Nattokinase, a Subtilisin-like Alkaline-Serine Protease, Reduces Mutacin Activity by Inactivating the Competence-Stimulating Peptide in Streptococcus mutans.

Streptococcus mutans is a major cariogenic organism because of its ability to form biofilms on tooth surfaces. Bacteriocins produced by S. mutans (known as mutacins) are indirect pathogenic factors that play a role in the persistence of this microbe in the oral environment. Nattokinase, a subtilisin-like alkaline serine protease, potently inhibits biofilm formation without affecting S. mutans growth. However, effective strategies utilizing nattokinase to control mutacin production by S. mutans are lacking. In this study, we evaluated the effect of nattokinase on mutacin activity in 46 strains of S. mutans with different mutacin genotypes isolated from the dental plaques of pediatric patients with caries. Nattokinase reduced the activity of mutacin against oral streptococci at a concentration of 1 mg/mL in all clinical isolates. Furthermore, nattokinase reduced the expression of non-lantibiotic mutacin structural genes (nlmABCD) and inactivated the extracellular competence-stimulating peptide involved in comDE activation, which regulates non-lantibiotic mutacin gene expression. These results suggest that nattokinase may reduce the virulence of S. mutans and could potentially be used as a new caries-preventive agent as an alternative to conventional drug treatments.

Roles of membrane vesicles from Streptococcus mutans for the induction of antibodies to glucosyltransferase in mucosal immunity.

Glucosyltransferase (Gtf) B and GtfC from Streptococcus mutans are key enzymes for the development of biofilm-associated diseases such as dental caries. Gtfs are involved in membrane vesicles (MVs) and function in the formation of biofilms by initial colonizers such as Streptococcus mitis and Streptococcus oralis on the tooth surface. Therefore, MVs may be important virulence factors and targets for the prevention of biofilm-associated disease. To clarify how GtfB encoded by gtfB and GtfC encoded by gtfC associate with MVs and whether MVs are effective as a mucosal immunogen to induce the production of antibodies against Gtfs, MVs from S. mutans UA159 wild-type (WT), gtfB-, gtfC- and gtfB-C- were extracted from culture supernatants by ultracentrifugation and observed by scanning electron microscopy. Compared with GtfB, GtfC was mainly contained in MVs and regulated the size and aggregation of MVs, and the biofilm formation of S. mutans. The intranasal immunization of BALB/c mice with MVs plus a TLR3 agonist, poly(I-C), was performed 2 or 3 times for 5 weeks, with an interval of 2 or 3 weeks. MVs from all strains caused anti-MV IgA and IgG antibody production. In quality analysis of these antibodies, the IgA and IgG antibodies produced by immunization with MVs from WT and gtfB- strains reacted with Gtfs in the saliva, nasal wash and serum but those produced by immunization with MVs from gtfC- and gtfB-C- strains did not. S. mutans MVs mainly formed by GtfC are an intriguing immunogen for the production of anti-Gtf antibodies in mucosal immunogenicity.

Neurochemical and behavioural impact of C18 fatty acids in male mice postweaning.

Dietary components, particularly essential fatty acids, affect the expression and maintenance of normal physiological phenotypes. However, the influence of C18 fatty acids that are abundantly present in the normal diet is unclear. We focused on the behavioural and neurochemical effects of C18 fatty acids during postweaning development in male mice. An AIN-93G diet supplemented with 8% stearic acid (C18:0), 3% oleic acid (C18:1), 3% linoleic acid (C18:2) or 3% α-linolenic acid (C18:3) was provided from four weeks of age for eight weeks. At 12 weeks of age, novel exploratory behaviour and social interaction tests were carried out. One week after the last behavioural test, the brain of each mouse was removed. The frequency of social interactive behaviour was decreased by approximately 70% in the C18:0 group compared to the basal diet group, but there was no difference in cumulative time. The frequency of social interaction showed a positive correlation to cumulative time in mice fed with the experimental diets except for C18:0. Dietary C18 fatty acids following weaning had no impact on brain fatty acid composition except for the C18:3 diet. Furthermore, the neurochemical properties to be especially noted were that choline acetyltransferase activity was absolutely higher in C18:0 diet-fed mice than in the other groups, especially in the frontal cortex where it was 1.7-fold higher than in the basal diet-fed group. The present results reveal a significant possibility of neurochemical and behavioural effects of dietary fatty acids, and saturated fatty acids are of special importance during the postweaning period.

Homozygous and heterozygous GH transgenesis alters fatty acid composition and content in the liver of Amago salmon (Oncorhynchus masou ishikawae).

Growth hormone (GH) transgenic Amago (Oncorhynchus masou ishikawae), containing the sockeye GH1 gene fused with metallothionein-B promoter from the same species, were generated and the physiological condition through lipid metabolism compared among homozygous (Tg/Tg) and heterozygous GH transgenic (Tg/+) Amago and the wild type control (+/+). Previously, we have reported that the adipose tissue was generally smaller in GH transgenic fish compared to the control, and that the Δ-6 fatty acyl desaturase gene was down-regulated in the Tg/+ fish. However, fatty acid (FA) compositions have not been measured previously in these fish. In this study we compared the FAs composition and content in the liver using gas chromatography. Eleven kinds of FA were detected. The composition of saturated and monounsaturated fatty acids (SFA and MUFA) such as myristic acid (14:0), palmitoleic acid (16:1n-7), and cis-vaccenic acid (cis-18:1n-7) was significantly (P<0.05) decreased in GH transgenic Amago. On the other hand, the composition of polyunsaturated fatty acids (PUFAs) such as linoleic acid (18:2n-6), arachidonic acid (20:4n-6), and docosapentaenoic acid (22:5n-3) was significantly (P<0.05) increased. Levels of serum glucose and triacylglycerol were significantly (P<0.05) decreased in the GH transgenics compared with +/+ fish. Furthermore, 3'-tag digital gene expression profiling was performed using liver tissues from Tg/Tg and +/+ fish, and showed that Mid1 interacting protein 1 (Mid1ip1), which is an important factor to activate Acetyl-CoA carboxylase (ACC), was down-regulated in Tg/Tg fish, while genes involved in FA catabolism were up-regulated, including long-chain-fatty-acid-CoA ligase 1 (ACSL1) and acyl-coenzyme A oxidase 3 (ACOX3). These data suggest that liver tissue from GH transgenic Amago showed starvation by alteration in glucose and lipid metabolism due to GH overexpression. The decrease of serum glucose suppressed Mid1ip1, and caused a decrease of de novo FA synthesis, resulting in a decrease of SFA and MUFA. This induced expression of ACSL1 and ACOX3 to produce energy through ß-oxidation in the GH transgenic Amago.

Lipid and fatty acid composition of mesocarp and seed of avocado fruits harvested at northern range in Japan.

The lipid and fatty acid composition of the mesocarp and seed of avocado fruit grown and harvested in Japan, which is located at the northern range of the avacado, was investigated and compared to an imported avocado purchased commercially. The potential of the avocado mesocarp as an agricultural product in Japan was also explored. Total lipids (TL) accounted for approximately 20% of the mesocarp. Further analysis showed that the neutral lipid (NL) fraction accounted for at least 95% of the TL, and almost 90% of NL was triacylglycerol. Monoenoic acids accounted for at least 65% of the total fatty acids, and oleic acid, which is regarded as an especially important functional component of avacado accounted for approximately 50% of the monounsaturated fatty acids. A comparison of the Japanese avocado cultivars and an imported avocado cultivar in the present study revealed no significant differences in the lipid and fatty acid compositions. Therefore, production of avocado fruit, which is rich in various nutritional components, is expected to be increased on a larger number of farms in Japan in the future. It is believed to be necessary to carry out further verification, such as the establishment of a cultivation technique adoptable to Japan, examination of optimal soil and land features, and cultivar selection.

Hypoxia-induced changes in tight junction permeability of brain capillary endothelial cells are associated with IL-1beta and nitric oxide.

We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.

Sphingosine 1-phosphate induces the production of glial cell line-derived neurotrophic factor and cellular proliferation in astrocytes.

Sphingosine 1-phosphate (S1P) is a platelet-derived bioactive sphingolipid that evokes a variety of biological responses. To understand the role of S1P in the central nervous system, we have examined the effect of S1P on the production of glial cell line-derived neurotrophic factor (GDNF) and growth regulation of cortical astrocytes from rat embryo. Moreover, we examined the possibility that the expression of GDNF is regulated differently in cultured astrocytes from the stroke-prone spontaneously hypertensive rat (SHRSP) than in those from Wistar kyoto rats (WKY). The mRNA expression was quantitated by RT-PCR based on the fluorescent TaqMan methodology. A new instrument capable of measuring fluorescence in real time was used to quantify gene amplification in astrocytes. GDNF protein was investigated by enzyme-linked immunosorbent assay. S1P induced the expression of GDNF mRNA and the production of GDNF protein in a dose-dependent manner in WKY astrocytes. Moreover, S1P increased cell numbers and induced the proliferation of astrocytes. In addition, the level of mRNA expression and protein production of GDNF was significantly lower in SHRSP than WKY astrocytes following exposure to S1P. These findings revealed that S1P augments GDNF protein production and cellular growth in astrocytes. Also, our results indicate that production in SHRSP astrocytes was attenuated in response to S1P compared with that observed in WKY. We conclude that S1P specifically triggers a cascade of events that regulate the production of GDNF and cell growth in astrocytes. Our results also suggest that the reduced expression of GDNF caused by S1P is a factor in the stroke proneness of SHRSP.