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Volume 128 (2000)
- Issue 6 Pages 891-
- Issue 5 Pages 719-
- Issue 4 Pages 545-
- Issue 3 Pages 345-
- Issue 2 Pages 153-
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Volume 128, Issue 3
Displaying 1-27 of 27 articles from this issue
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Dong Luo, Weimin Qi, Jie Ma, Yan Jun Wang, David Wishart2000 Volume 128 Issue 3 Pages 345-347
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated and characterized an anti-idiotype monoclonal antibody (AR 42.1) which is capable of mimicking a distinct and specific epitope of MUC-1 antigen. The cDNA sequences coding for the AR 42.1 variable regions were determined. We found significant amino acid homology between complementary determining regions 3 (CDR 3) in the heavy chain of AR 42.1 and the determinant epitope sequence of MUC-1. This 10 amino acid sequence may represent an “internal image” of the anti-idiotype antibody to the MUC-1 antigen, and could be used for development of a MUC-1 surrogate for immunotherapy.View full abstractDownload PDF (258K) -
Hiroyuki Motoshima, Kenji Inagaki, Takashi Kumasaka, Makio Furuichi, H ...2000 Volume 128 Issue 3 Pages 349-354
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Methionine γ-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent α, γ-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudonwnas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 Å resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine β-lyase and L-cystathionine γ-synthase from Escherichia coli.View full abstractDownload PDF (1912K) -
Shoji Odani, Yukimi Namba, Akio Ishii, Teruo Ono, Hiroshi Fujii2000 Volume 128 Issue 3 Pages 355-361
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUnlike other fatty acid-binding proteins, cutaneous (epidermal) fatty acid-binding proteins contain a large number of cysteine residues. The status of the five cysteine residues in rat cutaneous fatty acid-binding protein was examined by chemical and massspectrometric analyses. Two disulfide bonds were identified, between Cys-67 and Cys-87, and between Cys-120 and Cys-127, though extent of formation of the first disulfide bond was rather low in another preparation. Cys-43 was free cysteine. Homology modeling study of the protein indicated the close proximity of the sulfur atoms of these cysteine pairs, supporting the presence of the disulfide bonds. These disulfide bonds appear not to be directly involved in fatty acid-binding activity, because a recombinant rat protein expressed in Escherichia coli in which all five cysteines are fully reduced showed fatty acid-binding activity as examined by displacement of a fluorescent fatty acid analog by long-chain fatty acids. However, the fact that the evolutionarily distant shark liver fatty acid-binding protein also has a disulfide bond corresponding to the one between Cys-120 and Cys-127, and that fatty acid-binding proteins play multiple roles suggests that some functions of cutaneous fatty acid-binding protein might be regulated by the cellular redox state through formation and reduction of disulfide bonds. Although we cannot completely exclude the possibility of oxidation during preparation and analysis, it is remarkable that a protein in cytosol under normally reducing conditions appears to contain disulfide bonds.View full abstractDownload PDF (1199K) -
Kuniyo Inouye, Hiromi Tanaka, Hiroshi Oneda2000 Volume 128 Issue 3 Pages 363-369
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStates of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69°C for 10min. The activity was increased by adding NaCl, and was doubled with 3M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7M GdnHCl, whereas no change was observed even with 8M urea. Half of the inactivation was induced at 0.8M GndHCl and at 2M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I- and Cs+, and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp 154 is water-accessible, but it should be in a crevice not to contact with I- and Cs+. All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.View full abstractDownload PDF (628K) -
Shuuji Koyanagi, Kumiko Nagata, Toshihide Tamura, Sakura Tsukita, Nobu ...2000 Volume 128 Issue 3 Pages 371-375
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHelicobacter pylori, a microaerophilic Gram-negative spiral bacterium residing in the human stomach, contains a small size soluble cytochrome c. This cytochrome c was purified from the soluble fraction of H. pylori by conventional chromatographies involving octyl-cellulose and CM-Toyopearl. Its reduced form gave an alpha absorption band at 553 nm, and thus the cytochrome was named H. pylori cytochrome c-553. The cytochrome, giving a band below 10, 000 Da upon SDS-PAGE, was determined to have a mass of 8, 998 by time of flight mass spectroscopy. Its N-terminal peptide sequence was TDVKALAKS---, indicating that the nascent polypeptide was cleaved to produce a signal peptide of 19 amino acid residues and a mature protein composed of 77 amino acid residues. The cb-type cytochrome c oxidase oxidized ferrocytochrome c-553 of this bacterium actively (Vmax of about 250 s-1) with a small Km (0.9 μM). Analysis of the effect of the salt concentration on the oxidase activity indicated that oxidation of cytochrome c-553 is highly inhibited under high ionic conditions. The amino acid sequence of H. pylori cytochrome c-553 showed the closest similarity to that of Desulfovibrio vulgaris cytochrome c-553, and these sequences showed a weak relationship to that of the cytochrome c8-group among class I cytochromes c.View full abstractDownload PDF (1146K) -
Ayano Satoh, Miwa Hazuki, Kyoko Kojima, Jun Hirabayashi, Isamu Matsumo ...2000 Volume 128 Issue 3 Pages 377-381
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnnexins are structurally related proteins that bind phospholipids in a calcium-dependent manner. Recently, we showed that annexins IV, V, and VI also bind glycosaminoglycans in a calcium-dependent manner. Annexins are widely distributed from lower to higher eukaryotes, and the nematode Caenorhabditis elegans has been found to contain Nex-1, an annexin homologue. Here, we characterize the ligand-binding properties of Nex-1 using recombinant Nex-1. Nex-1 binds to liposomes containing phosphatidylserine. The apparent Kd was calculated by Biacore to be 4.4 nM. Compared to mammalian annexins, the Nex-1 phospholipid-binding specificities were similar whereas the Kd values were one order of magnitude larger. The Nex-1 glycosaminoglycan-binding specificities were investigated by affinity chromatography and solid-phase assays. Nex-1 binds to heparin, heparan sulfate, and chondroitin sulfate but not to chondroitin and chemically N- or O-desulfated heparin. Besides phospholipids, heparan sulfate and/or chondroitin (sulfate), probably on perlecan, could be endogenous ligands of Nex-1.View full abstractDownload PDF (1176K) -
Zilian Zhang, Setsuko Komatsu2000 Volume 128 Issue 3 Pages 383-389
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) activase catalyzes the activation of RuBisCO in vivo. Two full length cDNAs designated as OsrcaA 1 and OsrcaA 2 encoding two RuBisCO activase isoforms of 47 and 43 kDa, respectively, have been cloned and characterized. The two isoforms were 99% identical, the 47 kDa isoform having an additional 33 amino acids and a 5 amino acid substitution at the carboxyl terminus. The deduced amino acid sequences of OsrcaA 1 and OsrcaA 2 showed 73-89% identity with RuBisCO activase from other higher plants. Two highly conserved ATP binding sites were identified. The Osrca mRNAs, and the RuBisCO activase proteins of 43 and 47 kDa were specifically detected in leaf, but not in root or etiolated seedling tissues. During leaf development, the abundance of Osrea mRNAs increased from the 7 th to the 3 rd leaf, and reached a maximum in the 2 nd leaf, although the amounts of the 43 and 47 kDa RuBisCO activase remained almost unchanged among the six leaves, indicating the involvement of post-transcription control in the regulation of RuBisCO activase expression in rice. The co-immunoprecipitation of RuBisCO LSU and SSU with RuBisCO activase suggests that RuBisCO activase interacts with RuBisCO in vivo.View full abstractDownload PDF (1986K) -
Tadashi Fujii, Takao Narita, Hitosi Agematu, Naoki Agata, Kunio Isshik ...2000 Volume 128 Issue 3 Pages 391-397
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Lysine 6-aminotransferase (LAT) is an enzyme involved in L-lysine catabolism in a wide range of living organisms. LAT from Flavobacterium lutescens IFO 3084 was purified, and its structural gene (lat) was cloned, sequenced and expressed in Eseheriehia coli. Native PAGE analysis of purified LAT gave a single band corresponding to a molecular weight of about 110, 000. lat encoded a protein of 493 amino acids with a deduced molecular weight of 53, 200, which is very close to that of purified LAT determined on SDS-PAGE. Expression of lat in E. coli revealed that lat encodes a single subunit protein leading to LAT activity. These data suggested that LAT from F. lutescens IFO 3084, like most other aminotransferases, is derived from a single ORF and is active as a homodimer.View full abstractDownload PDF (1306K) -
Tomoko Makishima, Michihiro Yoshimi, Sohtaro Komiyama, Nobuyuki Hara, ...2000 Volume 128 Issue 3 Pages 399-405
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDAD 1 is a mammalian homologue of Saccharomyces cerevisiae Ost 2 p, a subunit of the oligosaccharyltransferase complex. Loss of its function induces apoptosis in hamster BHK 21 cells. By means of a two-hybrid method involving DAD 1 as bait, the C-terminal region of Mcl-1, one of the bcl-2 family, was isolated. Consistently, DAD 1 binds well to Mcl-1 in COS cells when overexpressed. On deletion analysis, the C-terminal domain of Mcl-1 containing BH2 (bcl-2 homologous domain) was found to be essential for the interaction with DAD 1. On the other hand, the C-terminal half of DAD 1 was concluded to be essential for the interaction with Mcl-1. Surprisingly, a ΔC-DAD 1 mutant lacking only 4 amino acid residues from the C-terminus did not complement the tsBN 7 mutation, while it interacted well with Mcl-1. In contrast, ΔN-DAD 1 lacking 20 amino acid residues from the N-terminus still exhibited the ability to complement the tsBN 7 mutation. Thus, the C-terminus of DAD 1 was suggested to play an important role in N-linked glycosylation and to complement the tsBN 7 mutation. Mcl-1 may be required for the inhibition of apoptotic cell death caused by a loss of DAD 1.View full abstractDownload PDF (2837K) -
Satoshi Iwasaki, Masakazu Kataoka, Mariko Sekiguchi, Yoji Shimazaki, K ...2000 Volume 128 Issue 3 Pages 407-414
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhorbol ester treatment induces the phosphorylation of SNAP-25 at Ser187 and the potentiation of Ca2+-induced dopamine (DA) and acetylcholine (Ach) release from PC 12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser187. DA and ACh release, assayed in low-K+ as well as high-K+ solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K+-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K+-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K+-dependent neurotransmitter release. The potentiation of high-K+-dependent DA release by phorbol 12, 13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K+-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC 12 cells.View full abstractDownload PDF (2788K) -
Seiichi Taguchi, Kei-ichiro Nishihama, Keiko Igi, Kotaro Ito, Hiroko T ...2000 Volume 128 Issue 3 Pages 415-425
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI 2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI 2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI 2. The results suggest that the P 1 reactive center site (position 70 of STI 2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated β-casein was used as a partner Gln-substrate. It is characteristic in that the same primary enzyme contact region of STI 2 is shared by both enzymes, MTG and proteases. For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI 2. Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) sitespecific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70 Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67 Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI 2 and its mutants. In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also, 70 Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between MTG and GTG.View full abstractDownload PDF (2374K) -
Differential Expression of Mouse α5 (IV) and α6 (IV) Collagen Genes in Epithelial Basement MembranesKenji Saito, Ichiro Naito, Tsugio Seki, Toshitaka Oohashi, Eiki Kimura ...2000 Volume 128 Issue 3 Pages 427-434
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe first completed the primary structure of the mouse α5 (IV) and α6 (IV) chains, from which synthetic peptides were produced and α chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [α1 (IV)]2 α2 (IV), α3 (IV) α4 (IV) α5 (IV), and [α5 (IV)]2 α6 (IV). In skin basement membrane two of the three forms, [α1 (IV)]2 α2 (IV) and [α5 (IV)]2 α6 (IV), were detected. The α3 (IV) α4 (IV) α5 (IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [α5 (IV)]2 α6 (lV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.View full abstractDownload PDF (2220K) -
Futoshi Suizu, Kozue Ueda, Takahiro Iwasaki, Maki Murata-Hori, Hiroshi ...2000 Volume 128 Issue 3 Pages 435-440
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRegulatory light chain of myosin II (MRLC) was identified as a novel substrate of p 90 ribosomal S 6 kinase (RSK)-2, a Ser/Thr protein kinase which is phosphorylated and activated by mitogen-activated protein kinase (MAPK) in vitro and in vivo. Phosphopeptide map of MRLC phosphorylated by RSK-2 was identical to that by myosin light chain kinase (MLCK). Phosphoserine was recovered by the phosphoamino acid analysis of MRLC phosphorylated by RSK-2. Further, phosphorylation using recombinant glutathione S-transferase (GST) fusion proteins of HeLa MRLC 2 revealed that RSK-2 phosphorylated wild-type MRLC 2 (GST-wtMRLC 2) but not its mutants GST-MRLC 2S 19 A or GST-MRLC 2T 18 A 19 A (alanine substituted for Ser 19 or both Ser 19 and Thr 18). These results revealed that RSK-2 phosphorylates MRLC at Ser 19 as did MLCK. Phosphorylation of myosin II by RSK-2 resulted in activation of actin-activated MgATPase activity of myosin II. Interestingly, RSK-2 activity to phosphorylate MRLC was suppressed by phosphorylation with MAPK. RSK-2 might be a mediator that regulates myosin II activity through the MAPK cascade.View full abstractDownload PDF (1834K) -
Hidefumi Uchiyama, Tetsuya Inaoka, Toyomi Ohkuma-Soyejima, Hiroko Toga ...2000 Volume 128 Issue 3 Pages 441-447
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProlyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42min at 60°C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.View full abstractDownload PDF (1456K) -
Sun Tack Kim, Si Myung Byun2000 Volume 128 Issue 3 Pages 449-454
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe designed and expressed a single-chain class II major histocompatibility complex molecule capable of forming a stable complex with an antigenic peptide. The peptide-binding preference of the single-chain (sc) human leukocyte antigen derived from DRB5'0101 (DR 51) was determined to be similar to that of the authentic one, which requires a bulky hydrophobic residue at position-1 (P 1) as a primary anchor. For modulation of the peptide-binding affinity, we modified binding pocket 1 of sc DR 51 by sitedirected mutagenesis. The relative binding affinity of the engineered sc DR 51 for several P 1-substituted peptides was measured by competition assaying with a fluorescence labeled peptide. The se DR 51 molecule showed high affinity to the self-peptide derived from myelin basic protein, 87-98 with Phe as the P1 residue (F 90 F). While reduction of pocket 1 volume (β G 86 V) decreased the affinity of F 90 F, it rather increased the affinity of the Ala-substituted peptide as to the P 1 residue (F 90 A). Through more extensive engineering in the peptide-binding groove of the sc DR 51 molecule, it is expected that we can construct sc DR 51 variants with various peptide ligand motifs.View full abstractDownload PDF (1189K) -
Torn Kawakami, Chikako Toda, Kenichi Akaji, Takayuki Nishimura, Teruak ...2000 Volume 128 Issue 3 Pages 455-461
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarhii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Prc-MIH consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys (Acm)7, 24. 27 Lys (Boc)19]-Prc-MIH (1-39)-SCH2CH2CO-Nle-NH2 and H-[Cys (Acm)40. 44, 53 Lys (Boc)42, 51, 67]-Prc-MIH (40-75)-NH2, were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in the presence of silver chloride, 3, 4-dihydro-3-hydroxy-4-oxo-1, 2, 3-benzotriazine, and N, N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH (1-75)-NH2. This product was converted to the native form of Prc-MIH (synthetic Prc-MIH) in a buffer which contained cysteine and cystine. The synthetic Prc-MIH showed the same behavior by RP-HPLC and biological activity assays as the natural Prc-MIH. The disulfide bond between Cys 7 and Cys 44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys 24 and Cys 40 and between Cys 27 and Cys 53 were determined by comparing the elution position of an enzymatic digest of the synthetic Prc-MIH with authentic chemically synthesized samples, which contained three types of possible disulfide linkages.View full abstractDownload PDF (654K) -
Nobukazu Komatsu, Masami Nakagawa, Tatsuya Oda, Tsuyoshi Muramatsu2000 Volume 128 Issue 3 Pages 463-470
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous studies demonstrated that ricin induces the apoptotic death of U 937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD+ and ATP levels decrease in ricin-treated U 937 cells and that this decrease is followed by the ricinmediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD+ and ATP levels and concomitantly protects U 937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells. However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis. Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis. Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.View full abstractDownload PDF (1884K) -
Takenori Takizawa, Chizuru Tatematsu, Masami Watanabe, Minoru Yoshida, ...2000 Volume 128 Issue 3 Pages 471-476
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe double-stranded RNA-activated-protein kinase PKR was originally identified as a ribosomal protein that regulates protein synthesis at the translational level. While PKR locates predominantly to the cytoplasm, nuclear or nucleolar species of PKR have been detected. Here, we demonstrate that PKR possesses three leucine-rich sequences resembling nuclear export signals (NESs). Enhanced green fluorescent protein (EGFP) fused to one of these sequences and transfected in COS-1 cells exhibited predominant cytoplasmic staining, which was abrogated by a leucine to alanine substitution. In addition, Leptomycin B (LMB), an inhibitor of NES-mediated nuclear export, inhibited the cytoplasmic localization of EGFP-NES, indicating the potential activity of these stretches as NESs. Although EGFP fused to a PKR with three NES mutations still located to the cytoplasm, an additional N-terminal deletion impaired the cytoplasmic predominance, suggesting that the N-terminal region is also required for localization. These results suggest that the cytoplasmic localization of PKR is regulated by NESs as well as the N-terminal sequence.View full abstractDownload PDF (2457K) -
J. G. Shirani Ranasinghe, Ming-Cheh Liu, Yoichi Sakakibara, Masahito S ...2000 Volume 128 Issue 3 Pages 477-480
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSprague-Dawley rats were used as an experimental model for investigating the effects of manganese poisoning on the serum levels of unsulfated and sulfated forms of dopamine and its biosynthetic precursors, L-Dopa and L-p-tyrosine. Groups of rats were treated daily with Mn2+ (20mg or 40mg; in the form of MnSO4) or Na+ (20mg; in the form of Na2SO4). High performance liquid chromatography (HPLC) analysis of the serum samples taken after a 50-day experimental period revealed that the serum level of dopamine sulfate increased by more than 10 times compared with untreated control rats or rats treated with sodium sulfate. In contrast, there was a dramatic decrease (by as much as 4.8 times) in the serum level of unsulfated dopamine in manganese-treated rats. The serum levels of L-Dopa sulfate and L-p-tyrosine sulfate were also markedly elevated, although not as much as those of dopamine sulfate. Meanwhile, the serum levels of unsulfated L-Dopa and L-p-tyrosine showed no dramatic changes. Atomic absorption spectrophotometric analysis revealed in general an accumulation of manganese in the four organ samples taken from manganese-treated rats. Compared with liver, heart, and kidney, the highest degree of manganese accumulation in manganese-treated rats appeared to be in brain. These results together suggested a role for manganese in stimulating the dopamine-sulfating sulfotransferases in brain, thereby leading to the depletion of dopamine in vivo.View full abstractDownload PDF (353K) -
Susann Teneberg, Anna Berntsson, Jonas Ångström2000 Volume 128 Issue 3 Pages 481-491
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe heat-labile enterotoxin from Escherichia coli (LT) is responsible for so-called traveller's diarrhea and is closely related to the cholera toxin (CT). Toxin binding to GM 1 at the epithelial cell surface of the small intestine initiates the subsequent diarrheal disease. However, LT has a broader receptor specificity than CT in that it also binds to N-acetyllactosamine-terminated structures. The unrelated lectin from Erythrina corallodendron (ECorL) shares this latter binding property. The findings that both ECorL and porcine LT (pLT) bind to lactose as well as to neolactotetraosylceramide suggests a common structural theme in their respective primary binding sites. Superimposing the terminal galactose of the lactoses in the respective crystal structures of pLT and ECorL reveals striking structural similarities around the galactose despite the lack of sequence and folding homology, whereas the interactions of the penultimate GlcNAcβ3 in the neolactotetraosylceramide differ. The binding of branched neolactohexaosylceramide to either protein reveals an enhanced affinity relative to neolactotetraosylceramide. The β3-linked branch is found to bind to the primary Gal binding pocket of both proteins, whereas the β6-linked branch outside this site provides additional interactions in accordance with the higher binding affinities found for this compound. While the remarkable architectural similarities of the primary galactose binding sites of pLT and ECorL point to a convergent evolution of these subsites, the distinguishing structural features determining the overall carbohydrate specificities are located in extended binding site regions. In pLT, Arg 13 is thus found to play a crucial role in enhancing the affinity not only for N-acetyllactosamine-terminated structures but also for GM 1 as compared to human LT (hLT) and CT. The physiological relevance of the binding of N-acetyllactosamine-containing glycoconjugates to LT and ECorL is briefly discussed.View full abstractDownload PDF (4660K) -
Chia-Hui Lin, Mingli Hsieh, Yueh-Chun Li, Shuan-Yow Li, Deborah L. Pea ...2000 Volume 128 Issue 3 Pages 493-498
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArginine methylation in RNA-binding proteins containing arginin- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells. We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates for the endogenous protein methyltransferases or recombinant yeast arginine methyltransferase [Li, C. et al. (1998) Arch. Biochem. Biophys. 351, 53-59]. In this study we fractionated the lymphoblastoid cells to locate the methyltransferases and the substrates in cells. Different sets of hypomethylated methyl-accepting polypeptides with wide range of molecular masses were present in cytosolic, ribosomal, and nucleus fractions. The methylated amino acid residues of the methyl-accepting proteins in these fractions were determined. In all three fractions, dimethylarginine was the most abundant methylated amino acid. The protein-arginine methyltransferase activities in the three fractions were analyzed using recombinant fibrillarin (a nucleolar RGG protein) as the methyl-accepting substrate. Fibrillarin methylation was strongest in the presence of the cytosolic fraction, followed by the ribosomal and then the nucleus fractions. The results demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells.View full abstractDownload PDF (2590K) -
Kohei Oda, Shin Ogasawara, Hiroshi Oyama, Ben M. Dunn2000 Volume 128 Issue 3 Pages 499-507
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (765K) -
Junji Ezaki, Mitsue Takeda-Ezaki, Eiki Kominami2000 Volume 128 Issue 3 Pages 509-516
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN 2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the β-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.View full abstractDownload PDF (2822K) -
Koichi Oba, Toshihiko Yanase, Isao Ichino, Kiminobu Goto, Ryoichi Taka ...2000 Volume 128 Issue 3 Pages 517-528
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAd 4 BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F 1 gene encoding Ad 4 BP in adrenocortical cells have been proposed in the previous studies: the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F 1 genes, and a possible autoregulatory mechanism of the rFTZ-F 1 gene by Ad 4 BP itself through binding to the Ad 4 (or SF-1) site in the first intron. In the present study, the transcriptional regulation of the human FTZ-F 1 gene in adrenocortical cells was investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F 1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was also found to be essential for the transcription of the hFTZ-F 1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad 4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F 1 gene, an autoregulatory mechanism through the Ad 4 site was found to be unlikely in the hFTZ-F 1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad 4 BP had little effect on hFTZ-F 1 gene transcription in non-steroidogenic CV-1 cells. Such Ad 4 BP-independent regulation of the hFTZ-F 1 gene was in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP 11 A gene, in which the proximal promoter activity is Ad 4 BP-dependent and the transactivation by Ad 4 BP is silenced by DAX-1. Even though the Ad 4 BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F 1 gene in our study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F 1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F 1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F 1 gene appears to be independent of both Ad 4 BP and DAX-1.View full abstractDownload PDF (1581K) -
Sumiko Kaneda, Takashi Yura, Hideki Yanagi2000 Volume 128 Issue 3 Pages 529-538
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS150 kDa oxygen-regulated protein (ORP 150) is one of the endoplasmic reticulum (ER)-resident stress proteins. We have cloned and sequenced the entire human ORP 150 gene covering over 15-kb. Analyses of transcription initiation sites and transcriptional regulatory sequences revealed that at least three distinct mRNA species were produced by alternative promoters: two of them starting from alternative exon 1 (1 A or 1 B), and the third one starting from exon 2, six nucleotides upstream of the first AUG initiation codon. Among them, the transcript that begins with exon 1 B was preferentially induced by hypoxia or tunicamycin treatment. A cis-acting segment involved in the stress-dependent induction was found at the 5'-end of exon 1 A, which could account for the selective induction of the transcription from exon 1 B. Furthermore, in vitro analyses of translation of the third mRNA suggested the constitutive expression of the cytosolic ORP 150 due to the lack of the signal peptide resulting from differential translation initiation.View full abstractDownload PDF (2372K) -
Ikuko Fujii-Taira, Yasuhiro Tanaka, Koichi J. Homma, Shunji Natori2000 Volume 128 Issue 3 Pages 539-542
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we identified two proteins with molecular masses of 200 and 210 kDa in basement membranes of Sarcophaga imaginal discs as substrates for cathepsin L [Homma, K. and Natori, S. (1996) Eur. J. Biochem. 240, 443-447]. Here we demonstrated that the same proteins were also present in the basement membranes of larval brains. These proteins were suggested to be digested by cathepsin L secreted from the larval brains in response to 20-HE. From the behavior of these proteins during metamorphosis, we concluded that the basement membranes of larval brains are degraded at the early pupal stage and synthesized again at the late pupal stage, coinciding with the timing of brain remodeling that takes place during metamorphosis. Possibly, the transient disappearance of the basement membranes makes brain remodeling easier, and cathepsin L is suggested to play a crucial role in the degradation of the basement membranes.View full abstractDownload PDF (1518K) -
2000 Volume 128 Issue 3 Pages 543
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (19K)
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