Seasoning ingredients in a medium-fat diet regulate lipid metabolism in peripheral tissues via the hypothalamic-pituitary axis in growing rats.

We fed rats noodle (N) -diet containing 30 wt.% instant noodle with a 26% fat-to-energy ratio for 30 days (N-group). Compared with rats that were fed the same amount of nutrients (C-group), the N-group showed lower liver triacylglycerol levels and higher fecal cholesterol levels. We then analyzed transcriptome of the hypothalamic-pituitary (HP), the liver and the white adipose tissue (WAT). Thyroid stimulating hormone (Tshb), and its partner, glycoprotein hormone genes were up-regulated in the HP of N-group. Sterol regulatory element binding transcription factors were activated in the liver of N-group, while an up-regulation of the angiogenic signal occurred in the WAT of N-group. N-group showed higher urine noradrenaline (NA) level suggesting that these tissue signals are regulated by NA and Tshb. The N-diet contains 0.326 wt.% glutamate, 0.00236 wt.% 6-shogaol and Maillard reaction products. Our results suggest that these ingredients may affect lipid homeostasis via the HP axis.

Supplemental feeding of a gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, alleviates spontaneous atopic dermatitis and modulates intestinal microbiota in NC/nga mice.

The present study investigated the antiallergic and anti-inflammatory effects of 10-hydroxy-cis-12-octadecenoic acid (HYA), a novel gut microbial metabolite of linoleic acid, in NC/Nga mice, a model of atopic dermatitis (AD). Feeding HYA decreased the plasma immunoglobulin E level and skin infiltration of mast cells with a concomitant decrease in dermatitis score. HYA feeding decreased TNF-α and increased claudin-1, a tight junction protein, levels in the mouse skin. Cytokine expression levels in the skin and intestinal Peyer's patches cells suggested that HYA improved the Th1/Th2 balance in mice. Immunoglobulin A concentration in the feces of the HYA-fed mice was approximately four times higher than that in the control mice. Finally, denaturing gradient gel electrophoresis of the PCR-amplified 16 S rRNA gene of fecal microbes indicated the modification of microbiota by HYA. Taken together, the alterations in the intestinal microbiota might be, at least in part, associated with the antiallergic effect of HYA.

A gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, ameliorates intestinal epithelial barrier impairment partially via GPR40-MEK-ERK pathway.

Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca(2+)]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease.

Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription.

Supplemental epilactose prevents metabolic disorders through uncoupling protein-1 induction in the skeletal muscle of mice fed high-fat diets.

Obesity is one of the major health problems throughout the world. The present study investigated the preventive effect of epilactose--a rare non-digestible disaccharide--on obesity and metabolic disorders in mice fed high-fat (HF) diets. Feeding with HF diets increased body weight gain, fat pad weight and adipocyte size in mice (P<0·01), and these increases were effectively prevented by the use of supplemental epilactose without influencing food intake (P<0·01). Caecal pools of SCFA such as acetic and propionic acids in mice fed epilactose were higher compared with mice not receiving epilactose. Supplemental epilactose increased the expression of uncoupling protein (UCP)-1, which enhances energy expenditure, to 2-fold in the gastrocnemius muscle (P=0·04) and to 1·3-fold in the brown adipose tissue (P=0·02) in mice fed HF diets. Feeding HF diets induced pro-inflammatory macrophage infiltration into white adipose tissue, as indicated by the increased expression of monocyte chemotactic protein-1, TNF-α and F4/80, and these increases were attenuated by supplemental epilactose. In differentiated myogenic-like C2C12 cells, propionic acid, but not acetic or n-butyric acids, directly enhanced UCP-1 expression by approximately 2-fold (P<0·01). Taken together, these findings indicate that the epilactose-mediated increase in UCP-1 in the skeletal muscle and brown adipose tissue can enhance whole-body energy expenditure, leading to effective prevention of obesity and metabolic disorders in mice fed HF diets. It is suggested that propionic acid--a bacterial metabolite--acts as a mediator to induce UCP-1 expression in skeletal muscles.

Anti-inflammatory and Intestinal Barrier-protective Activities of Commensal Lactobacilli and Bifidobacteria in Thoroughbreds: Role of Probiotics in Diarrhea Prevention in Neonatal Thoroughbreds.

We previously isolated the commensal bacteria lactobacilli and bifidobacteria from the Thoroughbred intestine and prepared the horse probiotics LacFi(TM), consisting of Lactobacillus ruminis KK14, L. equi KK 15, L. reuteri KK18, L. johnsonii KK21, and Bifidobacterium boum HU. Here, we found that the five LacFi(TM) constituent strains remarkably suppressed pro-inflammatory interleukin-17 production in mouse splenocytes stimulated with interleukin-6 and transforming growth factor-ß. The protective effects of the probiotic on impaired intestinal barrier function were evaluated in Caco-2 cells treated with tumor necrosis factor-α. Evaluation of transepithelial resistance showed that all the strains exhibited intestinal barrier protective activity, with significant suppression of barrier impairment by L. reuteri KK18. The LacFi(TM) constituent strains were detected in neonatal LacFi(TM)-administered Thoroughbred feces using polymerase chain reaction denaturing gradient gel electrophoresis and culture methods. These five strains were found to be the predominant lactobacilli and bifidobacteria in the intestinal microbiota of LacFi(TM)-administered Thoroughbreds. Administration of LacFi(TM) to neonatal Thoroughbreds decreased diarrhea incidence from 75.9% in the control group (n=29 neonatal Thoroughbreds) to 30.7% in the LacFi(TM)-administered group (n=101 neonatal Thoroughbreds) immediately after birth to 20 weeks after birth. LacFi(TM) treatment also prevented diarrhea especially at and around 4 weeks and from 10 to 16 weeks. The duration of diarrhea was also shorter in the probiotics-administered group (7.4 ± 0.8 days) than in the control group (14.0 ± 3.2 days). These results indicate that the LacFi(TM) probiotics regulates intestinal function and contributes to diarrhea prevention.

Differential modulation of innate immunity in vitro by probiotic strains of Lactobacillus gasseri.

BACKGROUND: Probiotics species appear to differentially regulate the intestinal immune response. Moreover, we have shown that different immune-modulatory abilities can be found among probiotic strains belonging to the same species. In this study, we further addressed this issue while studying L. gasseri, a species that induces relevant immune activities in human patients. RESULTS: We determined the ability of two strains of L. gasseri, OLL2809 and L13-Ia, to alter cell surface antigen expression, cytokine production and nuclear erythroid 2-related factor 2 (Nrf2)-mediated cytoprotection in murine bone marrow-derived dendritic cells (DCs) and MODE-K cells, which represent an enterocyte model. Differential effects of L. gasseri strains were observed on the expression of surface markers in mature DCs; nevertheless, both strains dramatically induced production of IL-12, TNF-α and IL-10. Distinctive responses to OLL2809 and L13-Ia were also shown in MODE-K cells by analyzing the expression of MHC II molecules and the secretion of IL-6; however, both L. gasseri strains raised intracellular glutathione. Treatment of immature DCs with culture medium from MODE-K monolayers improved cytoprotection and modified the process of DC maturation by down-regulating the expression of co-stimulatory markers and by altering the cytokine profile. Notably, bacteria-conditioned MODE-K cell medium suppressed the expression of the examined cytokines, whereas cytoprotective defenses were significantly enhanced only in DCs exposed to OLL2809-conditioned medium. These effects were essentially mediated by secreted bacterial metabolites. CONCLUSIONS: We have demonstrated that L. gasseri strains possess distinctive abilities to modulate in vitro DCs and enterocytes. In particular, our results highlight the potential of metabolites secreted by L. gasseri to influence enterocyte-DC crosstalk. Regulation of cellular mechanisms of innate immunity by selected probiotic strains may contribute to the beneficial effects of these bacteria in gut homeostasis.

Supplemental naringenin prevents intestinal barrier defects and inflammation in colitic mice.

Intestinal barrier defects are involved in the pathogenesis of inflammatory bowel disease. The present study investigated the ameliorative effects of naringenin, a citrus polyphenol, on intestinal tight junction (TJ) barrier defects and inflammation in a murine model of colitis. In Expt. 1, using a 2 × 2 fractional design, the mice were administered water or 2% dextran sulfate sodium (DSS) in combination with feeding control or naringenin-containing diets for 9 d (severe disease stage). DSS administration caused severe colon damage and inflammation, as indicated by body weight loss, increased clinical sores, colon shortening, and gene expressions of inflammatory cytokines [interferon-γ, interleukin (IL)-6, macrophage inflammatory protein-2, and IL-17A). DSS administration also impaired TJ barrier integrity in the colon, as indicated by increased colon permeability and plasma LPS-binding protein levels, resulting from the impaired colonic expression of TJ proteins, occludin, junctional adhesion molecule-A, and claudin-3. Supplemental feeding with naringenin totally or partially attenuated these symptoms, suggesting that naringenin ameliorates the DSS-induced colitis at least partially through protection of the TJ barrier. In Expt. 2, analyses were performed at different disease stages (d 3, 6, and 9) to more widely examine the ameliorative role of naringenin on the initiation and development of colitis. DSS administration moderately induced colon shortening at d 3 and 6 and increased the disease activity index (DAI) and inflammatory cytokine (IL-6 and IL-17A) expression without any significant increases in colonic permeability. Feeding naringenin attenuated the increased DAI and colon shortening and tended to suppress the increased cytokine expression. These findings suggest that the presence of an additional mechanism underlying the naringenin-mediated, anticolitic effect along with barrier protection.

Effects of a chicken collagen hydrolysate on the circulation system in subjects with mild hypertension or high-normal blood pressure.

We investigated the effects of a chicken collagen hydrolysate (CCH) on the circulation system in humans. A total of 58 subjects with either mild hypertension (systolic blood pressure (SBP) of 140-159 mmHg or diastolic blood pressure (DBP) 90-99 mmHg) or high-normal blood pressure (SBP 130-139 mmHg or DBP 85-89 mmHg) were assigned to two groups, one involving a placebo and the other, the test food (including CCH of 2.9 g/d). The parameters related to each subject's circulation system were monitored over the study period of 18 weeks. The Δbrachial-ankle pulse wave velocity (baPWV), an indicator of arterial stiffness and marker of vascular damage, was significantly lower in the test food group than in the placebo group during the treatment period. The blood pressure in the test food group was also significantly lower than that in the placebo group, while the serum nitrogen oxide was higher in the test food group after the treatment. These results suggest that CCH exerted modulatory effects on the human circulation system.

Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium.

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.

Mechanism of protection of transepithelial barrier function by Lactobacillus salivarius: strain dependence and attenuation by bacteriocin production.

Enhanced barrier function is one mechanism whereby commensals and probiotic bacteria limit translocation of foreign antigens or pathogens in the gut. However, barrier protection is not exhibited by all probiotic or commensals and the strain-specific molecules involved remain to be clarified. We evaluated the effects of 33 individual Lactobacillus salivarius strains on the hydrogen peroxide (H(2)O(2))-induced barrier impairment in human epithelial Caco-2 cells. These strains showed markedly different effects on H(2)O(2)-induced reduction in transepithelial resistance (TER). The effective strains such as UCC118 and CCUG38008 attenuated H(2)O(2)-induced disassembly and relocalization of tight junction proteins, but the ineffective strain AH43324 did not. Strains UCC118 and CCUG38008 induced phosphorylation of extracellular signal-regulated kinase (ERK) in Caco-2 cells, and the ERK inhibitor U0126 attenuated the barrier-protecting effect of these strains. In contrast, the AH43324 strain induced phosphorylation of Akt and p38, which was associated with an absence of a protective effect. Global transcriptome analysis of UCC118 and AH43324 revealed that some genes in a bacteriocin gene cluster were upregulated in AH43324 under TER assay conditions. A bacteriocin-negative UCC118 mutant displayed significantly greater suppressive effect on H(2)O(2)-induced reduction in TER compared with wild-type UCC118. The wild-type strain augmented H(2)O(2)-induced phosphorylation of Akt and p38, whereas a bacteriocin-negative UCC118 mutant did not. These observations indicate that L. salivarius strains are widely divergent in their capacity for barrier protection, and this is underpinned by differences in the activation of intracellular signaling pathways. Furthermore, bacteriocin production appears to have an attenuating influence on lactobacillus-mediated barrier protection.

Repeated exposure to water immersion stress reduces the Muc2 gene level in the rat colon via two distinct mechanisms.

The mucin family plays a number of important roles in intestinal homeostasis. Among its members, the gel-forming Muc2, produced in goblet cells, is a major component of mucus and contributes to intestinal barrier integrity. Whereas psychological stress is known to impair intestinal barrier, the effects of chronic or repeated stress on mucin expression and goblet cell differentiation have not been well documented. The present study first examined the effects of different levels of exposure (3 days, 1 and 2 weeks) to water immersion stress on intestinal mucin gene expression in rats, and then explored the mechanisms underlying the stress-induced decrease in the colonic Muc2 level. Repeated water immersion stress for 1 and 2 weeks decreased colonic Muc2 gene levels to 40% of that of non-stressed animals, while exposure to stress for 3 days induced only a 25% decrease. The goblet cell numbers counts in the colons of the 1- and 2-week stress groups, but not the 3-day stress group, were decreased to 85% of that in non-stressed animals. Cdx2 expression, a transcriptional factor related to Muc2 synthesis in the goblet cells, was decreased in all stress groups, whereas Rath1 and Klf4 expressions, transcriptional factors related to goblet cell differentiation in Notch signaling were decreased in the 1-week stress group. Collectively, the repeated exposure to water immersion stress decreases Muc2 synthesis in the goblet cells via decreased Cdx2 expression and subsequently reduces the goblet cell number via Notch signaling suppression.

Identification of a new IgE-binding epitope of peanut oleosin that cross-reacts with buckwheat.

Peanut and buckwheat induce a severe allergic reaction, anaphylaxis, which is considered to be mediated by immunoglobulin E (IgE). We identified in this study a new IgE-binding epitope of the peanut allergen that cross-reacted with buckwheat. The phosphate-buffered saline-soluble fraction of buckwheat inhibited the binding between IgE and the peanut allergen. A cross-reactive peptide was isolated from the α-chymotrypsin hydrolysate of peanut. Based on the amino acid sequence and mass spectrometric analysis data, the peptide was identified as Ser-Asp-Gln-Thr-Arg-Thr-Gly-Tyr (SDQTRTGY); this sequence is identical to amino acids 2-9 in the N-terminal hydrophilic domain of oleosin 3 which is located on the surface of the lipid storage body. Synthetic SDQTRTGY was found to bind with IgE in the sera of all eight peanut-allergic patients tested. Since many foods of plant origin contain oleosin, the possibility of an anaphylactic cross-reaction in allergic patients should always be considered.

Effect of D-alanine in teichoic acid from the Streptococcus thermophilus cell wall on the barrier-protection of intestinal epithelial cells.

D-Alanylation of teichoic acid (TA) affects various functions of Gram-positive bacteria, including immunomodulatory effects. We investigated in this study the impact of D-alanine (D-Ala) in TA from Streptococcus thermophilus ATCC 19258(T) on the barrier-protecting effect in human intestinal Caco-2 cells. ATCC 19258(T) suppressed the tumor necrosis factor-α-induced decrease in transepithelial electrical resistance (TER), an indicator of the barrier function. The D-alanylation of TA in ATCC 19258(T) was growth phase- and culture temperature-dependent. Treatment of ATCC 19258(T) with Mg(2+) decreased the dlt mRNA expression and D-Ala content in TA and also abolished the suppressive effect on the TER decrease. Supplementation with L-alanine (L-Ala) to the broth led to an increase of D-Ala in ATCC 19258(T) and of the intestinal barrier-protecting effect. Taken together, D-Ala in TA played an important role in the barrier-protecting effect of S. thermophilus in the intestinal epithelium, and these beneficial effects could be enhanced by exogenous L-Ala.

Effect of the oral intake of yogurt containing Bifidobacterium longum BB536 on the cell numbers of enterotoxigenic Bacteroides fragilis in microbiota.

Enterotoxigenic Bacteroides fragilis (ETBF) strains have been suggested to be associated with acute and persistent diarrheal disease, inflammatory bowel disease and colorectal cancer, although further epidemiological studies are needed for clarification. Here, a pilot study was performed to examine the effect of the oral administration of yogurt supplemented with a probiotic strain on the cell numbers of fecal ETBF in a healthy population. Among 420 healthy adults, 38 subjects were found to be ETBF carriers, giving a prevalence of approximately 9%. Among them, 32 subjects were enrolled in an open, randomized, parallel-group study to ingest yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536 (BB536Y group), for 8 weeks, with milk provided to the control group (milk group). The cell numbers of ETBF and the dominant species of the B. fragilis group were measured by a quantitative PCR method. Compared with the baseline values, there was a significant decrease in the cell number of ETBF at week 8 in the BB536Y group but not in the milk group. Linear mixed models analysis for longitudinal data revealed a significant difference in the changes of ETBF cell number between the two groups during the intervention phase. These results imply the potential of probiotic yogurt for eliminating ETBF in the microbiota, but its clinical significance needs to be evaluated in the future. This is the first report of a possible effect of probiotic intake on ETBF in the microbiota.

Kaempferol enhances intestinal barrier function through the cytoskeletal association and expression of tight junction proteins in Caco-2 cells.

Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. We investigated the promotive effect of kaempferol on tight junction (TJ) barrier integrity in human intestinal Caco-2 cell monolayers. Transepithelial electrical resistance (TER; a TJ integrity marker) across the monolayers rapidly and markedly increased during the first 6 h after kaempferol administration and remained elevated until 48 h without any changes in the lucifer yellow or dextran fluxes. Immunoblot analysis demonstrated that kaempferol promoted the actin cytoskeletal association of the TJ proteins, zonula occludens (ZO)-1, ZO-2, occludin, claudin-1, claudin-3, and claudin-4, which was associated with the increase in TER. Kaempferol-mediated ZO-2 and claudin-4 expression was relatively smaller or occurred later than the kaempferol-promoted cytoskeletal association. Confocal microscopy showed that kaempferol-induced assembly of occludin and claudin-3 occurred at the TJ at 6 h postadministration. Extraction of cholesterol with methyl-ß-cyclodextrin suppressed the kaempferol-mediated increase in TER. Sucrose density gradient centrifugation showed that the kaempferol treatment increased the TJ protein distributions in the cholesterol-rich lipid microdomain fraction. Taken together, these results indicate that the membrane lipid microdomain is involved in the kaempferol-mediated promotion of TJ protein assembly and intestinal TJ integrity.

Streptococcus thermophilus ST28 ameliorates colitis in mice partially by suppression of inflammatory Th17 cells.

The effects of Streptococcus thermophilus ST28 on cytokine production by murine splenocytes stimulated with transforming growth factor-ß plus interleukin- (IL-) 6 were evaluated. The addition of ST28 significantly repressed IL-17 production compared to ATCC 19258 (type strain). ST28 also decreased the number of Th17 cells in the stimulated splenocytes. The anti-inflammatory effects of ST28 administration were evaluated in mice with colitis induced by dextran sodium sulphate (DSS). Oral treatment of mice with ST28 ameliorated the intestinal lesions by DSS. Upon DSS treatment, IL-17 production in lamina propria lymphocytes (LPLs) was induced, but ST28 significantly decreased its production. ST28 also decreased the percentage of Th17 cells in LPL from DSS-induced colitis. The present results imply that ST28 suppresses the Th17 response in inflamed intestines and would be useful in the treatment of Th17-mediated diseases, such as inflammatory bowel disease.

Bifidobacterium kashiwanohense sp. nov., isolated from healthy infant faeces.

Strains HM2-1 and HM2-2(T) were isolated from the faeces of a healthy infant and were characterized by determining their phenotypic and biochemical features and phylogenetic positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive, obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile rods. They did not grow at 15 or 45 °C in anaerobic bacterial culture medium, and their DNA G+C content was in the range 56-59 mol%. In enzyme activity tests, strains HM2-1 and HM2-2(T) were positive for α/ß-galactosidases and α/ß-glucosidases but negative for ß-glucuronidase and cystine arylamidase. An analysis of the cell-wall composition of strains HM2-1 and HM2-2(T) revealed the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate phosphoketolase shows that isolates HM2-1 and HM2-2(T) are members of the genus Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium. Strain HM2-2(T) was found to be related to Bifidobacterium catenulatum JCM 1194(T) (97.4 % 16S rRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200(T) (97.2 %: 1472/1514 bp), Bifidobacterium dentium ATCC 27534(T) (96.7 %: 1459/1509 bp) and Bifidobacterium angulatum ATCC 27535(T) (96.5 %: 1462/1515 bp). The predominant cellular fatty acids of strains HM2-1 and HM2-2(T) were 16 : 0 and 18 : 1ω9c, with proportions greater than 18 % of the total. Phylogenetic analyses involving phenotypic characterization, DNA-DNA hybridization and partial 16S rRNA gene sequencing proves that the strains represent a novel species of the genus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed. The type strain is HM2-2(T) ( = JCM 15439(T) = DSM 21854(T)).

Quantification of pork, chicken and beef by using a novel reference molecule.

A standard plasmid was constructed as a novel reference molecule for use in real-time quantitative PCR assays to verify the identity of beef, pork, chicken, mutton, and horseflesh. The plasmid contained a target domain of the cytochrome b (cyt b) gene and an artificial DNA sequence. Primers CO-F and CO-R, and probe CO-P were specifically designed to detect the artificial sequence. The calculated R² values of the standard curves (10³-107 copies per reaction) for the five species ranged between 0.998 and 0.999 in the quantification analysis. The constructed plasmid provides a universal method for measuring the copy number of cyt b DNA in minced meat. This method would be a useful procedure for verifying food labels.

Degradation of interleukin 8 by the serine protease MucD of Pseudomonas aeruginosa.

We investigated the influence of the type III effector, ExoS, on the host epithelial cell response to Pseudomonas aeruginosa infection, and we found that disruption of the exoS gene caused a significant increase in the amount of interleukin-8 (IL-8) in the culture medium of Caco-2 cells. We show that IL-8 was degraded in the culture medium following infection of the cells with the wild-type (PAO1), but not the exoS knock-out (the ΔexoS) strain. Purified ExoS protein itself did not degrade IL-8. We next show that IL-8 degradation by PAO1 was inhibited by the addition of serine protease inhibitors. These results strongly suggest that a bacterial serine protease that degrades IL-8 is expressed and secreted into the culture medium of Caco-2 cells infected with PAO1, and that the expression of this protein is repressed in cells infected with the ΔexoS strain. The PAO1 genome encodes 28 different protease genes, including two serine proteases: PA3535 and mucD. PA3535 and mucD gene knock-outs were constructed (ΔmucD and ΔPA3535), and ΔmucD but not ΔPA3535 showed reduced IL-8 degradation. To understand the significance of IL-8 degradation, we next evaluated neutrophil infiltration in lungs excised from mice intranasally infected with the P. aeruginosa strains. Increased neutrophil infiltration was observed in PAO1-infected mice, but not in ΔexoS- or ΔmucD-infected mice. Taken together, our results suggest that P. aeruginosa escapes from phagocytic killing due to IL-8 degradation following the secretion of the MucD serine protease, whose expression appears to be influenced by ExoS.