Osteogenic differentiation of the mesenchymal progenitor cells, Kusa is suppressed by Notch signaling

Notch receptor plays a crucial role in proliferation and differentiation of many cell types. To elucidate the function of Notch signaling in osteogenesis, we transfected the constitutively active Notch1 (Notch intracellular domain, NICD) into two different osteoblastic mesenchymal cell lines, KusaA and KusaO, and examined the changes of their osteogenic potentials. In NICD stable transformants (KusaA(NICD) and KusaO(NICD)), osteogenic properties including alkaline phosphatase activity, expression of osteocalcin and type I collagen, and in vitro calcification were suppressed. Transient transfection of NICD attenuated the promoter activities of Cbfa1 and Ose2 element. KusaA was capable of forming trabecular bone-like tissues when injected into mouse abdomen, but this in vivo bone forming activity was significantly suppressed in KusaA(NICD). Osteoclasts were induced in the KusaA-derived bone-like tissues, but lacked in the KusaA(NICD)-derived tissues. These results suggest that Notch signaling suppresses the osteoblastic differentiation of mesenchymal progenitor cells.     

Type III Cu mutants of Myrothecium verrucaria bilirubin oxidase

Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.     

Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.     

Contribution of ventral and dorsal mesoderm to primitive and definitive erythropoiesis in the salamander Hynobius retardatus

Previously, we found that the conversion of hemoglobins (Hbs) from the larval to the adult type occurred within a single erythroid cell population in a salamander, Hynobius retardatus ("Hb switching" model), whereas the transition involves replacement of red-blood-cell (RBC) populations ("RBC replacement" model) in many amphibians (M. Yamaguchi, H. Takahashi, and M. Wakahara, 2000, Dev. Gene Evol. 210, 180-189). To further characterize the Hb transition, developmental changes in the erythropoietic sites have been intensively analyzed using larval- and adult-specific globin antibodies and globin and GATA-3 RNA probes. Cells of the ventral blood island (VBI) and the dorsolateral plate (DLP) in embryos differentiate in situ to erythroid cells that contain larval globin mRNA, suggesting that both the VBI and the DLP contribute to "primitive" erythropoiesis. In contrast, the expression pattern of the GATA-3 gene suggests that cells of the DLP may contribute to "definitive" hematopoiesis. In order to determine whether it is possible to define a definitive erythropoiesis in H. retardatus or not, further experiments were done: (1) when metamorphosing larvae were treated with phenylhydrazine to induce anemia and then bled at the postmetamorphic stage after recovery from the anemia, a precocious Hb transition was observed in these animals; (2) an RBC population expressing only adult Hb was confirmed by subtracting the number of RBCs expressing larval Hb from the total number of RBCs during metamorphosis. All these results support the existence of a definitive erythroid cell population that contributes only adult RBCs in this species.     

Synthesis of branched cyclomaltooligosaccharide carboxylic acids (cyclodextrin carboxylic acids) by microbial oxidation

Novel branched cyclomaltooligosaccharide carboxylic acid (cyclodextrin carboxylic acid) derivatives were synthesized by microbial oxidation using Pseudogluconobacter saccharoketogenes to oxidize five types of branched cyclodextrins, including maltosyl beta-cyclodextrin (maltosyl-beta-CyD). For each novel cyclodextrin carboxylic acid derivative synthesized, the hydroxymethyl group of the terminal glucose residue in the branched part of the molecule was regiospecifically oxidized to a carboxyl group to give the corresponding uronic acid. In addition, the physicochemical properties of cyclomaltoheptaosyl-(6-->1)-alpha-D-glucopyranosyl-(4-->1)-alpha-D-glucopyranosiduronic acid (GUG-beta-CyD) (1) and its sodium salt were studied more extensively, as these compounds are most likely to have a practical application.     

Monitoring of the refolding process for immobilized firefly luciferase with a biosensor based on surface plasmon resonance

In order to examine the possibility of the use of a surface plasmon resonance (SPR) sensor for real-time monitoring of the process of refolding of immobilized proteins, the refolding of firefly luciferase immobilized on a carboxymethyldextran matrix layer was analyzed. The SPR signal of the immobilized luciferase decreased after unfolding induced by GdnCl and increased gradually in the refolding buffer, while there was no signal change in the reference surface lacking the immobilized protein. The decrease in the SPR signal on unfolding was consistent with the difference between the refractive indices of the native and unfolded protein solutions. The effects of blocking of the excess NHS-groups of the matrix layer on the refolding yield were examined by means of an SPR sensor. The results were consistent with those obtained with the enzymatic activity assay, indicating that the changes in the SPR signal reflected the real-time conformational changes of the immobilized protein. Hence, an SPR biosensor might be used for monitoring of the process of refolding of immobilized proteins and as a novel tool for optimization of the refolding conditions. This is the first demonstration that SPR signal changes reflect the conformational changes of an immobilized protein upon unfolding and refolding.     

Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli

Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)His] as an antigen. From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1. Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein. First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed. Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C. Then, we constructed 13 cysteine-scanning mutants, F179C to T191C. Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding. These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B). TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.     

Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities.

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.     

To what extent does a minimal atherosclerotic plaque alter the arterial wall shear stress distribution? A model study by an electrochemical method

An electrochemical surface shear stress measurement was applied to a model of very thin unilateral arterial stenosis (height of 1/8 of the model pipe diameter with very smooth surface). Three dimensional wall shear stress distribution was measured under steady flow field from a relatively low Reynolds number, Re = 270, to a high Reynolds number, Re = 1200. There was a characteristic high and low wall shear distribution pattern around the stenosis. There were also remarkable high shear stress areas on the opposite wall and both side walls of the stenosis. It was clearly shown that three dimensional structure of the flow field, hence, the wall shear stress distribution, is affected by a minimal change on the arterial wall.