The role of osteoclast differentiation and function in skeletal homeostasis
Osteoclasts are giant multinucleated cells that differentiate from hematopoietic cells in the bone marrow and carry out important physiological functions in the regulation of skeletal homeostasis as well as hematopoiesis. Osteoclast biology shares many features and components with cells of the immune system, including cytokine-receptor interactions (RANKL-RANK), intracellular signalling molecules (TRAF6) and transcription factors (NFATc1). Although the roles of these molecules in osteoclast differentiation are well known, fundamental questions remain unsolved, including the exact location of the RANKL-RANK interaction and ...
Source: Journal of Biochemistry - January 7, 2016 Category: Biochemistry Authors: Ikeda, K., Takeshita, S. Tags: JB Special Reviews-Cell Fate Decision, and its Underlying Molecular Mechanisms Source Type: research
In vitro affinity maturation and characterization of anti-P24 antibody for HIV diagnostic assay
P24 antigen is the main structural protein of HIV-1, its detection provide a means to aid the early diagnosis of HIV-1 infection. The aim of this study was to improve the selectivity and sensitivity of the HIV P24 diagnostic assay by developing a cohort of 9E8 affinity-matured antibodies through in vitro phage affinity maturation which was performed by complementarity determining region (CDR)-hot spot mutagenesis strategy. Antibody 9E8-491 had an affinity constant of 5.64 x 10–11 M, which was 5.7-fold higher than that of the parent antibody (9E8). Furthermore, the affinity, sensitivity and specificity of 9E8-491 were...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Xia, L., Zhang, J., Cui, C., Bi, X., Xiong, J., Yu, H., An, Z., Luo, W., Xia, N. Tags: Regular Papers Source Type: research
Effect of FliG three amino acids deletion in Vibrio polar-flagellar rotation and formation
Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (PSA) into FliG from Vibrio alginolyticus, whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mut...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Onoue, Y., Kojima, S., Homma, M. Tags: Regular Papers Source Type: research
In vitro dihydrouridine formation by tRNA dihydrouridine synthase from Thermus thermophilus, an extreme-thermophilic eubacterium
In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant (dus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The dus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNAPhe transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity....
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Kusuba, H., Yoshida, T., Iwasaki, E., Awai, T., Kazayama, A., Hirata, A., Tomikawa, C., Yamagami, R., Hori, H. Tags: Regular Papers Source Type: research
Mutagenesis study to disrupt electrostatic interactions on the twofold symmetry interface of Escherichia coli bacterioferritin
Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified ...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Zhang, Y., Wang, L., Ardejani, M. S., Aris, N. F., Li, X., Orner, B. P., Wang, F. Tags: Regular Papers Source Type: research
Sulphation of acetaminophen by the human cytosolic sulfotransferases: a systematic analysis
This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithe...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Yamamoto, A., Liu, M.-Y., Kurogi, K., Sakakibara, Y., Saeki, Y., Suiko, M., Liu, M.-C. Tags: Regular Papers Source Type: research
Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice
RelB is activated by the non-canonical NF-B pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The RelbVenus/Venus mice developed without any abnormalities observed in the Relb–/– mice, allowing...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Seki, T., Yamamoto, M., Taguchi, Y., Miyauchi, M., Akiyama, N., Yamaguchi, N., Gohda, J., Akiyama, T., Inoue, J.-i. Tags: Regular Papers Source Type: research
Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47
In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ~146 kDa on sodium dodecyl sulphate—polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cy...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Piao, J., Tashiro, A., Nishikawa, M., Aoki, Y., Moriyoshi, E., Hattori, A., Kakeya, H. Tags: Regular Papers Source Type: research
Yeast cell-based analysis of human lactate dehydrogenase isoforms
In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Mohamed, L. A., Tachikawa, H., Gao, X.-D., Nakanishi, H. Tags: Regular Papers Source Type: research
Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea
Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activi...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Eram, M. S., Wong, A., Oduaran, E., Ma, K. Tags: Regular Papers Source Type: research
Dynamic and distinct histone modifications of osteogenic genes during osteogenic differentiation
In this study, we examined the dynamics of H3K9/K14 and H4K12 acetylation; H3K4 mono-, di- and tri-methylation; H3K9 di-methylation and H3K27 tri-methylation in osteogenic genes, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase, bone sialoprotein and osteocalcin, during C3H10T1/2 osteogenesis. H3 and H4 acetylation and H3K4 di-methylation were elevated, and H3K9 di-methylation and H3K27 tri-methylation were reduced in osteogenic genes during C3H10T1/2 osteogenesis. C3H10T1/2 osteogenesis could be modulated by altering the patterns of H3 and H4 acetylation and H3K27 tri-methylation. In a gluc...
Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Zhang, Y.-X., Sun, H.-L., Liang, H., Li, K., Fan, Q.-M., Zhao, Q.-H. Tags: Regular Papers Source Type: research
Maid is a negative regulator of transforming growth factor-{beta}-induced cell migration
Maternal Id-like molecule (Maid) is a dominant negative helix-loop-helix protein that has been implicated in regulating gene expression as well as cell-cycle progression. Overexpressed Maid was previously shown to inhibit certain cellular responses induced by transforming growth factor-β (TGF-β), such as TGF-β-induced cytostasis and cell motility, but not epithelial–mesenchymal transition (EMT). The role of endogenous Maid in regulating TGF-β signalling, however, has not been elucidated. We have found evidence that endogenous Maid negatively regulates TGF-β-induced cell motility. Maid knockdo...
Source: Journal of Biochemistry - October 30, 2015 Category: Biochemistry Authors: Motizuki, M., Saitoh, M., Miyazawa, K. Tags: Regular Papers Source Type: research
Characterization of hamster NAD+-dependent 3(17){beta}-hydroxysteroid dehydrogenase belonging to the aldo-keto reductase 1C subfamily
The cDNAs for morphine 6-dehydrogenase (AKR1C34) and its homologous aldo-keto reductase (AKR1C35) were cloned from golden hamster liver, and their enzymatic properties and tissue distribution were compared. AKR1C34 and AKR1C35 similarly oxidized various xenobiotic alicyclic alcohols using NAD+, but differed in their substrate specificity for hydroxysteroids and inhibitor sensitivity. While AKR1C34 showed 3α/17β/20α-hydroxysteroid dehydrogenase activities, AKR1C35 efficiently oxidized various 3β- and 17β-hydroxysteroids, including biologically active 3β-hydroxy-5α/β-dihydro-C19/C21...
Source: Journal of Biochemistry - October 30, 2015 Category: Biochemistry Authors: Endo, S., Noda, M., Ikari, A., Tatematsu, K., El-Kabbani, O., Hara, A., Kitade, Y., Matsunaga, T. Tags: Regular Papers Source Type: research
A cell-based screening for TAZ activators identifies ethacridine, a widely used antiseptic and abortifacient, as a compound that promotes dephosphorylation of TAZ and inhibits adipogenesis in C3H10T1/2 cells
Transcriptional co-activator with PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif (TAZ) regulates in cell proliferation and differentiation. In mesenchymal stem cells it promotes osteogenesis and myogenesis, and suppresses adipogenesis. TAZ activators are expected to prevent osteoporosis, obesity and muscle atrophy. TAZ activation induces epithelial-mesenchymal transition, confers stemness to cancer cells and leads to poor clinical prognosis in cancer patients. In this point of view, TAZ inhibitors should contribute to cancer therapy. Thus, TAZ attracts attention as a two-faced drug target. We screened for TAZ modulators by using hu...
Source: Journal of Biochemistry - October 30, 2015 Category: Biochemistry Authors: Kawano, S., Maruyama, J., Nagashima, S., Inami, K., Qiu, W., Iwasa, H., Nakagawa, K., Ishigami-Yuasa, M., Kagechika, H., Nishina, H., Hata, Y. Tags: Regular Papers Source Type: research
Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block
We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal...
Source: Journal of Biochemistry - October 30, 2015 Category: Biochemistry Authors: Kawakami, T., Yoshikawa, R., Fujiyoshi, Y., Mishima, Y., Hojo, H., Tajima, S., Suetake, I. Tags: Regular Papers Source Type: research
