Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC-characteristic genes-including nine transcription factor genes -using DNA microarray and real-time RT-PCR analyses: Most of the MSC-characteristic genes were down-regulated 24 h after incubation with osteogenesis-, chondrogenesis-or adipogenesisinduction medium, or 48-72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the selfrenewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic-and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules-including GATA6, TRPC4, FLG and TGM2-revealed that MSC-like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC-characteristic transcription factors are involved in MSC stemness regulation.

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.

Background and ObjectiveTheaflavins (TFs), the major polyphenol in black tea, have the ability to reduce inflammation and bone resorption. The aim of this study was to evaluate the effects of TFs on experimental periodontitis in rats.Material and MethodsThirty rats were divided into five groups: Control (glycerol application without ligation), Ligature (glycerol application with ligation), TF1 (1 mg/mL TF application with ligation), TF10 (10 mg/mL TF application with ligation), and TF100 (100 mg/mL TF application with ligation). To induce experimental periodontitis, ligatures were placed around maxillary first molars bilaterally. After ligature placement, 100 μL glycerol or TFs were topically applied to the rats daily, and rats were euthanized 7 days after ligature placement. Micro‐computed tomography was used to measure bone resorption in the left side of the maxilla, and quantitative polymerase chain reaction was used to measure the expression of interleukin (IL)‐6, growth‐regulated gene product/cytokine‐induced neutrophil chemoattractant (Gro/Cinc‐1, rat equivalent of IL‐8), matrix metalloproteinase‐9 (Mmp‐9), receptor activator of nuclear factor‐kappa Β ligand (Rankl), osteoprotegerin (Opg), and the Rankl/Opg ratio in gingival tissue. With tissue from the right side of the maxilla, hematoxylin and eosin staining was used for histological analysis, immunohistochemical staining for leukocyte common antigen (CD45) was used to assess inflammation, and tartrate‐resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts.ResultsThe TF10 and TF100 groups, but not the TF1 group, had significant inhibition of alveolar bone loss, reduction in inflammatory cell infiltration in the periodontium, and significantly reduced numbers of CD45‐positive cells and TRAP‐positive osteoclasts compared with the Ligature group. Correspondingly, the TF10 and TF100 groups had significantly downregulated gene expression of IL‐6, Gro/Cinc‐1(IL‐8), Mmp‐9, and Rankl, but not of Opg. Consequently, Rankl/Opg expression was significantly increased in the Ligation group but was attenuated in the TF10 and TF100 groups.ConclusionThe results of this study suggest that topical application of TFs may reduce inflammation and bone resorption in experimental periodontitis. Therefore, TFs have therapeutic potential in the treatment of periodontal disease.

In this communication, we summarized our recent experimental approaches to an unsettled issue, i.e., the nature and role of an acidic precursor in enamel mineralization. The objectives we specially focused our attention on are: the composition, structure and high resolution images of enamel crystals at various developmental stages, thermodynamic and kinetic consideration of octacalcium phosphate (OCP) vs hydroxyapatite (HA) precipitation in physiological media simulating the enamel fluid, reversible changes in the composition and structure of OCP, effects of fluoride at low concentrations and enamel proteins on OCP hydrolysis, and adsorption of enamel proteins onto OCP and fluoridated hydrolysates at neutral pH and room temperature. On the basis of all experimental evidence, we propose that enamel crystal growth comprises two events: the two-dimensional growth of an OCP-like precursor in a narrow outermost zone adjacent to the ameloblasts and the subsequent overgrowth of apatite units on the template under discrete fluid environment in the underlying region distant from the cell layer. The experimental data also support the concept that the whole process of enamel mineralization is modulated substantially through interaction between enamel proteins and crystals including the acidic precursor.

We conducted three-dimensional (3D) reconstruction of oral tongue squamous cell carcinoma (OTSCC) using serial histological sections to visualize the architecture of invasive tumors. Fourteen OTSCC cases were collected from archival paraffin-embedded specimens. Based on a pathodiagnostic survey of whole cancer lesions, a core tissue specimen (3 mm in diameter) was dissected out from the deep invasion front using a paraffin tissue microarray. Serial sections (4 μm thick) were double immunostained with pan-cytokeratin and Ki67 antibodies and digitized images were acquired using virtual microscopy. For 3D reconstruction, image registration and RGB color segmentation were automated using ImageJ software to avoid operator-dependent subjective errors. Based on the 3D tumor architecture, we classified the mode of invasion into four types: pushing and bulky architecture; trabecular architecture; diffuse spreading; and special forms. Direct visualization and quantitative assessment of the parenchymal-stromal border provide a new dimension in our understanding of OTSCC architecture. These 3D morphometric analyses also ascertained that cell invasion (individually and collectively) occurs at the deep invasive front of the OTSCC. These results demonstrate the advantages of histology-based 3D reconstruction for evaluating tumor architecture and its potential for a wide range of applications.

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We previously reported that mutation of the transforming growth factor-beta3 (TGF-beta3) gene caused cleft palate in homozygous null (−/−) mice. TGF-beta3 is normally expressed in the medial edge epithelial (MEE) cells of the palatal shelf. In the present study, we investigated the mechanisms by which TGF-beta3 deletions caused cleft palate in 129 × CF-1 mice. For organ culture, palatal shelves were dissected from embryonic day 13.5 (E13.5) mouse embryos. Palatal shelves were placed singly or in pairs on Millipore filters and cultured in DMEM/F12 medium. Shelves were placed in homologous (+/+ vs +/+, −/− vs −/−, +/− vs +/−) or heterologous (+/+ vs −/−, +/− vs −/−, +/+ vs +/−) paired combinations and examined by macroscopy and histology. Pairs of −/− and −/− shelves failed to fuse over 72 hours of culture whereas pairs of +/+ (wild-type) and +/+ or +/− (heterozygote) and +/−, as well as +/+ and −/− shelves, fused within the first 48 hour period. Histological examination of the fused +/+ and +/+ shelves showed complete disappearance of the midline epithelial seam whereas −/− and +/+ shelves still had some seam remnants. In order to investigate the ability of TGF-beta family members to rescue the fusion between −/− and −/− palatal shelves in vitro, either recombinant human (rh) TGF-beta1, porcine (p) TGF-beta2, rh TGF-beta3, rh activin, or p inhibin was added to the medium in different concentrations at specific times and for various periods during the culture. In untreated organ culture −/− palate pairs completely failed to fuse, treatment with TGF-beta3 induced complete palatal fusion, TGF-beta1 or TGF-beta2 near normal fusion, but activin and inhibin had no effect. We investigated ultrastructural features of the surface of the MEE cells using SEM to compare TGF-beta3-null embryos (E 12. 5-E 16.5) with +/+ and +/− embryos in vivo and in vitro. Up to E13.5 and after E15.5, structures resembling short rods were observed in both +/+ and −/− embryos. Just before fusion, at E14.5, a lot of filopodia-like structures appeared on the surface of the MEE cells in +/+ embryos, however, none were observed in −/− embryos, either in vivo or in vitro. With TEM these filopodia are coated with material resembling proteoglycan. Interestingly, addition of TGF-beta3 to the culture medium which caused fusion between the −/− palatal shelves also induced the appearance of these filopodia on their MEE surfaces. TGF-beta1 and TGF-beta2 also induced filopodia on the −/− MEE but to a lesser extent than TGF-beta3 and additionally induced lamellipodia on their cell surfaces. These results suggest that TGF-beta3 may regulate palatal fusion by inducing filopodia on the outer cell membrane of the palatal medial edge epithelia prior to shelf contact. Exogenous recombinant TGF-beta3 can rescue fusion in −/− palatal shelves by inducing such filopodia, illustrating that the effects of TGF-beta3 are transduced by cell surface receptors which raises interesting potential therapeutic strategies to prevent and treat embryonic cleft palate.