Interaction of type I collagen (COL(I)) withTransient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-␤ receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-␣2␤1 integrin interaction facilitates differentiation and down-regulates TGF-␤ receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.Cells of osteoblast lineage exert various functions depending upon their differentiation stages to maintain bone formation. Production of type I collagen (COL(I)1 ) is one of the earlier events during this process, followed by sequential expression of alkaline phosphatase (ALP) and osteocalcin. Mineralization of newly formed matrix takes place thereafter (1). To form bones with structural integrity and physical strength, it appears to be of critical importance to maintain the sequential development of these multiple osteoblastic phenotypes.Using osteoblastic cells from bone or of clonal origin, various hormones and cytokines are shown to affect the differentiation process and functional properties of these cells (1, 2). In addition to these factors, matrix COL(I) is required for the differentiation of osteoblasts (3, 4). We have demonstrated that the effect of COL(I) on the development of osteoblastic properties is mediated by the interaction of COL(I) with cell-surface ␣2␤1 integrin and that the interaction also causes down-regulation of transforming growth factor (TGF)-␤ receptors without affecting the expression of the receptor mRNA (5), suggesting the translocation of TGF-␤ receptors from cell surface to intracellular compartments. Because TGF-␤ potently stimulates matrix protein synthesis but inhibits differentiation of osteoblasts (5), the down-regulation of its receptors may serve to facilitate further maturation of osteoblasts and mineralization of newly formed matrix. However, the mechanism and the intracellular pathways that control the differentiation and TGF-␤ receptor down-regulation of osteoblastic cells by COL(I)-␣2␤1 integrin interaction remain unclear.Accumulating evidence indicates that stimulation of ␤1 integrin by matrix proteins initiates intracellular signaling pathways in many types of cells (6 -9). One of the initial events triggered by the stimulation of ␤1 integrin is the association of its cytoplasmic domain with focal adhesion kinase (FAK), a cytosolic non-receptor tyrosine kinase, followed by tyrosine phosphorylation and activation of FAK (10). FAK-deficient mice display severe defects of mesodermal development in embryogenesis (11). The phenotype of FAK-deficient mice is strikingly similar to the fibronectin-deficient phenotype (12), suggesting that FAK uniquely mediates matrix-in...

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The magA gene from Magnetospirillum sp. strain AMB-1 is required for the synthesis of bacterial magnetic particles (BMPs). This gene has been cloned, sequenced and found to encode a protein which is homologous to the Escherichia coli potassium efflux membrane-binding protein, KefC. By using the firefly luciferase gene (luc) cloned downstream of the magA promoter, the effect of iron on regulation of magA expression was investigated, and transcription of magA was found to be enhanced by low concentrations of iron. Intracellular localization of the MagA protein was studied using magA-luc fusion proteins. The luc gene was cloned downstream of the magA hydrophilic C-terminal domain. Detection of luciferase activity in the cytoplasm, cell membrane, and magnetic particle membrane subcellular fractions confirmed that the MagA fusion protein was localized in the cell membrane. The fusion protein was also detected on the surface of the lipid bilayer covering the magnetic particles. These results suggest that MagA is a membrane-bound protein, the expression of which is enhanced at low iron concentrations.

Dock2 has been shown to be indispensable for chemotaxis of mature lymphocytes as a critical Rac activator. However, the functional expression of Dock2 in immature hematopoietic cells is unclear. In this study, we demonstrate that Dock2 is broadly expressed in bone marrow (BM) hematopoietic compartment, including hematopoietic stem/progenitor cell (HSC/HPC) fraction. Response of Dock2-/- HPCs to CXCL12 in chemotaxis and actin polymerization in vitro was impaired, although alpha4 integrin activation by CXCL12 was not altered. Myelosuppressive stress on HSCs in vivo, such as consecutive 5-FU administration and serial bone marrow transplantation, did not show hematopoietic defect in Dock2-/- mice. Long-term engraftment of transplanted Dock2-/- BM cells was severely impaired in competitive reconstitution. However, this was not intrinsic to HSCs but originated from the defective competition of Dock2-/- lymphoid precursors. These results suggest that Dock2 plays a significant role in BM lymphopoiesis, but is dispensable for HSC engraftment and self-renewal.

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Luciferase‐bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB‐1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5′ and 3′ terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB‐1. In addition, we produced bifunctional active‐fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N‐terminus and the C‐terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed‐batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 × 109 cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed‐batch culture of AMB‐1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB‐1 cultivated by fed‐batch culture and wild type of AMB‐1. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 704–709, 2000.

The thermal stability of PQQ glucose dehydrogenases (PQQGDHs) which were chimeras with more than 95% made up of the N-terminal region of Escherichia coli PQQGDH and the rest made up of the C-terminal region of Acinetobacter calcoaceticus PQQGDH was investigated. Among the chimeric PQQGDHs, E97A3 (E. eoli 97% and A. calcoaceticus 3%) and E95A5 were found to possess higher thermal stability than parental E. coli PQQGDH. Further detailed characterization of the thermal stability was carried out, focusing on E97A3. E97A3 showed a more than 3-fold and 12-fold increase in half life time at 40°C, compared with the PQQGDHs of E. coli and A. calcoaceticus, respectively. Using transition state theory, the increase in the free energy of inactivation observed in E97A3 was compared with those of the E. coli and ,4. calcoaceticus parental enzymes. The region responsible for this stabilization was also discussed.

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a beta 1-3 glucan with beta 1-6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8+ CTL but not by CD4+ T cells or NK1.1+ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.