Effects of intake of Lactococcus cremoris subsp. cremoris FC on constipation symptoms and immune system in healthy participants with mild constipation: a double-blind, placebo-controlled study.

This study evaluated the effect of Lactococcus cremoris subsp. cremoris FC (FC) on constipation symptoms and the immune system in healthy participants with mild constipation. Eighty-three participants were randomised into four groups with different doses: 50, 75, and 100 mg of freeze-dried FC (test) or corn starch (placebo). Defaecation frequency significantly increased in all test groups compared to the placebo group. Stool appearance and volume were improved considerably within the groups administered 50 mg and 75 mg of FC. The abundances of total bacteria, Bifidobacterium spp., and Lactobacillus group in the faeces showed increasing trends in the test groups. Regarding immunological parameters, the naive T cell counts in the blood were significantly higher at a dose of 75 mg of FC in the test group than in the placebo group. These results suggest that FC intake improves defaecation and some immunological parameters, especially naive T cell counts, in healthy adults.

Comprehensive evaluation of the bactericidal activities of free bile acids in the large intestine of humans and rodents.

In addition to functioning as detergents that aid digestion of dietary lipids in the intestine, some bile acids have been shown to exhibit antimicrobial activity. However, detailed information on the bactericidal activities of the diverse molecular species of bile acid in humans and rodents is largely unknown. Here, we investigated the toxicity of 14 typical human and rodent free bile acids (FBAs) by monitoring intracellular pH, membrane integrity, and viability of a human intestinal bacterium, Bifidobacterium breve Japan Collection of Microorganisms (JCM) 1192T, upon exposure to these FBAs. Of all FBAs evaluated, deoxycholic acid (DCA) and chenodeoxycholic acid displayed the highest toxicities. Nine FBAs common to humans and rodents demonstrated that α-hydroxy-type bile acids are more toxic than their oxo-derivatives and ß-hydroxy-type epimers. In five rodent-specific FBAs, ß-muricholic acid and hyodeoxycholic acid showed comparable toxicities at a level close to DCA. Similar trends were observed for the membrane-damaging effects and bactericidal activities to Blautia coccoides JCM 1395T and Bacteroides thetaiotaomicron DSM 2079T, commonly represented in the human and rodent gut microbiota. These findings will help us to determine the fundamental properties of FBAs and better understand the role of FBAs in the regulation of gut microbiota composition.

NKG2D functions as an activating receptor on natural killer cells in the common marmoset (Callithrix jacchus).

The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

Classification of chitosanases by hydrolytic specificity toward N¹,N4-diacetylchitohexaose.

The hydrolytic specificities of chitosanases were determined using N¹,N4-diacetylchitohexaose [(GlcN)2-GlcNAc-(GlcN)2-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N4-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N4-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the ß-glycosidic linkages in partially N-acetylated chitosan.

An adhesin-like protein, Lam29, from Lactobacillus mucosae ME-340 binds to histone H3 and blood group antigens in human colonic mucus.

A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

Administration of Cholic Acid Inhibits Equol Production from Daidzein in Mice.

Equol (Eq) is a metabolite of soy isoflavone daidzein (De) produced by the intestinal microbiota. The clinical effectiveness of soy isoflavone is considered to depend on the individual ability of Eq production. Previous studies have demonstrated that habitual dietary patterns may influence the production of Eq. For example, high Eq producers consumed less fat as a percentage of energy than low Eq producers. However, the inhibitory factors of Eq production are unknown. Recently, it was reported that bile acids induced by high-fat diet consumption may be a host-related factor controlling the composition of the intestinal microbiota. In this study, we investigated the effect of cholic acid (CA) administration, a mimic of the microbiota altered by a high-fat diet, on Eq production in mice. CA administration significantly decreased the levels of the De metabolites Eq, dihydrodaidzein, and O-desmethylangolensin in the serum of mice. However, CA administration did not affect the total molar concentration of De and its metabolites. Moreover, CA administration increased the levels of secondary bile acids, particularly deoxycholic acid (DCA), which has strong antibacterial activity in the cecum contents of mice. Thus, CA administration may increase the levels of DCA, a secondary bile acid, resulting in inhibition of Eq production. These findings may help to reveal the factors inhibiting Eq production and enhance the clinical effectiveness of isoflavone intake.

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats.

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4ß-hydroxycholesterol (4ßOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4ßOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4ßOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.

Cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Lactobacillus plantarum LA 318 recognizes human A and B blood group antigens.

Lactobacillus plantarum LA 318 is a potential probiotic strain isolated from normal human intestinal tissue that shows high adhesion to human colonic mucin mediated by the bacterial cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report the adhesion mechanism of the lactobacilli is in part due to GAPDH binding to human ABO-type blood group antigens expressed on human colonic mucin (HCM). After periodate oxidation of HCM, adhesion of L. plantarum LA 318 bacterial cells significantly decreased compared to normal HCM. A BIACORE binding assay of GAPDH to blood group antigens was then performed. High binding was observed to A and B group antigens, while binding to H group antigen was lower (P<0.01). No interaction was observed between GAPDH and various monosaccharides. Furthermore, GAPDH binding to the B-trisaccharide biotinyl polymer (BP)-probe [Galalpha1-3 (Fucalpha1-2) Gal-] was significantly higher as compared to B-disaccharide, Lewis D-trisaccharide, 3-fucosyl-N-acetylglucosamine and alpha-N-acetylneuraminic acid BP-probes. The data suggests the trisaccharide structure is important in binding to the blood group antigens. The binding of GAPDH to HCM significantly decreased after incubation with NAD+. This suggests that the NAD binding domain on GAPDH may be related to binding to HCM.

A novel mode of stimulating platelet formation activity in megakaryocytes with peanut skin extract.

We report in this study novel biochemical activities of peanut skin extract (PEXT) on thrombocytopoiesis. Peanut skin, derived from Arachis hypogaea L., is a traditional Chinese medicine that is used to treat chronic hemorrhage. We have shown that oral administration of PEXT increases the peripheral platelet levels in mice. Recently, we reported a liquid culture system that is useful for investigating megakaryocytopoiesis and thrombocytopoiesis from human CD34+ cells. In this liquid culture system, PEXT was shown to enhance the formation of CD41+/DAPI- cells (platelets), but had no effect on the formation of CD41+/DAPI+ cells (megakaryocytes) or on the DNA content. Furthermore, PEXT selectively stimulated proplatelet formation from cultured mature megakaryocytes and phorbol 12-myristate 13 acetate (PMA)-induced formation of platelet-like particles from Meg01 cells. Despite having no influence on the formation of megakaryocyte colony forming units (CFUs), PEXT increased the size of megakaryocytes during their development from CD34+ cells. PEXT showed no effect on the GATA-1 and NF-E2 mRNA levels, which are known to play an important role in thrombocytopoiesis and, based on the results of a pMARE-Luc (pGL3-MARE-luciferase) assay, had no influence on NF-E2 activation in Meg01 cells. These results suggest that PEXT accelerates proplatelet formation from megakaryocytes but does not influence the development of hematopoietic stem cells into megakaryocytes.

Whole-Body Counter Evaluation of Internal Radioactive Cesium in Dogs and Cats Exposed to the Fukushima Nuclear Disaster.

As a result of the 2011 nuclear incident that occurred at the Fukushima Nuclear Power Plant, a large number of abandoned dogs and cats were left within the disaster zone. A small number of these animals were rescued and cared for at shelters. Prior to the dispersal of these animals to their owners or fosterers, we evaluated the degree of internal radiocesium contamination using a specially designed whole-body counter. We conducted 863 non-invasive measurements of gamma rays due to internal radioactive cesium for 68 dogs and 120 cats at one shelter. After plotting graphs of 137Cs density we generated exponential functions of decay from seven dogs and six cats. From the regression formulae, we were able to determine the biological half-lives as 38.2 days for dogs and 30.8 days for cats. We found that in dogs there was a correlation between the biological half-life of radioactive cesium and age. Using our data, we estimated whole-body densities for each cat and dog at the time when they were rescued. We found that there were deviations in the data distributions among the different species, likely due to the timing of rescue, or living habits prior to rescue. A significant correlation was found when extracted feline reproductive organs were analyzed; the coefficients for the estimation of whole-body densities were approximately 7-fold higher than those based on the extracted feline reproductive organs. This may be due to the fact that majority of the radioactive cesium accumulates within muscular tissue with less distribution in other organs. It is possible to plan the appropriate management period in an animal shelter based on the use of the biological half-life of radioactive cesium calculated in this study. We believe that the correlations we uncovered in this work would be of great use for the management of companion animals in the event of a future nuclear accident.

Isolation of lactic acid bacteria bound to the porcine intestinal mucosa and an analysis of their moonlighting adhesins.

The adhesion of lactic acid bacteria (LAB) to the intestinal mucosa is one of the criteria in selecting for probiotics. Eighteen LAB were isolated from porcine intestinal mucin (PIM): ten strains of Lactobacillus, six strains of Weissella, and two strains of Streptococcus. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for phosphate-buffered saline (PBS) extracts from the LAB, many bands were detected in half of the samples, while a few and/or no clear bands were detected in the other half. All six of the selected LAB showed adhesion to PIM. L. johnsonii MYU 214 and MYU 221 showed adhesion at more than 10%. W. viridescens MYU 208, L. reuteri MYU 213, L. mucosae MYU 225, and L. agilis MYU 227 showed medium levels of adhesion at 5.9-8.3%. In a comprehensive analysis for the adhesins in the PBS extracts using a receptor overlay analysis, many moonlighting proteins were detected and identified as candidates for adhesins: GroEL, enolase, and elongation factor Tu in MYU 208; peptidase C1, enolase, formyl-CoA transferase, phosphoglyceromutase, triosephosphate isomerase, and phosphofructokinase in MYU 221; and DnaK, enolase, and phosphoglycerate kinase in MYU 227. These proteins in the PBS extracts, which included such things as molecular chaperones and glycolytic enzymes, may play important roles as adhesins.

NKG2D⁺ IFN-γ⁺ CD8⁺ T cells are responsible for palladium allergy.

Nickel, cobalt, and chromium are well known to be causal agents of allergic contact dermatitis. Palladium (Pd) can also cause allergic disease and exposure results from wide use of this metal in dental restorations and jewelry. Metal allergy is categorized as a delayed-type hypersensitivity, and metal-responsive T cell clones have been isolated from allergic patients. However, compared to nickel, little is known about the pathology of allergic disease mediated by Pd, and pathogenic T cells are poorly understood. To identify the pathogenic T cells that are responsible for onset of Pd allergy, we enriched metal-responsive lymphocytes by sequential adoptive transfer of involved lymph node cells. Here we show that sequential adoptive transfer gradually increased the incidence and the intensity of Pd allergy, and CD8⁺ T cells are responsible for the disease as CD8⁺ T cell-depleted mice and ß2-microglobulin-deficient mice did not develop Pd allergy. In addition, we found that draining lymph node cells skewed toward CD8⁺ T cells in response to Pd challenge in 8th adoptive transferred recipient mice. The CD8⁺ T cells expressed NKG2D, a costimulatory molecule involved in the production of IFN-γ. NKG2D ligand was also induced in Pd-injected tissues. Furthermore, both NKG2D ligand-transgenic mice, where NKG2D is downmodulated, and IFN-γ-deficient mice showed impaired Pd allergy. Taken together, these results indicate that IFN-γ-producing NKG2D⁺ CD8⁺ T cells are responsible for Pd allergy and suggest that NKG2D is a potential therapeutic target for treatment of metal allergy.

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Adhesion tests are complex, time-consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.

Food preservative potential of gassericin A-containing concentrate prepared from a cheese whey culture supernatant from Lactobacillus gasseri LA39.

Gassericin A (GA) is a circular bacteriocin produced by Lactobacillus gasseri LA39. In this study, GA-containing concentrate was prepared using a cross-flow membrane filtration device (30 kDa cut-off) from the culture supernatant of Lb. gasseri LA39 cultivated in a cheese whey-based food-grade medium. The bacteriocin activity titer in the concentrate was 16 times as high as that of the culture supernatant and was completely maintained through each incubation at 4°C for 3 months, 37°C for 2 months, 60°C for 5 h, and 100°C for 30 min. The GA-containing concentrate was used with glycine powder to make custard creams, and then four representative strains of custard cream spoilage bacteria (Bacillus cereus, Lactococcus lactis subsp. lactis, Achromobacter denitrificans and Pseudomonas fluorescens) were individually inoculated at c. 10(3) colony forming units/g in the custard creams. Throughout 30 days of incubation at 30°C, all of the inoculated bacteria were completely inhibited by the combination of 5% (w/w) of the GA-containing concentrate and 0.5% (w/w) glycine. This is the first highly practical application of GA to foods as a biopreservative, and the concentration method and the bacteriocin concentrate would contribute to biopreservation of several foods.