Characterization of a pseudomonad 2-nitrobenzoate nitroreductase and its catabolic pathway-associated 2-hydroxylaminobenzoate mutase and a chemoreceptor involved in 2-nitrobenzoate chemotaxis

Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the beta5-alpha2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.     

Sonic hedgehog expression during early tooth development in Suncus murinus

Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.     

Intestinal Microbiota is Different in Women with Preterm Birth: Results from Terminal Restriction Fragment Length Polymorphism Analysis

Preterm birth is a leading cause of perinatal morbidity and mortality. Studies using a cultivation method or molecular identification have shown that bacterial vaginosis is one of the risk factors for preterm birth. However, an association between preterm birth and intestinal microbiota has not been reported using molecular techniques, although the vaginal microbiota changes during pregnancy. Our aim here was to clarify the difference in intestinal and vaginal microbiota between women with preterm birth and women without preterm labor. 16S ribosomal ribonucleic acid genes were amplified from fecal and vaginal DNA by polymerase chain reaction. Using terminal restriction fragment length polymorphism (T-RFLP), we compared the levels of operational taxonomic units of both intestinal and vaginal flora among three groups: pregnant women who delivered term babies without preterm labor (non-PTL group) (n = 20), those who had preterm labor but delivered term babies (PTL group) (n = 11), and those who had preterm birth (PTB group) (n = 10). Significantly low levels of Clostridium subcluster XVIII, Clostridium cluster IV, Clostridium subcluster XIVa, and Bacteroides, and a significantly high level of Lactobacillales were observed in the intestinal microbiota in the PTB group compared with those in the non-PTL group. The levels of Clostridium subcluster XVIII and Clostridium subcluster XIVa in the PTB group were significantly lower than those in the PTL group, and these levels in the PTL group were significantly lower than those in non-PTL group. However, there were no significant differences in vaginal microbiota among the three groups. Intestinal microbiota in the PTB group was found to differ from that in the non-PTL group using the T-RFLP method.     

Dietary supplementation with fructooligosaccharides attenuates allergic peritonitis in mice

Fructooligosaccharides (FOS) are a prebiotic supplement, which can enhance immunological responses in the host to activate mucosal immunity probably through regulation of gastrointestinal microflora. Nonetheless, the therapeutic potential of prebiotics on allergic pathologies has not been fully elucidated. Therefore, the purpose of this study was to evaluate the preventive and therapeutic effects of dietary supplementation with FOS on a murine model of allergic peritonitis induced by ovalbumin (OVA). Male C3H/HeN mice were intraperitoneally administrated with OVA (1 μg) bi-weekly (Day 0-42, total four times) and were fed a diet containing 0 or 2.5% FOS ad libitum (Day 7-43). At Day 43, mice were killed and several parameters were evaluated. As results, supplementation with FOS alleviated OVA-related peritoneal inflammation characterized by trafficking of polymorphonuclear leukocytes such as eosinophils and neutrophils in the peritoneal cavity. Also, FOS significantly suppressed the protein level of interleukin (IL)-5 and eotaxin in the peritoneal lavage fluid elicited by OVA. In addition, a FOS-supplemented diet significantly reduced the serum allergen specific-IgG(1) level, whereas it significantly increased total IgA levels in the cecal contents as compared with a control diet in the presence of OVA. These results suggest that dietary supplementation with FOS can prevent/ameliorate allergic peritoneal inflammation induced by OVA. The efficacy can at least partially be associated with the regulation of Ig class switching and inhibition of the local expression of IL-5 and eotaxin.     

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.     

Quantitative regulation of histone variant H2A.Z during cell cycle by ubiquitin proteasome system and SUMO-targeted ubiquitin ligases

Quantitative control of histones and histone variants during cell cycle is relevant to their epigenetic functions. We found that the level of yeast histone variant H2A.Z in the G2/M-phase is actively kept low by the ubiquitin proteasome system and SUMO-targeted ubiquitin ligases. Overexpression of H2A.Z induced defects in mitotic progression, suggesting functional importance of this quantitative control.     

Biotransformation of isoeugenol and eugenol by cultured cells of Eucalyptus perriniana.

Three new biotransformation products, eugenyl beta-rutinoside, and isoeugenyl beta-gentiobioside and beta-rutinoside, together with eugenyl beta-glucoside and beta-gentiobioside, and isoeugenyl beta-glucoside, were isolated from jar fermentor culture of Eucalyptus perriniana following administration of eugenol and isoeugenol, respectively. This is the first report of rhamnosylation in a biotransformation catalysed by cultured cells of E. perriniana.     

Isolation of two human fibroblastic cell populations with multiple but distinct potential of mesenchymal differentiation by ceiling culture of mature fat cells from subcutaneous adipose tissue

Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.