A knowledge of beta cell-specific gene expression provides a basis for identifying proteins potentially involved in beta cell function and pathology. To identify candidate beta cell-specific genes, we applied the PCR-based subtractive hybridization technique of representational difference analysis (RDA) to the mouse SV40-transformed endocrine cell lines, betaTC3 and alphaTC1. Following three successive subtractions of alphaTC1 complementary DNA from betaTC3 complementary DNA, difference products were cloned into pUC19 and nucleotide sequences determined. Comparison of 91 sequences against the databases identified 11 known and 8 novel genes. Known genes included previously reported beta cell-specific genes, insulin I/II and islet amyloid polypeptide, as well as other non-beta cell-specific genes such as those for insulin-like growth factor II, selenoprotein P, neuronatin, prohormone convertase, and type 1 protein kinase A regulatory subunit. By Northern blot hybridization, expression of the majority of known and novel genes was restricted to betaTC3 cells. Novel genes BA-12, -13, -14, and -18 were expressed not only in betaTC3 cells, but also in normal pancreatic islets and a limited number of other tissues. The deduced amino acid sequence of BA-14 showed significant homology with members of the cadherin superfamily indicating that BA-14 may encode a cadherin-like molecule potentially involved in beta cell adhesion events during islet ontogeny. In betaTC3 cells, none of the novel genes were regulated at the RNA level by high glucose. However, in parallel studies, transcription of BA-12 was significantly increased by both sodium butyrate and nicotinamide, suggesting that this gene may play a role in pancreatic beta cell growth and/or differentiation. In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate beta cell growth and differentiation.

Bone marrow stromal cells (BMSCs) include cells with multidirectional differentiation potential described as mesenchymal stem cells. For clinical use, it is important to develop a way to isolate BMSCs from bone marrow in a closed system without centrifugation. After screening 200 biomaterials, we developed a device containing a nonwoven fabric filter composed of rayon and polyethylene. The filter selectively traps BMSCs among mononuclear cells in bone marrow based on affinity, not cell size. The cells are then recovered by the retrograde flow. Using canine and human bone marrow cells, the biological properties of BMSCs isolated by the device were compared with those obtained by conventional methods using centrifugation. The total number isolated by the device was larger, as was the number of CD106(+)/STRO-1(+) double-positive cells. The cells showed osteogenic, chondrogenic, and adipogenic differentiation potential in vitro. Finally, the direct transplantation of cells isolated by the device without in vitro cultivation accelerated bone regeneration in a canine model of osteonecrosis in vivo. The proposed method is rapid and efficient, does not require a biological clean area, and will be useful for the clinical application of mesenchymal stem cells in bone marrow.

The relationship between TiN microstructures and diffusion barrier properties of TiN against Cu was investigated. TiN deposited by a chemical vapor deposition (CVD) method is composed of columnar grains grown normal to the sidewall on the side of the trench. On the other hand, the gram boundaries of sputter-deposited TiN tilt upward from the normal direction to the sidewall, and the tilt angle depends on the sputtering conditions. Voids between TiN grains are observed on the side of the trench and the size of the voids depends on the deposition conditions. In the Cu/CVD-TiN (the upper/lower layer) and Cu/conventional sputtered TiN system, no Cu is detected in surrounding SiO, films, either outside the sidewall or underneath the bottom of the trench after annealing at 400°C for 3 mm. However, in the Cu/long-throw sputtered TiN system, where TiN is composed of columnar grains with void regions between grains, and the tilt angle of the grain boundaries from the normal direction to the sidewall is 27°, some Cu is detected outside the sidewall, even when the TiN thickness on the side of the trench is equal to or thicker than that of CVD-TiN or conventionally sputtered TiN. The diffusion barrier property of TiN in Cu metallization depends on the TiN grain structures rather than on the TiN thickness on the side of the trench. InfroductionCu has been considered as a promising metallization

This study compared pain intensity and psychosocial characteristics between patients with burning mouth syndrome (BMS) and those with trigeminal neuralgia (TN). Data from 282 patients with BMS and 83 patients with TN were analyzed. Patients reported duration of illness: duration ≤ 6 months was defined as acute illness and > 6 months as chronic illness. Present pain intensity and worst pain intensity during the past 6 months were reported using a 0-10 numeric rating scale (NRS). In addition, depression and somatization scores were evaluated on questionnaires. Patients with chronic BMS reported significantly higher pain intensity and had worse psychosocial characteristics than did those with acute BMS. Pain intensity was higher in TN patients than in BMS patients, although neither pain intensity nor psychosocial characteristics significantly differed between patients with acute and chronic illness.Logistic regression analysis of BMS and TN patients revealed that the odds ratio for worst pain was significantly lower for BMS patients than for TN patients and that the odds ratio for somatization score was 3.8 times higher in BMS patients. These findings suggest that BMS patients may require pain control targeting the central nervous system or psychosocial characteristics. (J Oral Sci 54, 321-327, 2012)

Mutations in the triple PDZ domain-containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic β β β β cells. Using a yeast two-hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for h armonin-interacting, a nkyrin r epeat-containing p rotein. This interaction was confirmed in an over-expression system and in mammalian cells, and shown to be mediated by the three C-terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co-localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic β β β β -cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 ( js ) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co-localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.