p130Cas-dependent actin remodelling regulates myogenic differentiation

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.     

Distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa: age-related progression

Age-related changes were examined in the distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa. The olfactory mucosa of female ICR mice at postnatal ages from 10 days to 16 months were investigated histologically by hematoxylin and eosin staining, high-iron diamine-Alcian blue (HID-AB) staining, and immunohistochemistry for olfactory marker protein (OMP), βIII tubulin (βIIIT), and Ki67. The lesions in the neuroepithelium and Bowman’s glands were quantitatively assessed by morphometric analyses of sections stained with anti-OMP antibody or HID-AB. The first appearance of neuroepithelial abnormality was observed in the dorsomedial portion of the olfactory mucosa in 5-month-old mice. The distribution and severity of lesions progressed with increasing age. In mildly affected epithelium in which OMP-positive olfactory receptor neurons (ORNs) were present but in smaller amounts, the numbers of βIIIT-positive and Ki67-positive neuroepithelial cells tended to be increased, indicating that neurogenesis was upregulated in these areas. In contrast, severely affected epithelium in which OMP-positive ORNs were virtually absent showed high variability in the numbers of βIIIT- and Ki67-positive cells among the areas examined, probably reflecting differences in the capacity of the basal cells remaining in the affected area to generate new neuronal cells. Histological analysis with HID-AB revealed that spontaneous lesions in Bowman’s glands also occurred in aged mouse olfactory mucosa. Lesions in the neuroepithelium and underlying Bowman’s glands tended to be spatially co-localized, suggesting a close association between pathogeneses in these two structures. Moreover, lesions in Bowman’s glands were associated with changes in the biochemical composition of mucus on the olfactory mucosa. This information should prove useful in improving the understanding of the pathogenetic mechanisms underlying age-related changes in the peripheral olfactory system. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (nos. 14770886, 16790987 and 18799002; K. Kondo) and a grant from the Japanese Ministry of Health, Labour, and Welfare (Comprehensive Research on Aging and Welfare, no. H13-choju-012; K. Nibu).     

Synthesis of puromycin derivatives with backbone-elongated substrates and associated translation inhibitory activities

We have synthesized a series of 5′-phosphorylated and 5′-cytidylyl-(3′–5′)-cytidylyl-(3′–5′)-puromycin derivatives that have backbone-elongated substrates. All the synthesized puromycin derivatives showed good solubility in water and were applied to translation inhibitory assay in a reconstituted Escherichia coli translation system.     

Critical Role of Heparin Binding Domains of Ameloblastin for Dental Epithelium Cell Adhesion and Ameloblastoma Proliferation

AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.The extracellular matrix provides structural support for cells and regulates cell proliferation, migration, differentiation, and apoptosis for tissue development and homeostasis (1). The extracellular matrix also plays a crucial role in pathological processes and diseases, such as wound healing, tumorigenesis, and cancer development (2, 3). AMBN (ameloblastin), also known as amelin and sheathlin, is a tooth-specific extracellular matrix and the most abundant non-amelogenin enamel matrix protein (4–6). AMBN is expressed primarily by ameloblasts, which are differentiated from the oral ectoderm and form a polarized single cell layer underlying the enamel matrix. In a previous study, we created Ambn-null mice and demonstrated that AMBN is required for cell attachment and polarization and for maintaining the differentiation state of ameloblasts and is essential for enamel formation (3). Overexpression of Ambn in transgenic mice causes abnormal enamel crystallite formation and enamel rod morphology (7). These results suggest that enamel formation and rod morphology are influenced by temporal and spatial expressions of AMBN and imply that the AMBN gene locus may be involved in the etiology of a number of cases of undiagnosed hereditary amelogenesis imperfecta (8). Further, it was reported that recombinant AMBN enhances pulpal wound healing and reparative dentine formation following pulpotomy procedures, suggesting that it functions as a signal molecule in epithelial-mesenchymal interactions (9).We previously reported that about 20% of Ambn-null mice developed an odontogenic tumor of dental epithelium origin in the buccal vestibule of the maxilla (3). The epithelial cells of odontogenic tumors express enamel matrix proteins, including AMEL (amelogenin), ENAM (enamelin), and TUFT (tuftelin), but not AMBN, indicating that AMBN deficiency is probably the primary cause of tumorigenesis seen in those mice. An ameloblastoma appearing in the jaw is the most frequently encountered odontogenic tumor and is characterized by benign but locally invasive behavior with a high rate of recurrence. Since abnormal proliferation and growth of ameloblastoma cells easily destroys surrounding bony tissues, wide excision is required to treat this disorder. It is also reported that ameloblastomas rarely metastasize to other parts of the body, such as the lungs and regional lymph nodes (10, 11). Associations of AMBN mutations were reported in ameloblastomas, adenomatoid odontogenic tumors, and squamous odontogenic tumors (12). These results suggest that AMBN regulates odontogenic tumor formation.In the present study, we investigated the mechanism of AMBN in dental epithelial cell adhesion and ameloblastoma proliferation. We found that AMBN has heparin binding domains, which are essential for AMBN binding to dental epithelial cells. We demonstrate that overexpression of recombinant AMBN inhibits proliferation of human ameloblastoma cells. This inhibition requires the heparin binding sites of AMBN and is accompanied by dysregulation of Msx2, p21, and p27. These results suggest that AMBN suppresses ameloblastoma cell proliferation by regulating cellular signaling through the heparin binding domains.     

Purification and molecular characterization of a novel diadenosine 5′,5′′′-P1,P4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K_m values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.     

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.     

Contribution of a single hydrogen bond between betaHis81 of MHC class II I-E(k) and the bound peptide to the pH-dependent thermal stability

To determine the energetic contribution of the hydrogen bond between betaHis81 of the major histocompatibility complex class II (MHC II) molecule, I-E(k), and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond-disrupted mutant, I-E(k)-Hb betaH81Y, in which the betaHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I-E(k)-Hb betaH81Y mutant was lower than that of the I-E(k)-Hb wild-type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I-E(k)-Hb betaH81Y mutant as compared with that of the I-E(k)-Hb wild-type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by betaHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.     

Reconstitution of CD8+ T cells by retroviral transfer of the TCR alpha beta-chain genes isolated from a clonally expanded P815-infiltrating lymphocyte

Gene transfer of TCR alphabeta-chains into T cells may be a promising strategy for providing valuable T lymphocytes in the treatment of tumors and other immune-mediated disorders. We report in this study the reconstitution of CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from an infiltrating T cell into P815. Analysis of the clonal expansion and Vbeta subfamily usage of CD8(+) TIL in the tumor sites demonstrated that T cells using Vbeta10 efficiently infiltrated and expanded clonally. The TCR alpha- and beta-chain sequences derived from a tumor-infiltrating CD8(+)/Vbeta10(+) single T cell clone (P09-2C clone) were simultaneously determined by the RT-PCR/single-strand conformational polymorphism method and the single-cell PCR method. When P09-2C TCR alphabeta-chain genes were retrovirally introduced into CD8(+) T cells, the reconstituted T cells positively lysed the P815 tumor cells, but not the A20, EL4, or YAC-1 cells, in vitro. In addition, the CTL activity was blocked by the anti-H2L(d) mAb. Furthermore, T cells containing both TCR alpha- and beta-chains, but not TCR beta-chain alone, accumulated at the tumor-inoculated site when the reconstituted CD8(+) T cells were adoptively transferred to tumor-bearing nude mice. These findings suggest that it is possible to reconstitute functional tumor-specific CD8(+) T cells by transfer of TCR alphabeta-chain genes derived from TIL, and that such T cells might be useful as cytotoxic effector cells or as a vehicle for delivering therapeutic agents.