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Volume 129, Issue 1
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Tamotsu Zako, Kenkichi Harada, Teruhisa Mannen, Satoshi Yamaguchi, Ats ...2001 Volume 129 Issue 1 Pages 1-4
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn 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.View full abstractDownload PDF (1169K) -
Toshiro Takai, Ko Okumura, Chisei Ra, Toyokazu Yokota, Yasushi Okumura2001 Volume 129 Issue 1 Pages 5-12
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInterfering with the binding of IgE to high-affinity IgE receptor α chain (FcεRIα) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human FcεRIα inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human FcεRIα Fabs without papain -digestion of the whole antibody. Four Fabs with different C-termini of CHI were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-FcεRIα binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human FcεRIα will provide useful information for the design of therapeutic reagents for allergy and asthma.View full abstractDownload PDF (2326K) -
Shunji Ishiwata, Jun Katayama, Hiroki Shindo, Yasuko Ozawa, Kunihiko I ...2001 Volume 129 Issue 1 Pages 13-17
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSQueuosine is a modified nucleoside located at the first position of the tRNA anticodon, which is synthesized by tRNA-guanine transglycosylase (TGT). Although the levels of queuosine in cancer cells have been reported to be lower than those in normal cells, the expression levels of TGT remain to be determined. We determined the expression levels of a subunit of TGT (TGT60KD). Contrary of our expectations, the results revealed higher levels of expression of TGT60KD than that in normal cells, and the level of queuosine in the tRNA fraction corresponded with that of TGT60KD expression. These results suggest the possibilities that the expression levels of TGT60KD regulate TGT activity and the levels of queuosine, and that TGT60KD plays significant roles in carcinogenesis. To our knowledge, this is a first report of increased expression levels of TGT60KD in human cancer cells.View full abstractDownload PDF (996K) -
Yasushi Kamisaka, Naomi Noda2001 Volume 129 Issue 1 Pages 19-26
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExogenous fluorescent phosphatidic acid (PA) and phosphatidylcholine (PC) were transported into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora [Kamisaka et al. (1999) Biochim. Biophys. Acta 1438, 185-198]. We further investigated the processes of fluorescent PA and PC transport into lipid bodies in this fungus by changing culture conditions. Lowering incubation temperature decreased lipid body labeling by 1-pahnitoyl, 2-[5-(5, 7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-PA (C5-DMB-PA), but fluorescence did not accumulate in organelles other than lipid bodies. C5-DMB-PC transport into lipid bodies was blocked at temperatures below 15°C and fluorescence accumulated in intracellular membranes, presumably endoplasmic reticulum membranes. The low-temperature block of C5-DMB-PC transport enabled us to do pulse-chase experiments in which fungal cells were pulse-labeled at 15°C with C5-DMB-PC and chased at 30°C. The results clearly depicted transport of C5-DMB-PC and its derivatives from intracellular membranes to lipid bodies. Transport was temperature-dependent and ATP-dependent, although microtubules and actin filaments were not substantially involved. Experiments using 14C-labeled fatty acids and glycerol instead of C5-DMB-PC under the same conditions suggested that transport depicted by fluorescence agreed with metabolism and transport of PC containing native fatty acids. Furthermore, the transport mechanism preferred PC containing unsaturated fatty acids such as linoleic acid. This study dissect lipid transport of PA and PC into lipid bodies and reveal regulatory steps for lipid body formation in this fungus.View full abstractDownload PDF (2466K) -
Takashi Yoneya, Tomoyuki Tahara, Kenji Nagao, Yoko Yamada, Terumi Yama ...2001 Volume 129 Issue 1 Pages 27-34
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComplementary DNAs encoding a previously unidentified mouse Notch ligand and its human ortholog were isolated. The new Notch ligand contains a signal sequence, a DSL domain, eight epidermal growth factor-like repeats, a transmembrane domain, and an intracellular region, all of which are characteristics of members of the Delta protein family. The new protein was therefore designated Delta-4. Several previously unidentified sequences in both the extracellular and intracellular regions were shown to be conserved among vertebrate Delta proteins. The tissue distribution of Delta-4 mRNA resembles that previously described for Notch-4 (Int-3) transcripts. However, in situ hybridization with mouse lung revealed that Delta-4 mRNA is abundant in squamous alveolar cells that neighbor endothelial cells; Notch-4 expression is largely restricted to the latter cell type. Soluble forms of the extracellular portion of Delta-4 inhibit the apparent proliferation of human aortic endothelial cells, but not human pulmonary arterial endothelial cells.View full abstractDownload PDF (2078K) -
Rumi Ishisaka, Tomoko Kanno, Jitsuo Akiyama, Tamotsu Yoshioka, Kozo Ut ...2001 Volume 129 Issue 1 Pages 35-41
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt. c). Treatment of ML with Ca2+ in the presence of inorganic phosphate (Pi), in contrast, released mitochondrial Cyt. c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2, 2'-azobis-(2-amidinopropane) dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.View full abstractDownload PDF (1709K) -
Yuichi Machida, Kiyohito Murai, Katsuhide Miyake, Shinji Iijima2001 Volume 129 Issue 1 Pages 43-49
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yeast SWI/SNF complex is involved in remodeling of chromatin structure during transcriptional modulation. One of the key subunits of this complex, called SWI2/SNF2, has a DNA-dependent ATPase activity. Two different types of mammalian homolog of yeast SWI2/SNF2, called BRM and BRG1, were recently identified. They are closely similar in structure but have distinct functions. We investigated the expression of BRM and BRG1 during differentiation of neural precursor cells (NPCs) cultured in vitro. The expression of BRM was very low in NPCs and was induced to a high level during differentiation to neurons and astrocytes. In contrast, BRG1 was constantly expressed throughout differentiation. These phenomena were also observed in differentiation of P19 embryonal carcinoma cells to neural cells. Immunocytochemical analyses revealed that the expression of BRM started even in the undifferentiated nestin-positive cells. These results indicate that BRM may have an important role in neural cell differentiation.View full abstractDownload PDF (2155K) -
Tsuyoshi Hirota, Satoshi Kagiwada, Takaoki Kasahara, Toshiyuki Okano, ...2001 Volume 129 Issue 1 Pages 51-59
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMelatonin is secreted from the pineal gland in a circadian manner. It is well established that the synthesis of melatonin shows a diurnal rhythm reflecting a daily change in serotonin N-acetyltransferase (NAT) activity, and the overall secretion of melatonin requires a cellular release process, which is poorly understood. To investigate the possible involvement of Golgi-derived vesicles in the release, we examined the effect of brefeldin A (BFA), a reversible inhibitor of Golgi-mediated secretion, on melatonin secretion of cultured chick pineal cells. We show here that treatment with BFA completely disassembles the Golgi apparatus and reduces melatonin secretion. In more detailed time course experiments, however, the inhibition of melatonin secretion is only observed after the removal of BFA in parallel with the reassembly of the Golgi apparatus. This inhibition of melatonin secretion is not accompanied by accumulation of melatonin in the cells. These observations indicate that chick pineal melatonin is released independently of the Golgi-derived vesicles, and suggest inhibition of melatonin synthesis after the removal of BFA. By measuring the activities and mRNA levels of melatonin-synthesizing enzymes, we found that the removal of BFA specifically inhibits NAT activity at the protein level. On the other hand, BFA causes no detectable phase-shift of the chick pineal oscillator regulating the circadian rhythm of melatonin secretion. The results presented here suggest that the Golgi-mediated vesicular transport is involved in neither the melatonin release nor the time-keeping mechanism of the circadian oscillator, but rather contributes to the regulation of NAT activity.View full abstractDownload PDF (3422K) -
Alexei Cherepanov, Esengül Yildirim, Simon de Vries2001 Volume 129 Issue 1 Pages 61-68
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOligonucleotide-directed mutagenesis is a widely used method for studying enzymes and improving their properties. The number of mutants that can be obtained with this method is limited by the number of synthetic 25-30mer oligonucleotides containing the mutation mismatch, becoming impracticably large with increasing size of a mutant library. To make this approach more practical, shorter mismatching oligonucleotides (7-12mer) might be employed. However, the introduction of these oligonucleotides in dsDNA poses the problem of sealing a DNA nick containing 5'-terminal base pair mismatches. In the present work we studied the ability of T4 DNA ligase to catalyze this reaction. It was found that T4 DNA ligase effectively joins short oligonucleotides, yielding dsDNA containing up to five adjacent mismatches. The end joining rate of mismatching oligonucleotides is limited by the formation of the phosphodiester bond, decreasing with an increase in the number of mismatching base pairs at the 5'-end of the oligonucleotide substrate. However, in the case of a 3 bp mismatch, the rate is higher than that obtained with a 2 bp mismatch. Increasing the matching length with the number of mismatching base pairs fixed, or moving the mismatching motif downstream with respect to the joining site increases the rate of ligation. The ligation rate increases with the molar ratio [oligonucleotide:dsDNA]; however, at high excess of the oligonucleotide, inhibition of joining was observed. In conclusion, 9mer oligonucleotides containing a 3 bp mismatch are found optimal substrates to introduce mutations in ds DNA, opening perspectives for the application of T4 DNA ligase in mutagenesis protocols.View full abstractDownload PDF (1875K) -
Shoji Odani, Katsuya Baba, Yutaka Tsuchida, Yutaka Aoyagi, Shuji Wakui ...2001 Volume 129 Issue 1 Pages 69-76
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo fatty acid-binding proteins (FABP), FABP-1 and FABP-2, were purified from the liver cytosol of the teleost, Lateolabrax japonicus (Japan sea bass), and characterized. The complete primary structure of FABP-2 was determined by protein analysis to be the following: MDFSGTWQVY AQENYEEFLR AMELPADVIK MAKDIKPITE IKQSGNDFVV TSKTPGKTVT NSFTIGKEAD ITTMDGKKIR CVVNLEGGKL VCNTGKFCHI QELRGGEMVE TLTMGSTTLI RKSKKM. Partial peptide sequences of FABP-1 were also determined. Phylogenetic analysis indicates that FABP-2 is a homologue of mammalian hepatic FABP, whereas FABP-I is most similar to the members of mammalian cardiac FABP subfamily. L. japonicus FABP-2 contains three cysteine residues, and a disulfide bond is identified between Cys-81 and Cys-92. A theoretical model of FABP-2 generated by a homology modeling method indicates close proximity of the two cysteine residues in the three-dimensional structure. This is a rather rare case of cytosolic protein having a disulfide bond under the normally reducing conditions of the cytosol, though the presence or absence of disulfide bonds does not seem to affect the ligand-binding ability.View full abstractDownload PDF (1986K) -
Bok-Soo Lee, Yong-Man Kim, Hyung-Sik Kang, Hwan Mook Kim, Kwang-Ho Pyu ...2001 Volume 129 Issue 1 Pages 77-86
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitric oxide (NO) has diverse effects on immune responses and hepatic functions. In BNL CL.2 cells, the marine embryonic liver cells, inducible nitric oxide synthase (iNOS) mRNA expression appeared after 3h of treatment with IFN-γ and LPS. Interestingly, mRNA and protein expression of iNOS was down-regulated by sodium nitroprusside (SNP) and diethylamine dinitric oxide in a time- and dose-dependent manner, but not by H2O2. TNF-α gene expression was also dramatically reduced by SNP, but IL-6 gene expression was inhibited much less. IFN-γ and LPS-induced chloramphenicol acetyltransferase activity of iNOS promoter constructs was inhibited by SNP- Electrophoretic mobility shift assay showed that SNP inhibited IFN-γ plus LPS-induced Oct-1 binding activity, and the inhibition was reversed by DTT. Mutation in the Oct-1 site completely abolished iNOS promoter activity. In addition, supershift assay and Southwestern analysis demonstrated that the Oct-1 binding activity was inhibited by SNP. Taken together, these results indicate that NO suppresses IFN-γ plus LPS-induced iNOS expression, and that Oct-1 is an important element in this process.View full abstractDownload PDF (5937K) -
Shigeyuki Nada, Satoshi Murakami, Shizuka Okamoto, Yoshiyuki Kubo, Aki ...2001 Volume 129 Issue 1 Pages 87-91
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tet-racycline/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-scan-ning 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 cysteinescanning 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.View full abstractDownload PDF (1943K) -
Shigeko Araki, Shoji Yamada, Sachiko Abe, Hatsue Waki, Kazuo Kon, Saki ...2001 Volume 129 Issue 1 Pages 93-100
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have reported the existence of a triphosphonoglycosphingolipid, EGL-I, in the eggs of a sea gastropod, Aplysia kurodai [Yamada, S., Araki, S., Abe, S., Kon, K., Ando, S., and Satake, M. (1995) J. Biochem. 117, 794-799]. We have now isolated a novel glycosphingolipid, named EGL-II, from the eggs of Aplysia. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and proton magnetic resonance spectrometry, its structure was revealed to be as follows: Galα1→3 (GlcNAcα1→2) Galα1→3 (3-O-MeGalα1→2) Galα1→3[6'-O-(2-aminoethylphosphonyl) Galα1→2] (2-aminoethylphosphonyl→6) Galβ1→4 (2-aminoethylphosphony1→6) Glcβ1→1ceramide. The major aliphatic components of the ceramide are pahnitic acid, stearic acid, and anteisononadeca-4-sphingenine.View full abstractDownload PDF (2608K) -
Masayuki Nakanishi, Atsushi Iwata, Chizuko Yatome, Yukio Kitade2001 Volume 129 Issue 1 Pages 101-105
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat, values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8μM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101nM for the parasite enzyme and 47nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.View full abstractDownload PDF (1144K) -
Hiromi Sakaguchi, Miki Watanabe, Chikako Ueoka, Eiko Sugiyama, Tamotsu ...2001 Volume 129 Issue 1 Pages 107-118
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe glycosaminoglycan (GAG)-protein linkage regions of various proteoglycans share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residues in the core proteins. In previous analysis we demonstrated unique modifications by epimerization, sulfation and phosphorylation of the component sugars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. This will be useful for investigation of their biological roles, which are largely unknown, but they have been implicated in biosynthesis. A variety of linkage region-derived hexasaccharides was first prepared as reducing sugar chains from peptide chondroitin/dermatan sulfate of whale cartilage, shark cartilage, and bovine aorta by means of chondroitinase digestion in conjunction with β-elimination in the absence of reducing reagents, but involving a mild alkali, 0.5M LiOH, at 4°C to prevent peeling reactions. The structures of these oligosaccharides were determined by the combination of HPLC, enzymatic digestion, matrix-assisted laser desorption ion-ization time-of-flight (MALDI-TOF) mass spectrometry, and 1H NMR spectroscopy, which revealed eleven different hexasaccharides including a novel structure, ΔHexAα1-3GalNAcβ1-4IdoAα1-3Gal (4-O-sulfate) β1-3Galβ1-4Xyl (ΔHexA and IdoA represent unsaturated hexuronic acid and L-iduronic acid, respectively). These oligosaccharides were labeled with a fluorophore, 2-aminobenzamide, to prepare analytical probes using the recently developed procedure [Kinoshita and Sugahara (1999) Anal. Biochem. 269, 367-378]. The fluorophore-tagged hexasacharides of low picomoles were well separated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry. The principle of the method should be applicable to the analysis of the linkage region oligosaccharides derived from heparin and heparan sulfate as well.View full abstractDownload PDF (1536K) -
Nikolaos Gaitatzis, Astrid Hans, Rolf Müller, Stefan Beyer2001 Volume 129 Issue 1 Pages 119-124
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyxothiazol is synthesized by the myxobacterium Stigmatella aurantiaca DW4/3-1 via a combined polyketide synthase/polypeptide synthetase. The biosynthesis of this secondary metabolite is also dependent on the gene product of mtaA. The deduced amino acid sequence of mtaA shows similarity to 4'-phosphopantetheinyl transferases (4'-PP transferase). This points to an enzyme activity that converts inactive forms of the acyl carrier protein domains of polyketide synthetases (PKSs) and/or the peptidyl carrier protein domains of nonribosomal polypeptide synthetases (NRPSs) of the myxothiazol synthetase complex to their corresponding holo-forms. Heterologous co-expression of MtaA with an acyl carrier protein domain of the myxothiazol synthetase was performed in Escherichia coli. The proposed function as a 4'-PP transferase was confirmed and emphasizes the significance of MtaA for the formation of a catalytically active myxothiazol synthetase complex. Additionally, it is shown that MtaA has a relaxed substrate specificity: it processes an aryl carrier protein domain derived from the enterobactin synthetase of E. coli (ArCP) as well as a peptidyl carrier protein domain from a polypeptidesynthetase of yet unknown function from Sorangium cellulosum. Therefore, MtaA should be a useful tool for activating heterologously expressed PKS and NRPS systems.View full abstractDownload PDF (2065K) -
Shinji Mizuarai, Ken-ichiro Ono, Jun You, Masamichi Kamihira, Shinji I ...2001 Volume 129 Issue 1 Pages 125-132
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCationic lipid vesicle-mediated gene transfer has become common forin vitro gene delivery. However, the transfection efficiency is often impaired by serum. DDAB (dimethyldioctadecyl ammonium bromide) lipid vesicle-mediated gene transfer, which we previously reported, has the same problem. To overcome this obstacle, we here report a novel transfection vehicle using protamine-modified DDAB lipid vesicles. While free protamine was simply added to the DNA/lipid complex in the previous study, in the present method the protamine is chemically conjugated to stearic acid and incorporated into DDAB lipid vesicles. Gene transfer was not significantly inhibited in 10% serum-containing medium by this method for the transfection of cultured cells. Protamine-modified DDAB lipid vesicles also enhanced virus transduction efficiency in the presence of serum using a replication-defective retroviral vector. Furthermore, the vesicles allowed efficient gene transfer for avian embryos in vivo. These results indicate that the method is useful for the production of transgenic animals and gene therapy.View full abstractDownload PDF (2636K) -
Morimichi Mizuno, Yoshinori Kuboki2001 Volume 129 Issue 1 Pages 133-138
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteo-pontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the α2β1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-α2β1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.View full abstractDownload PDF (1648K) -
Miwako Kajihara, Shimako Kawauchi, Makoto Kobayashi, Hajime Ogino, Sat ...2001 Volume 129 Issue 1 Pages 139-146
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLarge Maf proteins, which are members of the basic leucine zipper (b-Zip) superfamily, are involved in the determination and control of cellular differentiation. The expression patterns of various vertebrate large Maf mRNAs were described previously. Here, we report the cloning of a novel zebrafish large Maf cDNA, SMaf1 (Somite Maf1), and other zebrafish large Mafs, the N-terminus domains of which possess transactivational activity. We also analyzed the expression patterns of SMaf1 and SMaf2 (Somite Maf2)/Krml2 as well as MafB/Val and c-Maf during zebrafish embryogenesis. In particular, the robust expression of the novel SMaf1 mRNA, which overlapped that of MyoD, in somitic cells during somitogenesis was noteworthy. In addition, the expression patterns of SMaf2 and MafB in the blood-forming regions, and those of c-Maf and MafB in the lens cells showed spatial redundancy, although the temporal appearance of these genes at these sites differed. These data indicate that SMafs may play important roles in somitogenesis, and that Maf proteins may have overlapping and yet specific functions as to the determination and differentiation of cell lineages.View full abstractDownload PDF (2668K) -
Masayuki Ikuzawa, Tomofumi Inokuchi, Ken-ichiro Kobayashi, Shigeki Yas ...2001 Volume 129 Issue 1 Pages 147-153
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo pepsinogens (Pg C and Pg A) were isolated from the stomach of adult Xenopus laevis by Q-Sepharose, Sephadex G-75, and Mono-Q column chromatographies. Autolytic conversion and activation of the purified Pgs into the pepsins were examined by acid treatment. We determined the amino acid sequences from the NH2-termini of Pg C, pepsin C, Pg A, and pepsin A. Based on the sequences, the cDNAs for Pg C and Pg A were cloned from adult stomach RNA, and the complete amino acid sequences of the Pg C and Pg A were predicted. In addition, a Pg A cDNA was cloned from the stomach of adult bullfrog Rana catesbeiana, and the primary structure of the Pg A was predicted. Molecular phylogenetic analysis showed that such anuran Pg C and Pg A belong to the Pg C group and the Pg A group in vertebrates, respectively. The molecular properties of Pg C and Pg A, such as size, sequences of the activation peptide and active site, profile of autolytic activation, and pH dependency of proteolytic activity of the activated forms, pepsin C and pepsin A, resemble those of Pgs found in other vertebrates. However, the hemoglobin-hydrolyzing activity of Xenopus pepsin C is completely inhibited in the presence of equimolar pepstatin, an inhibitor of aspartic proteinases. Thus, the Xenopus pepsin C differs significantly from other vertebrate pepsins C in its high susceptibility to pepstatin, and closely resembles A-type pepsins.View full abstractDownload PDF (1868K) -
Sakae Kitada, Akio Ito2001 Volume 129 Issue 1 Pages 155-161
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of α and β subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the N-terminal basic matrix targeting signals (MTS). Basic residues in MTS and acidic sites in MPP are required for effective processing. To elucidate whether the enzyme recognizes the MTS through electrostatic interaction, we investigated the effects of various salts on MPP activity. Decreases in the activity depended on the ionic strength and increases in the Michaelis constant value correlated clearly with the ionic strength, indicating a lower affinity of the enzyme for the substrate. Direct determination of the affinity between MPP and a MTS peptide using surface plasmon resonance showed a decrease in the association rate with high salt and that dissociation constant values were decreased. The effect of salt on the processing activity towards a variety of precursors was confirmed using five precursors with different sequences and lengths of MTS. Thus, we propose that electrostatic interactions are indispensable for the association between various MTS and MPP.View full abstractDownload PDF (2430K) -
Javed Mahmood, Hiroko Takita, Yasutaka Ojima, Masahiro Kobayashi, Taka ...2001 Volume 129 Issue 1 Pages 163-171
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new biocompatible glass, which is composed of CaO, P2O5, SiO2, and Al2O3 (abbreviated CPSA) and is characterized by higher elasticity than previous bioglass products, was molded into fibers with a diameter of 9μm. With CPSA fibers, two geometrically different structures, balls and bundles (each 20mg in weight), were prepared, combined with 2.2μg of rhBMP-2 (a gift from Yamanouchi Co., Japan) and implanted subcutaneously into rats. The histology showed remarkably higher bone formation in the ball-CPSA/BMP at 2 and 4 weeks than in the bundle-CPSA/BMP. The ball-CPSABMP showed 10 times higher alkaline phosphatase (ALP) activity at the second week and 5 times higher osteocalcin content at the fourth week than the bundle-CPSA/BMP. Vascular development in the implants was evaluated by mRNA expression of Flt-1 and KDR, two receptors for vascular endothelial growth factor (VEGF). Both receptors showed higher expression in the case of the ball, while they were not detected in the bundle. It is concluded that the BMP-induced bone formation depends highly upon the porous vasculature-inducing geometry of the matrix, which can be constructed with the new CPSA fibers.View full abstractDownload PDF (2810K) -
Hideaki Ura, Kazuaki Harata, Ikuo Matsui, Seiki Kuramitsu2001 Volume 129 Issue 1 Pages 173-178
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe determined the crystal structure of the liganded form of α-aminotransferase from a hyperthermophile, Pyrococcus horikoshii. This hyperthermophilic enzyme did not show domain movement upon binding of an acidic substrate, glutamate, except for a small movement of the α-helix from Glu16 to A1a25. The ω-carboxyl group of the acidic substrate was recognized by Tyr70* without its side-chain movement, but not by positively charged Arg or Lys. Compared with the homologous enzymes from Thermus thermophilus HB8 and Escherichia coli, it was suggested that the more thermophilic the enzyme is, the smaller the domain movement is. This rule seems to be applicable to many other enzymes already reported.View full abstractDownload PDF (1401K) -
Yin Gao, Kapil Mehta2001 Volume 129 Issue 1 Pages 179-183
Published: 2001
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe provide evidence that in vitro protein cross-linking can be accomplished in three concerted steps: (i) a change in protein conformation; (ii) formation of interchain disulfide bonds; and (iii) formation of interchain isopeptide cross-links. Oxidative refolding and thermal unfolding of ribonuclease A, lysozyme, and protein disulfide isomerase led to the formation of cross-linked dimers/oligomers as revealed by SDS-polyacrylamide gel electrophoresis. Chemical modification of free amino groups in these proteins or unfolding at pH<7.0 resulted in a loss of interchain isopeptide cross-linking without affecting interchain disulfide bond cross-linking. Furthermore, preformed interchain disulfide bonds were pivotal for promoting subsequent interchain isopeptide crosslinks; no dimers/oligomers were detected when the refolding and unfolding solution contained the reducing agent dithiothreitol. Similarly, the Cys326Ser point mutation in protein disulfide isomerase abrogated its ability to cross-link into homodimers. Heterogeneous proteins become cross-linked following the formation of heteromolecular interchain disulfide bonds during thermal unfolding of a mixture of of ribonuclease A and lysozyme. The absence of glutathione and glutathione disulfide during the unfolding process attenuated both the interchain disulfide bond cross-links and interchain isopeptide cross-links. No dimers/oligomers were detected when the thermal unfolding temperature was lower than the midpoint of thermal denaturation temperature.View full abstractDownload PDF (1851K)
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