Differential expression of cyclophilin A and EMMPRIN in developing molars of rats.

A complex and intricate cascade of gene expression is essential for late stage tooth development. This study was performed to detect molecules involved in dental hard tissue formation and tooth eruption by comparing gene expression in cap stage molar germs (before eruptive movement and dental hard tissue formation) with that in root formation stage molar germs (after eruptive movement and dental hard tissue formation). DD-PCR revealed that cyclophilin A (Cyp-A), a potent chemoattractant for monocytes as well as a ligand for extracellular matrix metalloproteinase inducer (EMMPRIN) was expressed differentially in the two stages molar germs. The levels of Cyp-A and EMMPRIN mRNA were significantly higher at the root formation stage than at the cap and crown stages of the molar germs. Immunofluorescence showed that Cyp-A and EMMPRIN were expressed strongly in the follicular cells overlaying the occlusal region of the molar germs at the root formation stage. In contrast, their immunoreactivity was weak in the follicular tissues and was not region-specific in molar germs at the cap stage. In addition, the MCP-1 and CSF-1 mRNA levels increased in parallel to that of Cyp-A mRNA and the increased number of osteoclasts at the occlusal region. Immunoreactivity against Cyp-A and EMMPRIN was also observed in the fully differentiated ameloblasts and odontoblasts. This study suggests that Cyp-A and EMMPRIN play roles in the maturation of dental hard tissue and the formation of an eruption pathway.

Relaxin is up-regulated in the rat ovary by orthodontic tooth movement.

Relaxin (Rln) is an ovarian hormone that stimulates osteoclastic and osteoblastic activities and connective tissue turnover. To investigate the expression of Rln during orthodontic tooth movement, rats were implanted with orthodontic appliances that connected a spring from the upper incisors to the first molar with a 70 cN force. Rats in each group were killed 6, 48, and 144 h after activating the appliance, and the levels of Rln1 and Rln3 expression in the ovary were determined by real-time RT-PCR, northern blots, western blots, and immunofluorescence analyses. The amount of tooth movement induced by the orthodontic force increased in a time-dependent manner. The levels of Rln1 mRNA increased by 12-, 41-, and 263-fold at 6, 48, and 144 h, respectively, after orthodontic tooth movement. The time-dependent increase in the concentration of Rln 1 protein in the ovary was also confirmed by western blotting. Rln 1 was localized in the granulosa cells of the ovarian follicles, and the immunoreactivity against Rln 1 was increased by the movement. In contrast, the concentration of Rln 3 was below the level of detection. The results of this study suggest that local changes in periodontal tissues induced by orthodontic tooth movement may affect Rln1 expression in the ovary. However, further studies are needed to decipher the mechanisms involved and the possible contribution of the increased level of expression of Rln 1 to the tooth movement.

Change of Bacillus cereus flavonoid O-triglucosyltransferase into flavonoid O-monoglucosyltransferase by error-prone polymerase chain reaction.

The attachment of sugar to flavonoids enhances their solubility. Glycosylation is performed primarily by uridine diphosphate-dependent glycosyltransferases (UGTs). The UGT from Bacillus cereus, BcGT-1 transferred three glucose molecules into kaempferol. The structural analysis of BcGT-1 showed that its substrate binding site is wider than that of flavonoid monoglucosyltransferase of plant. In order to create monoglucosyltransferase from BcGT-1, error-prone polymerase chain reaction (PCR) was performed. We analyzed 150 clones. Among them, two mutants generated only kaempferol O-monoglucoside, albeit with reduced reactivity. Unexpectedly, the two mutants harbored mutations in the amino acids located outside of the active sites. Based on the modeled structure of BcGT-1, it was proposed that the local change in the secondary structure of BcGT-1 caused the alteration of triglucosyltransferase into monoglucosyltransferase.

Production of genistein from naringenin using Escherichia coli containing isoflavone synthase-cytochrome P450 reductase fusion protein.

Isoflavonoids are a class of phytoestroegens. Isoflavonone synthase (IFS) is responsible for the conversion of naringenin to genistein. IFS is a cytochrome P450 (CYP), and requires cytochrome P450 reductase (CPR) for its activity. Additionally, the majority of cytochrome P450s harbor a membrane binding domain, making them difficult to express in Escherichia coli. In order to resolve these issues, we constructed an in-frame fusion of the IFS from red clover (RCIFS) and CPR from rice (RCPR) after removing the membrane binding domain from RCIFS and RCPR reductase. The resultant fusion gene, RCIFS-RCPR, was expressed in E. coli. The conversion of naringenin into genistein was confirmed using this E. coli transformant. Following the optimization of the medium and cell density for biotransformation, 60 microM of genistein could be generated from 80 microM of naringenin. This fusion protein approach might be applicable to express other P450s in E. coli.