Estrogenic Prenylated Flavonoids in Sophora flavescens.

Sophora flavescens is a medicinal herb distributed widely in Japan and it has been used to treat various diseases and symptoms. To explore its pharmacological use, we examined the estrogenic activity of four prenylated flavonoids, namely kurarinone, kushenols A and I, and sophoraflavanone G, which are characterized by the lavandulyl group at position 8 of ring A, but have variations in the hydroxyl group at positions 3 (ring C), 5 (ring A) and 4' (ring B). These prenylated flavonoids were examined via cell proliferation assays using sulforhodamine B, Western blotting, and RT-PCR, corresponding to cell, protein, and transcription assays, respectively, based on estrogen action mechanisms. All the assays employed here found weak but clear estrogenic activities for the prenylated flavonoids examined. Furthermore, the activities were inhibited by an estrogen receptor antagonist, suggesting that the activities were likely being mediated by the estrogen receptors. However, there were differences in the activity, attributable to the hydroxyl group at position 4', which is absent in kushenol A. While the estrogenic activity of kurarinone and sophoraflavanone G has been reported before, to the best of our knowledge, there are no such reports on kushenols A and I. Therefore, this study represents the first report of their estrogenic activity.

Growth Inhibitory Mechanism of Contact-independent Antifungal TM-I-3 Bacillus sporothermodurans Strain against Aspergillus fumigatus and Cladosporium cladosporioides.

We previously identified a new Bacillus sporothermodurans strain, TM-I-3, which produces volatile compounds that show potent inhibitory activity against certain types of fungi, including Aspergillus fumigatus and Cladosporium cladosporioides. Non-contact antifungals derived from this bacterium may provide multidirectional inhibition and may be useful in disease prevention. This study is aimed at identifying the stage of fungal growth that is inhibited by TM-I-3 to elucidate the mechanism of its contact-independent antifungal activity. We evaluated mycelial growth and the gross fungal colony areas after 7 days in each experimental group varying the time in the proximity of TM-I-3 for 24 hours each. The fungal growth inhibition assay showed that TM-I-3 inhibited spore germination: the lag phase in the sigmoid growth curve. The present study demonstrated that TM-I-3 might be an effective fungistatic agent against pathogenic and allergenic fungi.

The Bacteriological Properties of Bacillus Strain TM-I-3 and Analysis of the Volatile Antifungal Compounds Emitted by this Bacteria.

Previously, we isolated and examined a bacterial strain designated as TM-I-3, belonging to the genus Bacillus, from soil in Nagasaki, Japan. This bacterium was able to inhibit the growth of molds, without coming into direct contact with them. Non-contact antifungals are capable of providing multidirectional inhibition and may contribute to disease prevention. In this study, we revealed the bacteriological properties of TM-I-3 and evaluated the antifungal activity of the compounds emitted from this bacterium. In addition, we analyzed the antimicrobial substances released from TM-I-3 using GC/MS to elucidate the mechanism of its action. Antimicrobial compounds from strain TM-I-3 were identified as acetic acid, propanoic acid, isovaleric acid, 2-methylbutanoic acid, and benzaldehyde, which are all reported to have antimicrobial activity. TM-I-3 demonstrated possible efficacy in inhibiting the growth of Aspergillus fumigatus, Cladosporium cladosporioides and Penicillium expansum, which may lead to inhibition of common fungal contaminants of household products and prevention of some pulmonary diseases.

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28.

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.

Decomposition of insoluble and hard-to-degrade animal proteins by enzyme E77 and its potential applications.

Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.

Properties of a high malic acid-producing strains of Saccharomyces cerevisiae isolated from sake mash.

We characterized high malic acid-producing strains of Saccharomyces cerevisiae isolated from sake mash. We compared the gene expression of these strains with those of the parental strain by DNA microarray, and found that stress response genes, such as HSP12, were commonly upregulated in the high malate-producing strains, whereas thiamine synthesis genes, such as THI4 and SNZ2, were downregulated in these strains.

Development of vasoactive intestinal polypeptide-immunoreactive nerves in the major cerebral arteries of the quail anterior circulation.

Development of cerebral perivascular nerves immunoreactive for vasoactive intestinal polypeptide (VIP) was investigated in the Japanese quails, using immunohistochemistry and quantitative analysis. VIP-immunoreactive (VIP-IR) nerves supplying the anterior circulation appeared on the cerebral carotid artery (CCA) at embryonic day 10 and on the cerebroethmoidal artery (CEA) after hatching. Nerves from the CCA increased greatly in number and spread progressively during successive embryonic stages, while those from the CEA were sparse all through the post-hatching stages, mostly remained limited to this vessel wall. The distribution of VIP-IR nerves to the respective major arteries of the anterior circulation from the two vascular routes was basically similar among post-hatching day (P) 15, P20, P30 and P50. Likewise, no clear statistical difference was observed with regard to the nerve density of the corresponding arteries in the four age groups. These findings suggest that VIP-IR innervation of the quail anterior circulation usually attains its mature pattern at the third week after hatching.

Structural and mechanistic exploration of acid resistance: kinetic stability facilitates evolution of extremophilic behavior.

Kinetically stable proteins are unique in that their stability is determined solely by kinetic barriers rather than by thermodynamic equilibria. To better understand how kinetic stability promotes protein survival under extreme environmental conditions, we analyzed the unfolding behavior and determined the structure of Nocardiopsis alba Protease A (NAPase), an acid-resistant, kinetically stable protease, and compared these results with a neutrophilic homolog, alpha-lytic protease (alphaLP). Although NAPase and alphaLP have the same number of acid-titratable residues, kinetic studies revealed that the height of the unfolding free energy barrier for NAPase is less sensitive to acid than that of alphaLP, thereby accounting for NAPase's improved tolerance of low pH. A comparison of the alphaLP and NAPase structures identified multiple salt-bridges in the domain interface of alphaLP that were relocated to outer regions of NAPase, suggesting a novel mechanism of acid stability in which acid-sensitive electrostatic interactions are rearranged to similarly affect the energetics of both the native state and the unfolding transition state. An acid-stable variant of alphaLP in which a single interdomain salt-bridge is replaced with a corresponding intradomain NAPase salt-bridge shows a dramatic >15-fold increase in acid resistance, providing further evidence for this hypothesis. These observations also led to a general model of the unfolding transition state structure for alphaLP protease family members in which the two domains separate from each other while remaining relatively intact themselves. These results illustrate the remarkable utility of kinetic stability as an evolutionary tool for developing longevity over a broad range of harsh conditions.

Degradation of PrP(Sc) by keratinolytic protease from Nocardiopsis sp. TOA-1.

A keratinolytic alkaline proteae (NAPase) from Nocardiopsis sp. TOA-1 degraded a scrapie prion without any chemical or physical treatment. Optimal temperature and pH were 60 degrees C and above pH 10.0. The scrapie prion was completely degraded within 3 min under optimal conditions.

Purification and some properties of a keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA-1.

A novel alkaliphilic Nocardiopsis sp., strain TOA-1, was isolated from a tile-joint of a bathroom. Strain TOA-1 produced a variety of alkaline hydrolytic enzymes. An alkaline protease, designated NAPase, was purified and characterized. NAPase had a very high keratinolytic activity and high stability under acidic conditions.

Occurrence of an Affinity Site apart from the Active Site on the Raw-Starch-Digesting but Non-Raw-Starch-Adsorbable Bacillus subtilis 65 alpha-Amylase.

alpha-Cyclodextrin specifically inhibited raw starch digestion by Bacillus subtilis 65 alpha-amylase. The raw starch digestibility and alpha-cyclodextrin-Sepharose 6B adsorbability of this alpha-amylase were simultaneously lost when the specific domain corresponding to the affinity site essential for raw starch digestion was deleted by proteolysis. Occurrence of the affinity site on raw-starch-digesting enzymes was proven also with bacterial amylase.