste disorders (102 young [18–25 years] and 82 older [65–89 years] participants). Filter paper discs (5 mm dia.) were soaked in aqueous MSG solutions (1, 5, 10, 50, 100 and 200 mM), then placed on three oral sites innervated by different taste nerves. The lowest concentration participants correctly identified was defined as the recognition threshold (RT) for MSG. This test showed good reproducibility for inter- and intra-observer variability. We concluded that: (1) The RT of healthy controls differed at measurement sites innervated by different taste nerves; that is, the RT of the anterior tongue was higher than that of either the posterior tongue or the soft palate in both young and older individuals. (2) No significant difference in RT was found between young adults and older individuals at any measurement site. (3) The RT of patients with taste disorders was higher before treatment than that of the healthy controls at any measurement site. (4) The RT after treatment in these patients improved to the same level as that of the healthy controls. (5) The cutoff values of RT, showing the highest diagnostic accuracy (true positives + true negatives), were 200 mM MSG for AT and 50 mM MSG for PT and SP. The diagnostic accuracy at these cutoff values was 0.92, 0.87 and 0.86 for AT, PT and SP, respectively. Consequently, this umami taste sensitivity test is useful for discriminating between normal and abnormal umami taste sensations.     

Isolation and Identification of a Microorganism Producing Bilirubin Oxidase

For the purposes of decoloring raw sewage and use as an analytical tool in clinical fields, we tried to obtain microorganisms producing an enzyme which is reactive to bilirubin. One strain of microorganism (MT-1) showed strong ability to produce the enzyme.

The morphological and physiological characteristics of strain MT-1 were studied. This strain was found to belong to Myrothecium verrucaria.

For the production of the enzyme, this strain was aerobically cultured at 25°C in a jar fermentor which contained potato-glucose medium. The highest activity was obtained after 62-hr cultivation.

This enzyme was also produced by other strains belonging to Myrothecium.     

A New Enzyme “Bilirubin Oxidase” Produced by Myrothecium verrucaria MT-1

The thermal degradation kinetics of pectin methylesterase (PME) from carrot and lettuce were studied. Fresh extracts were exposed to temperatures from 55 to 70 °C until the enzyme was inactivated. A model based on the presence of two forms of the enzyme, one active and one non-active, is proposed. The natural variability of the PME activity was taken into the model in the form of normally distributed random effects. The common model parameters obtained (cleavage constant (0.0395±0.0062 s^?1), degradation constant (0.556±0.112 s^?1), cleavage energy of activation (469±23 kJ mol^?1) and degradation energy of activation (488±18 kJ mol^?1)) show that the PME degradation kinetics of the two vegetables can be explained with a single set of parameters.     

Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2′-modified-4′-thionucleosides

Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2′-modified-4′-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2′-fluoro-4′-thionucleoside and 2′-fluoronucleoside units as well as only 2′-fluoronucleoside units, while those prepared as combination of 2′-OMe nucleoside/2′-OMe-4′-thionucleoside and 2′-fluoronucleoside units did not show significant activity. Measurement of T_m values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity.     

Stage- and cell-specific expression of Dnmt3a and Dnmt3b during embryogenesis

DNA methylation is essential for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, contribute to the creation of DNA methylation patterns in embryos. We demonstrated that the Dnmt3a and Dnmt3b proteins are expressed at different stages of embryogenesis. Dnmt3b is specifically expressed in totipotent embryonic cells, such as inner cell mass, epiblast and embryonic ectoderm cells, whilst Dnmt3a is significantly and ubiquitously expressed after E10.5. The difference in the expression stages of the Dnmt3a and Dnmt3b proteins may contribute to their distinct functions during the embryogenesis.     

Autoxidation of oxymyoglobin. An overall stoichiometry including subsequent side reactions

Oxymyoglobin (MbO2) is oxidized easily to metmyoglobin (metMb) with generation of the superoxide anion, which can be converted by the spontaneous dismutation into H2O2, this being also a potent oxidant of MbO2. In the presence of sodium azide in stoichiometric amounts, however, the rate of autoxidation of MbO2 increased rapidly with increasing concentration of the anion, but soon reached a saturating level, the extent of which was about twice that of the normal autoxidation in buffer alone. Quantitative analysis has revealed that this enhancement is not due to the nucleophilic displacement of O2- from MbO2 by the anion (Satoh, Y., and Shikama, K. (1981) J. Biol. Chem. 256, 10272-10275), but is due to the additional oxidation of MbO2 by H2O2 freed from the metMb being occupied by the anion at the sixth coordination position. Based on these novel results and stoichiometric considerations, it is possible to propose a new view that H2O2 produced from O2- can be eliminated or decomposed mostly, if not completely, by the metMb resulting from the normal autoxidation reaction of MbO2, presumably via the formation of the ferryl species.     

Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1

Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.     

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.     

A method for estimating nucleotide diversity from AFLP data

A method for estimating the nucleotide diversity from AFLP data is developed by using the relationship between the number of nucleotide changes and the proportion of shared bands. The estimation equation is based on the assumption that GC-content is 0.5. Computer simulations, however, show that this method gives a reasonably accurate estimate even when GC-content deviates from 0.5, as long as the number of nucleotide changes per site (nucleotide diversity) is small. As an example, the nucleotide diversity of the wild yam, Dioscorea tokoro, was estimated. The estimated nucleotide diversity is 0.0055, which is larger than estimations from nucleotide sequence data for Adh and Pgi.