[Show abstract][Hide abstract] ABSTRACT: Adipose tissue-derived stem cells (ADSCs) isolated from adult tissue have pluripotent differentiation and self-renewal capability. The tissue source of ADSCs can be obtained in large quantities and with low risks, thus highlighting the advantages of ADSCs in clinical applications. Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to affect ADSC differentiation in mice and rats; however, few studies have been performed on dogs. We aimed to examine the in vitro effect of VPA on canine ADSCs. Three days of pretreatment with VPA decreased the proliferation of ADSCs in a dose-dependent manner; VPA concentrations of 2 mM and above inhibited the proliferation of ADSCs. In parallel, VPA increased p16 and p21 mRNA expression, suggesting that VPA attenuated the proliferative activity of ADSCs by activating p16 and p21. Furthermore, the effects of VPA on adipogenic, osteogenic or neurogenic differentiation were investigated morphologically. VPA pretreatment markedly promoted neurogenic differentiation, but suppressed the accumulation of lipid droplets and calcium depositions. These modifications of ADSCs by VPA were associated with a particular gene expression profile, viz., an increase in neuronal markers, that is, NSE, TUBB3 and MAP2, a decrease in the adipogenic marker, LPL, but no changes in osteogenic markers, as estimated by reverse transcription-PCR analysis. These results suggested that VPA is a specific inducer of neurogenic differentiation of canine ADSCs and is a useful tool for studying the interaction between chromatin structure and cell fate determination.
Journal of Veterinary Medical Science 08/2013; · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aqueous extracts of Rhizopus oryzae (Aq-ROU) have a broad range of physiological activity. Here we identified a new physiological effect of Aq-ROU in rat hepatocyte cell line RLN-10. Aq-ROU induced the accumulation of nitrite, a stable metabolite nitric oxide (NO), in cell culture medium and induced potent diaminofluorescein-FM diacetate staining in the cells. Real-time reverse transcriptase (RT)-PCR analysis showed marked inducible NO synthase gene expression. Additionally, markedly enhanced expression of p22(phox) and temporally increased expression of NADPH oxidase1 indicated that superoxide was produced. Nuclear translocation of nuclear factor-kappa (NF-κ) B p65 increased remarkably following Aq-ROU and following lipopolysaccharide treatment, a potent activator of NF-κB. Ammonium pyrrolidine-1-carbodithioate, an inhibitor of NF-κB, inhibited NO production following Aq-ROU treatment. Our data indicate that Aq-ROU induces NO production and potentially the production of superoxide, which may contribute to the broad range of physiological effects observed for Aq-ROU ingested by animals.
Bioscience Biotechnology and Biochemistry 07/2013; · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.
Bioscience Biotechnology and Biochemistry 05/2013; · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adlercreutzia equolifaciens DSM 19450(T) was isolated from human feces and is able to metabolize daidzeins (soybean isoflavonoids) to equol. Here, we report the finished and annotated genome sequence of this organism.
[Show abstract][Hide abstract] ABSTRACT: Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish.
PLoS ONE 01/2011; 6(8):e23184. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that the dilating effect of nitric oxide (NO) on the fetal ductus arteriosus (DA) is age dependent and more marked in the premature stages in rats, but the factors that mediate this effect are poorly understood. The purpose of this study is to determine the changes in the expression of NO synthase (NOS) mRNA in the fetal DA and to examine the effect of an 11-beta-hydroxylase inhibitor of corticosterone synthesis, namely, metyrapone, on NOS expression. NOS 3 mRNA expression was observed in 17.5-day-old rat fetuses; thereafter, its level significantly increased and reached its peak on day 19.5 and then decreased until the end of the gestation period (day 21.5). To inhibit corticosterone synthesis, a constant infusion of metyrapone was administered to rats; this significantly decreased the fetal plasma corticosterone concentration as well as NOS 3 mRNA expression in the DA in a time-dependent manner. These results indicate that NO is generated by NOS 3 in the DA and that the age-dependant expression of NOS 3 in the premature DA is attributable to corticosterone-associated activity.
Journal of Veterinary Medical Science 05/2010; 72(5):555-60. · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously reported that nitric oxide (NO) is first detected in the uterus of a pregnant rat on gestational day 13.5 (GD13.5) and that NO levels peak on GD17.5. In addition, NO production in the uterus is mainly derived from the decidua and not the myometrium. The aim of the present study was to reveal the role of NO that peaked on GD17.5 of gestation in the decidua. To inhibit NO production, pregnant rats were continuously administered by an nitric oxide synthase inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME) for 48 h. In the control group, saline was infused instead of L-NAME. After treatment, the decidua were obtained from GD13.5, GD17.5 and GD21.5 rats. Apoptosis and activated caspase-3-positive cells were observed by transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemistry, respectively. The caspase-3 enzyme activity was also measured in the cell lysate from the decidua. The numbers of TUNEL-positive cells and activated caspase-3-positive cells each increased and the amount of caspase-3 activity also increased significantly in rats on GD17.5 than in rats in the control group, but no changes were observed in rats on GD13.5 and GD21.5. Furthermore, enzyme activity regarding the initiator caspases, caspase-8 and -9, upstream factors for caspase-3 in the caspase cascade, was measured simultaneously on GD17.5 under the same treatment. Caspase-8 and -9 enzyme activities increased significantly in the control group; an increment of caspase-8 activity was especially prominent. The present results indicate that an inhibitor of NO production caused apoptosis through typical apoptotic signals in the decidua on GD17.5, suggesting that an NO peak in the decidua is essential to cell survival and the maintenance of uterine formation.
Experimental Biology and Medicine 04/2010; 235(4):455-62. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) has been reported as a key mediator in enhancing hepatocyte proliferation during liver regeneration. Juvenile hepatocytes have a strong ability to proliferate while still in their undifferentiated state but the mechanism of NO production and its contribution to hepatocyte proliferation are not yet fully understood. The present study was designed to investigate NO production in the normal liver and its contribution to hepatocyte proliferation in juvenile rats. Endogenous NO production was evaluated quantitatively using a spin trap followed by electron paramagnetic resonance spectroscopy with the Fe-N, N-diethyldithiocarbamate complex as an NO-trapping reagent in the rat liver. NO production in the liver significantly peaked at 3 weeks after birth, but NO synthase (NOS) 3 expression did not change between 2 to 5 weeks after birth, while NOS 1 and NOS 2 mRNA were not detected. Hepatocyte proliferation, measured by the incorporation of 5-bromo-2'-deoxyuridine into the DNA, was found to decline significantly when endogenous NO production was inhibited by the administration of the NOS inhibitor N(G)-nitro- (L)-arginine methyl ester. These findings indicate that endogenous NO production peaked at 3 weeks after birth and hepatocyte proliferation declined significantly when NO production was inhibited. Thus, this study provides a novel insight into the contribution of NO to hepatic growth and liver maturation in juveniles.
Journal of Veterinary Medical Science 02/2010; 72(7):861-7. · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We clarified nitric oxide (NO) production in the rat uterus by electron paramagnetic resonance spectroscopy and with Fe-N-(dithiocarboxy) sarcosine complex (an NO-trapping reagent). We examined changes in NO production in the whole uterus, decidua, and myometrium (gestational days 13.5-21.5). The expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative reverse transcription-polymerase chain reaction. The uterine NO levels were low on day 13.5, peaked on day 17.5, and thereafter decreased significantly. The NO production levels in the decidua and myometrium were the same on day 13.5, but the levels in the decidua were 2- to 4-fold higher than those in the myometrium from day 15.5 onwards. The NOS-2 mRNA expression pattern correlated well with changes in the NO levels in the decidua, whereas the NOS-3 mRNA was expressed constantly during gestation. Thus NOS-2-generated NO in the decidua contributed significantly to uterine NO levels.
Bioscience Biotechnology and Biochemistry 10/2009; 73(10):2163-6. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To monitor changes in NO production over time in the fetal placenta of rats, we used electron paramagnetic resonance spectroscopy with the Fe-N-(dithiocarboxy) sarcosine (Fe-DTCS) complex as an NO-trapping reagent. The expression of nitric oxide synthase (NOS) isoforms was examined in parallel using quantitative RT-PCR. NO production was first detected on day 13.5 of gestation. NO levels reached a peak on day 15.5, then decreased significantly during the last few days of gestation. The pattern of expression of NOS II mRNA was in good agreement with changes in NO levels, whereas NOS III mRNA expression did not change markedly during gestation. Thus, it appears that NO levels in the placenta are NOS II-dependent and differ at different gestational stages.
Journal of Veterinary Medical Science 05/2009; 71(4):495-8. · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.
DNA Research 07/2008; 15(3):151-61. · 4.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of the endocrine-disrupting chemical 3,3′,4,4′,5-pentachlorobiphyenl (PCB 126) on intestinal microbiota after oral administration, and the improvement of intestinal microbiota and feces quantity by the subsequent administration of Lactobacillus acidophilus or Lactobacillus reuteri was investigated. All the rats were given 100 μg/kg bodyweight of PCB 126. The changes in bacterial counts were confirmed using a culture method. The administration of PCB 126 tended to decrease the bacterial counts of lactobacilli (109.6−1010.2 to 108.8−109.2) and bifidobacteria (105.3−106.1 to 103.6−104.2), and to increase those of Enterobacteriaceae (108.2−109.1 to 109.4−1010.3) and staphylococci (106.6−107.4 to 107.2−108.4) compared to no PCB 126 administration. After administration of PCB 126, L. acidophilus or L. reuteri orally administered to rats caused Enterobacteriaceae and staphylococci counts to decrease, suggesting that the intestinal microbiota was improved by the lactobacilli. The administration of L. acidophilus and L. reuteri improved the balance of intestinal microbiota, and defecation volume returned to its normal level. L. acidophilus and L. reuteri have a remedial effect on intestinal microbiota affected by PCB 126 and can function to lessen accumulated PCB 126 volume.
[Show abstract][Hide abstract] ABSTRACT: Cell-surface Toll-like receptors (TLRs) initiate innate immune responses, such as inducible nitric oxide synthase (iNOS) induction, to microorganisms' surface pathogens. TLR2 and TLR4 play important roles in gastric mucosa infected with Helicobacter pylori (H. pylori), which contains lipopolysaccharide (LPS) as a pathogen. The present study investigates their physiological roles in the innate immune response of gastric epithelial cells to H. pylori-LPS. Changes in the expression of iNOS, TLR2, and TLR4, as well as downstream activation of mitogen-activated protein kinases and nuclear factor-kappaB (NF-kappaB), were analyzed in normal mouse gastric mucosal GSM06 cells following stimulation with H. pylori-LPS and interferon-gamma. Specific inhibitors for mitogen-activated protein kinases, NF-kappaB, and small interfering RNA for TLR2 or TLR4 were employed. The immunohistochemistry of TLR2 was examined in human gastric mucosa. H. pylori-LPS stimulation induced TLR2 in GSM06 cells, but TLR4 was unchanged. TLR2 induction resulted from TLR4 signaling that propagated through extracellular signal-related kinase and NF-kappaB activation, as corroborated by the decline in TLR4 expression on small interfering RNA treatment and pretreatment with inhibitors. The induction of iNOS and the associated nitric oxide production in response to H. pylori-LPS stimulation were inhibited by declines in not only TLR4 but also TLR2. Increased expression of TLR2 was identified in H. pylori-infected human gastric mucosa. TLR4 signaling initiated by H. pylori-LPS and propagated via extracellular signal-regulated kinase and NF-kappaB activation induced TLR2 expression in gastric epithelial cells. Induced TLR2 cooperated with TLR4 to amplify iNOS induction. This positive correlation may constitute a mechanism for stimulating the innate immune response against various bacterial pathogens, including H. pylori-LPS.
[Show abstract][Hide abstract] ABSTRACT: In this study, the main changes in bacterial floral diversity in the gastrointestinal tract of a Thoroughbred foal were monitored by using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). The foal died of catarrhal enteritis of the cecum and large colon. Diarrheal feces and gastrointestinal contents were compared with normal feces. The closest relatives of the bacterium in the samples were Lactobacillus johnsonii (100% similarity), uncultured Bacteroides sp. (92.5% similarity), Bacteroides fragilis (96.3% similarity), and Enterococcus faecium/Enterococcus durans (100% similarity); these were detected by PCR-DGGE using a universal primer set. Monitoring revealed that the numbers of Escherichia coli/Shigella sonnei (97.9% similarity) were significantly higher in the diarrheal feces. Thus, PCR-DGGE is a useful tool for monitoring the main changes in bacterial floral diversity occurring in the gastrointestinal tracts of Thoroughbreds.
Journal of Equine Veterinary Science - J EQUINE VET SCI. 01/2007; 27(1):14-19.
[Show abstract][Hide abstract] ABSTRACT: Protection against in vivo infection with Salmonella and enhancement of in vitro super- oxide production by peripheral blood neutrophils are two reported effects o ft reatment with aque- ous extracts o ft he fungus Rhizopus oryzae U-1 (Aq-ROU). Here, we report that Aq-ROU also has antiproliferative activity and can induce apoptosis in a human promyelocytic leukemia cell line, HL-60. During Aq-ROU-induced apoptosis, HL-60 cells undergo genomic DNA fragmentation characteristic of apoptosis after just 6 hr of treatment. Using phos- phatidylserine (PS) as an indicator of apoptosis, we also found that the proportion o fa poptotic cells in- creased significantly after 9 hr of treatment. Indeed, induction of apoptosis by Aq-ROU reached 43.3% after 24 hr of treatment, which is comparable to the effects of a known apoptosis-inducer, actinomycin D. Moreover, the activities of caspase-3, -8, and -9 increased in parallel with Aq-ROU treatment, with a peak of activity 9 hr after the initial treatment. Taken together, the results suggest that R. oryzae contains one or more water-soluble factor that can reliably an de fficiently induce apoptosis in human cells via activation of caspase-3.
Journal of Health Science - J HEALTH SCI. 01/2007; 53(6):760-765.
[Show abstract][Hide abstract] ABSTRACT: The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.
Bioscience Biotechnology and Biochemistry 06/2004; 68(5):1027-34. · 1.27 Impact Factor