Masashi Kitamura’s research while affiliated with Josai University and other places

Two novel aromatic polyketides, penicanesins J and K (1, 2), were isolated from the marine-derived fungus Didymella aeria, along with the known compound integrastatin B (3). The structures of the new compounds were determined by NMR spectroscopy and synthetic methods. The isolated compounds were tested for monoamine oxidase (MAO) B inhibition, anti-amyloid beta (Aβ) aggregation, and protective activity against H2O2-induced cell death in human neuroblastoma SH-SY5Y cells. Integrastatin B (3) showed potential activity for inhibition of Aβ aggregation and protection against H2O2-induced cell death.

Background/aim: Alzheimer's disease (AD) is the most common form of dementia worldwide. Nattokinase is a serine protease extracellularly produced by natto, a fermented product of Bacillus subtilis var. natto. In this study, we investigated the therapeutic effects of nattokinase in a rat model of AD induced by aluminum and D-galactose. Materials and methods: Forty Wistar rats were randomly divided into four groups: normal, vehicle, and orally administered nattokinase (NK65 and NK130 groups). Except for the normal group, all groups were treated with AlCl3 and D-galactose for 10 weeks. The NK65 and NK130 groups additionally received 65 mg/kg/day and 130 mg/kg/day nattokinase, respectively. We analyzed β-amyloid levels in the cerebrospinal fluid (CSF), and the spatial reference test was evaluated using the Morris water maze test. After the Morris water maze test, rats of all groups were subjected to micro-computed tomography (μCT) to assess constructional changes in the brain. Aluminum concentration and β-amyloid levels were analyzed by histochemical staining in all brain tissues. Results: Oral administration of nattokinase in the AD rat model increased free-form β-amyloid levels in the CSF and improved aluminum and amyloid plaque accumulation in the brain. Brain μCT images showed enhanced brain volume with fewer constructional changes after treatment with nattokinase. In the behavioral tests, both the escape latency time in the spatial reference test and the time taken to cross the platform area in the spatial probe test improved partially. Conclusion: The results suggest that nattokinase has potential therapeutic applications in the treatment of AD.

This study proposes a sequential liquid dispensing method using a centrifugal microfluidic device operating at a constant rotational speed for the multiplexed genetic detection of nucleic acid targets across multiple samples in a single operation. A pair of passive valves integrated into each microchamber enabled the liquid to fill towards the center of rotation against the centrifugal force, facilitating the complete removal of air inside the microchamber. Liquid manipulation can be achievable without any surface coating of the device by exploiting the inherent hydrophobicity of the polymer. Furthermore, design guidelines for the optimization of microfluidic devices are clarified. Consequently, our proposed method allows direct liquid dispensing into the reaction chambers without cross-contamination while simultaneously metering the sample/reagent volume for the colorimetric loop-mediated isothermal amplification (LAMP) reaction. In addition, we demonstrated the simultaneous detection of four foodborne pathogens (Salmonella spp., Vibrio parahaemolyticus, Campylobacter spp., and norovirus genogroup II (GII)) across four samples in a centrifugal microfluidic device within 60 min. Furthermore, the device exhibited high quantitation (R² > 0.98) of the DNA concentration in the sample. Our proposed method enables a more compact design by eliminating the need for metering chambers and offers a point-of-care testing platform with high simplicity as it operates at a constant rotational speed.

The emergence of coronavirus disease 2019 (COVID-19), a novel identified pneumonia resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has significantly impacted and posed significant challenges to human society. The papain-like protease (PLpro) found in the nonstructural protein 3 of SARS-CoV-2 plays a vital role in viral replication. Moreover, PLpro disrupts the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 from host proteins. Consequently, PLpro has emerged as a promising drug target against SARS-CoV-2 infection. Computational studies have reported that ciclesonide can bind to SARS-CoV-2 PLpro. However, the inhibitory effects of ciclenoside on the PLpro have not been experimentally evaluated. Here, we evaluated the inhibitory effects of synthetic glucocorticoids (sGCs), including ciclesonide, on SARS-CoV-2 PLpro in vitro assay. Ciclesonide significantly inhibited the enzymatic activity of PLpro, compared with other sGCs and its IC50 was 18.4 ± 1.89 µM. These findings provide insights into the development of PLpro inhibitors. Fullsize Image

A novel tricyclic polyketide, curvulanone (1), was isolated from the marine-derived fungus Curvularia aeria. The structure of 1 was determined by NMR and single-crystal X-ray crystallography. 1 had a cyclopentabenzopyranone with 3-acetic acid structure that is rarely found in natural compounds. Monoamine oxidase and sirtuin 1 inhibitory test was exhibited and 1 showed their inhibitory activity. Fullsize Image

Nattokinase is a protease produced by Bacillus subtilis var. natto that exhibits various beneficial biological effects. Thus, a reliable assay to determine nattokinase levels is needed. In this study, we developed novel mouse monoclonal antibodies (mAbs) that recognize nattokinase, and created a specific and sensitive enzyme-linked immunosorbent assay (ELISA) to measure nattokinase levels. The ELISA was developed using a combination of new mouse antinattokinase mAbs used as capture antibodies coated onto 96-well plates, with a peroxidase-conjugated antibody used for detection. This ELISA enabled detection of nattokinase at 1 ng/mL. We believe that the novel mAbs developed in this study will be useful in future for elucidating nattokinase function.

Camellia sinensis leaves are one of the most economically important crops, and various tea cultivars are available in Japan. In the present study, we compared the constituents of Camellia sinensis var. sinensis cv. Yabukita (“Yabukita”), a standard tea cultivar, and those of C. sinensis var. sinensis cv. Sayamakaori (“Sayamakaori”), grown in Saitama Prefecture, using proton nuclear magnetic resonance ( ¹ H-NMR) metabolomics technology. Principal component analysis and hierarchical cluster analysis of ¹ H-NMR spectroscopic data of the MeOH extracts of C. sinensis leaves revealed differences in the metabolic profiles of each tea cultivar. Chromatographic separation based on ¹ H-NMR spectral data showed that kaempferol 3- O-β-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranoside was a distinct component of “Sayamakaori”. This compound can be used as maker for the identification of “Sayamakaori” and the maintenance of its quality.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases worldwide with public health concern, yet no antiviral therapies have been developed. In this study, we aimed to screen crude drugs, which are components of Japanese traditional medicine, ''Kampo'' to see their effects on HuNoV infection using a reproducible HuNoV cultivation system, stem-cell derived human intestinal organoids/enteroids (HIOs). Among the 22 crude drugs tested, Ephedra herba significantly inhibited HuNoV infection in HIOs. A time-of-drug addition experiment suggested that this crude drug more preferentially targets post-entry step than entry step for the inhibition. To our knowledge, this is the first anti-HuNoV inhibitor screen targeting crude drugs, and Ephedra herba was identified as a novel inhibitor candidate that merits further study.