Tatsuya Takizawa

Comparative Genome Analysis of Lactobacillus reuteri and Lactobacillus fermentum Reveal a Genomic Island for Reuterin and Cobalamin Production

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 1112T 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 1112T has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B12). 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 13C3-glycerol demonstrated that L. reuteri JCM 1112T could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112T 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.

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/β-galactosidases and α/β-glucosidases but negative for β-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)).

Onset of the Constrictive Effect of Indomethacin on the Ductus arteriosus in Fetal Rats

The time of onset of the constrictive effect of indomethacin on the ductus arteriosus (DA) in fetal rats was assessed by measurement of the caliber of the DA after maternal treatment with indomethacin on days 19-21 of gestation. The day following overnight mating was regarded as day 0 of gestation. Observation was performed by direct exposure of the DA by hand shaving of intact frozen fetuses. On days 20 and 21, the DA was significantly constricted 3 h after maternal treatment with 1 mg/kg of indomethacin. When the DA was examined at 19 1/2 and 19 2/3 days of gestation (3 h after indomethacin exposure), it was significantly constricted at 19 2/3 days but not at 19 1/2 days. Higher doses of indomethacin (10 and 100 mg/kg) induced a significant constriction of the DA at day 19 1/2, but not at the beginning of the same day (1.00 a.m.). These results suggest that the onset of the susceptibility of the DA to the constrictive effect of indomethacin occurs in the first half of day 19 of gestation.

Increased constriction of the ductus arteriosus with combined administration of indomethacin and L-NAME in fetal rats.

We studied age-related changes and the caliber of the ductus arteriosus (DA) after two-pathway inhibition of prostaglandin E2 and nitric oxide (NO) by the combined administration of indomethacin, a cyclooxygenase inhibitor, and NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in fetal rats. Pregnant rats from day 18 to 21 of gestation were used. They were administered indomethacin orally (3 mg/kg) 3 h before cesarean section, and then L-NAME (50 mg/kg) was injected intraperitoneally 3 h before the rats were killed. Using rapid-freezing and shaving methods, the caliber of the DA in fetal rats was measured. Compared with the indomethacin alone group, indomethacin plus L-NAME further constricted the DA after indomethacin and L-NAME were simultaneously administered 3 h before the rats were sacrificed. The extent of the final DA constriction was almost equal to the addition of each effect of indomethacin and L-NAME. We concluded that the magnitude of DA constriction following indomethacin plus L-NAME was due to the additive effects of these agents, suggesting a possible method to treat patent DA in premature infants.

Nitric oxide induces vascular endothelial growth factor expression in the rat placenta in vivo and in vitro.

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.

Molecular phylogeny and diversity of Myanmar and Bhutan mithun based on mtDNA sequences.

The mithun (Bos frontalis), synonymous with mithan and gayal, is considered to be a domesticated form of gaur (B. gaurus). However, there has been a controversy concerning its origin. In an effort to address this issue, the mitochondrial cytochrome b (cytb) genes of 20 mithun from Myanmar and 13 from Bhutan were sequenced to trace its maternal origin. Seven cytb haplotypes were found in the 33 mithun, and the phylogenetic tree for these haplotypes clearly showed three embranchments involving five gaur types, a B. indicus type, and a B. taurus type. Sixteen Myanmar and 12 Bhutan mithun had gaur haplotypes, while a B. indicus haplotype was found in three Myanmar and one Bhutan mithun. The B. taurus haplotype was detected in a single Myanmar animal. These results demonstrated that the principal maternal origin of mithun was gaur and suggested that it was directly domesticated from gaur. However, some introgression of domestic cattle existed in current mithun populations. The presence of cattle mtDNA raised the question of how many cattle nuclear genes might have been integrated into the gene pool of mithun.

Effect of Acute Maternal Alcohol Consumption on the Fetal Ductus arteriosus in the Rat

This study was conducted to determine whether acute alcohol consumption in near-term pregnant rats results in constriction of the fetal ductus arteriosus (DA). Twenty milliliters per kilogram of 30% (v/v) alcohol was administered via a stomach tube 0.5, 2, 4, 6 and 12 h prior to cesarean section and fetal sacrifice, at 1.00 p.m. on the 20th gestational day (day 20.5), in experimental groups A1-A5, respectively. Controls were given water alone. The calibers of the DA and pulmonary artery (PA) were measured using the whole-body freezing method with direct exposure of the DA and PA by shaving the frozen chests of the fetuses. The DA was significantly constricted 30 min after alcohol treatment. The constriction was also observed in groups A2-A4, but not in group A5. The PA was also less markedly but significantly constricted 30 min after alcohol administration, but groups A2-A5 showed normal PA caliber. It is concluded that alcohol has a constrictive effect on the DA when administered close to the end of gestation in the rat.

Changes in nitric oxide production levels and expression of nitric oxide synthase isoforms in the rat uterus during pregnancy.

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.

An insertion/deletion variant of a thymine base in exon 2 of the porcine beta 3-adrenergic receptor gene associated with loin eye muscle area.

An insertion/deletion variant of a thymine base (T5 and T6) in exon 2 of porcine beta 3-adrenergic receptor (ADRB3) gene has been described. In the current study, we made an association study between the ADRB3 polymorphisms and production traits in 735 Duroc pigs. The allele frequencies for the T5 and T6 alleles in our study population were 0.433 and 0.567, respectively. Any associations between ADRB3 genotype and average daily weight gain during test period, or backfat thickness and intramuscular fat content were not detected in either sex. However the size of the loin eye muscle area (EMA) was significantly associated with ADRB3 genotypes in gilts. T6-homozygous gilts had a higher mean of EMA (40.6 +/- 0.6 cm(2)) than T5-homozygous (38.1 +/- 0.4 cm(2), P = 0.002) and heterozygous (38.8 +/- 0.3 cm(2), P = 0.034) gilts. This association was not detected in males. In addition, a multiple traits animal model best linear unbiased predictor (BLUP) analysis revealed that the T6-homozygous genotype had positive effects on breeding value of EMA. Accordingly, we suggest that ADRB3 polymorphism has the potential to be an important genetic marker for prediction of EMA in Duroc pigs.

Association of an SNP marker in exon 24 of a class 3 phosphoinositide-3-kinase (PIK3C3) gene with production traits in Duroc pigs.

A C↔T single nucleotide polymorphism (SNP) on exon 24 of the porcine class 3 phosphoinositide-3-kinase (PIK3C3) gene is considered a possible genetic marker for selecting backfat (BF) thickness and carcass fat, although only one study has published results on its effects by performing experiments on a single resource family. We analyzed the association of this PIK3C3 polymorphism with production traits in 739 Duroc pigs. The C allele frequency was 67.9% in our study population. PIK3C3 polymorphism showed significant effects on average daily weight gain (ADG), BF thickness, intermuscular fat content (IMF), and the size of the loin eye muscle area (EMA). The C alleles increased ADG, BF and IMF, and decreased EMA. The predicted differences in traits between the homozygous pigs of the C and T alleles were 40 g/day for DG, 1.2 mm for BF, 0.44% for IMF, and 1.6 cm(2) for EMA. Furthermore, the statistical models for estimating the breeding values of each trait had lower Akaikes information criterion values when adding PIK3C3 genotype information. We therefore confirmed that the polymorphism in PIK3C3 (C2604T) has the potential to be a genetic marker for production traits in Duroc pigs.