Immunolocalization of FGF-2, -7, -8, -10 and FGFR-1-4 during regeneration of the rat submandibular gland.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play important roles in the development of the submandibular gland. Although regeneration of submandibular glands follows a similar process to their development, it is unknown how FGFs and FGFRs are distributed during regeneration of submandibular gland. The aim of this study was to determine the localization of FGFs and FGFRs during such regenerative processes. After 7 days' obstruction, the submandibular glands were collected at days 0, 1, 3, 7, 11 and 14 after duct release to study regeneration. The regenerative processes of the submandibular gland were investigated by immunohistochemistry for FGF-2, 7, 8, 10 and FGFR-1-4. Immunohistochemical staining revealed that FGF-2 was moderately expressed in the epithelial cells of duct-like structures (DLS) and newly formed acinar cells (NFAC) at days 0-7, and strongly in intercalated duct (ICD) at control gland and Day 7-14. FGF-7 was localized moderately in NFAC and DLS. FGF-8 was localized moderately in the epithelial cells of DLS during regeneration. Strong positive immunoreactions for FGF-10 were found in NFAC and the epithelial cells of DLS during regeneration, as well as the ICD and lateral surfaces of the maturing acinar cells (MAC). FGFR-1 was expressed moderately in the ICD, and weakly in the NFAC and MAC. Positive immunoreactions for FGFR-2 were not observed during regeneration. Additionally, FGFR-4 was detected strongly in the ICD and slightly in NFAC. These findings suggest that FGF-2, -7, -8 and -10 play important roles in NFAC, MAC, and DLS through FGFR-1 and -4 during regeneration of submandibular gland.

Runx1 and Runx3 Are Downstream Effectors of Nanog in Promoting Osteogenic Differentiation of the Mouse Mesenchymal Cell Line C3H10T1/2.

Previously, we reported that the transcription factor Nanog, which maintains the self-renewal of embryonic stem cells (ESCs), promotes the osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 through a genome reprogramming process. In the present study, to clarify the mechanism underlying the multipotency of mesenchymal stem cells (MSCs) and to develop a novel approach to bone regenerative medicine, we attempted to identify the downstream effectors of Nanog in promoting osteogenic differentiation of mouse mesenchymal cells. We demonstrated that Runx1 and Runx3 are the downstream effectors of Nanog, especially in the early and intermediate osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2.

Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control green fluorescent protein (GFP) gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process.

Immunohistological study of small Rho GTPases and ß-catenin during regeneration of the rat submandibular gland.

Our study immunohistochemically evaluated the localization patterns of small Rho GTPases and ß-catenin during regeneration of the rat submandibular gland. After 7 days of obstruction, regenerating glands were collected at days 0, 3, 7, 11 and 14 after duct release to study regeneration. RhoA was detected strongly and RhoC was detected weakly in the cytoplasm of newly formed acinar cells from day 3 to 7, and both RhoA and RhoC at the basal site and cytoplasm were detected moderately from day 11 to 14. RhoB was detected strongly and moderately in the cytoplasm of newly formed and matured acinar cells, respectively, and detected strongly in duct-like structures (DLSs) and intercalated ducts (ICDs). Rac1 was detected at the cell-cell and subcellular region, but ß-catenin was not observed in newly formed acinar cells. Rac1 immunolabeling gradually reduced, and the ß-catenin staining pattern became stronger. p-Rac1, a phosphorylated form of Rac1, was observed in the cytoplasm of newly formed acinar cells. At apical and subcellular region of DLSs and ICDs, Rac1 and ß-catenin were detected. These findings suggest that RhoA and RhoC might be involved in the actin cytoskeleton at the basolateral site of regenerating acinar cells, and RhoB might play a unique role in regenerating acinar cells and in DLSs and ICDs. Rac1 and ß-catenin at the cell-cell region might play important roles in cell-cell adhesion and the differentiation of regenerating acinar cells, as well as actin reconstruction at apical and subcellular regions of DLSs and ICDs.

Nasopharyngeal carriage of drug-resistant Streptococcus pneumoniae in children with acute otitis media evaluated by polymerase chain reaction-based genotyping of penicillin-binding proteins.

A polymerase chain reaction (PCR)-based genotyping of the penicillin-binding protein (PBP) genes pbp1a, pbp2x and pbp2b was used to characterize Streptococcus pneumoniae isolated from the nasopharynx of children with acute otitis media (AOM). Mutations were observed in pbp1a, pbp2x and pbp2b genes in 36.5% of the strains. Decreased susceptibility to beta-lactam antibiotics was closely associated with the frequency of mutations in the three PBP genes. Of penicillin-intermediately-resistant S. pneumoniae strains, 54.5% appeared to be genetically similar to penicillin-resistant S. pneumoniae strains. Of penicillin-susceptible S. pneumoniae strains, 33.3% had mutations in the pbp2x gene and showed relatively high MICs to cephalosporins. Strains with mutations in the three PBP genes were often isolated from children < or = 2 years old. Evaluation of mutations in PBP genes using PCR will prove useful for studying the epidemiology of antibiotic resistance.