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Volume 125 (1999)
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Volume 125, Issue 2
Displaying 1-28 of 28 articles from this issue
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Tapas K. Kundu, M. R. S. Rao1999 Volume 125 Issue 2 Pages 217-222
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCpG islands are stretches of DNA sequence that are enriched in the (CpG)n repeat and are present in close association with all housekeeping genes as well as some tissue-specific genes in the mammalian genome. Methylation of CpG islands strongly influences both structural organization and function of chromatin. The presence of a CpG island in a given chromosomal domain can, by itself, give rise to relatively open and active chromatin. Recently, several histone acetyltransferases, histone deacetylases, and chromatin remodeling factors have been found to be part of the transcription machinery. It is becoming increasingly clear that CpG islands and their methylation status may influence the function or recruitment of these newly discovered chromatin remodeling factors, especially the histone deacetylases. In addition, CpG islands may also play a significant role in the reorganization of chromatin during mammalian spermiogenesis.View full abstractDownload PDF (836K) -
Fumiaki Yamao1999 Volume 125 Issue 2 Pages 223-229
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA growing number of cellular functions have been shown to be regulated through protein degradation. The selective degradation of many short-lived proteins in eukaryotic cells is mediated by the ubiquitin system, by which proteins covalently ligated to ubiquitin are targeted for degradation. The selectivity of the destruction is ensured by the substrate specificity in the ubiquitination steps composed of a series of enzymatic reactions. Ubiquitin-ligase (E3), in conjunction with ubiquitin-conjugating enzyme (E2), has been implicated as playing an essential role in the substrate recognition. The substantial character, however, of the ligase was not clear until several recent studies demonstrated ligases that exert key roles in irreversible steps of the cell-cycle control. In this review, attention is focused on the molecular basis of target recognition of ubiquitination, particularly as exemplified in the ubiquitin-ligases in the cell-cycle control mechanisms.View full abstractDownload PDF (948K) -
Shunji Ohsako, Yoshiki Takamatsu1999 Volume 125 Issue 2 Pages 230-235
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yeast UBC9 and hus5 gene products have been identified as putative E2 members of the ubiquitin-conjugating enzyme (UBC) family and have been shown to play an essential role in cell cycle progression. We have identified a Drosophila Ubc9/Hus5 homologue (termed dUBC9) in an attempt to identify proteins that interact with the amino-terminal transcriptional repression domain of the Groucho corepressor by use of the yeast two-hybrid system. The predicted dUBC9 protein consists of 159 amino acids and shows 85, 68, and 54% amino acid sequence identities with human UBC9 homologue, Schizosaccharomyces pombe Hus5, and Saccharomyces cerevisiae Ubc9 proteins, respectively. Expression of dUBC9 cDNA complements a temperature-sensitive ubc9-1 mutation of S. cerevisiae to fully restore normal growth, indicating that the dUBC9 protein can act as a substitute for the yeast Ubc9 protein. The dUBC9 transcripts were about 1.2 kb and were detected at all stages of Drosophila development and in ovaries and Schneider cells. However, an increased level was observed in early embryos and ovaries. The dUBC9 gene is present as a single copy in the genome and localized in segment 21C-D on the left arm of the second chromosome.View full abstractDownload PDF (2770K) -
Yo-Ichiro Kamimura, Yasuo Kawasaki, Takeshi Ohara, Akio Sugino1999 Volume 125 Issue 2 Pages 236-244
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we have purified and characterized DNA helicase III from the yeast Saccharomyces cerevisiae [Shimizu, K. and Sugino, A. (1993) J. Biol. Chem. 268, 9578-9584]. Here, we have further characterized DNA helicase III activity. It was found that the combined action of the helicase III, yeast DNA topoisomerase I (yTop I), and yeast RPA protein on a covalently closed, circular DNA generates a highly underwound DNA species that has been called form I* or form U. Furthermore, these underwound structures can be accessed by yeast DNA polymerase I (α)-primase to initiate DNA synthesis. These reactions mimic in vivo initiation of chromosomal DNA replication. In order to clone the gene encoding DNA helicase III, a partial amino acid sequence of the purified DNA helicase III polypeptide was determined. Using a mix oligonucleotides synthesized based on the amino acid sequence of the helicase, we cloned the gene encoding the helicase III and found it to be identical to YER176W (HEL1) on chromosome V. The amino acid sequence predicted from the nucleotide sequence of the gene has conserved DNA helicase domains that are highly homologous to those of DNA helicases required for DNA replication. However, complete deletion of the gene from the chromosome did not result in any growth defect, suggesting that the gene product is not required for DNA synthesis or that it is functionally substituted by other helicase(s). Furthermore, the deletion strain does not exhibit sensitivity to any DNA-damaging reagents, although it is hypersensitive to calcofluor white, hygromycin, and papulacandin.View full abstractDownload PDF (5647K) -
Nobuko Hasegawa, Takayoshi Torii, Isao Nagaoka, Kiyoo Nakayasu, Hiroak ...1999 Volume 125 Issue 2 Pages 245-252
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman serum sulfotransferase activities were measured in normal controls and patients with macular corneal dystrophy (MCD), an inherited disorder characterized by the decreased sulfation of keratan sulfate in the corneal stroma and serum, using two kinds of acceptor: partially desulfated keratan sulfate and a trisaccharide with a GlcNAc residue at the nonreducing terminal, GIcNAcβ1-3Galβ1-4GlcNAc. When partially desulfated keratan sulfate was used as the acceptor, only sulfotransferase activity which transfers sulfate to position 6 of the Gal residues was detected. In contrast, when GlcNAcβ1-3Galβ1-4GlcNAc was used as the acceptor, sulfotransferase activity which transfers sulfate to position 6 of the nonreducing terminal GlcNAc residue could be detected. Although keratan sulfate levels in the sera of MCD patients determined by ELISA were much lower than those in normal controls, there were no detectable differences in either the sulfotransferase activity responsible for the sulfation of position 6 of Gal residues or that responsible for the sulfation of position 6 of nonreducing end GlcNAc residues between normal controls and MCD patients. These results suggest that the sulfotransferase involved in the sulfation of keratan sulfate, which is assumed to be deficient in MCD patients, may not be secreted into the serum, and that direct measurement of the sulfotransferase activity present in affected tissues such as the cornea instead of serum may be necessary to confirm the postulated deficiency in the biosynthesis of keratan sulfate in MCD.View full abstractDownload PDF (1046K) -
Hiroko Shirotani, Fuminori Tokunaga, Takehiko Koide1999 Volume 125 Issue 2 Pages 253-262
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInherited antithrombin deficiency is associated with a predisposition for familial venous thromboembolic disease. Pleiotropic effect-type mutants of antithrombin that have an amino acid replacement in a distal hinge region including strands 1C, 4B, and 5B of the polypeptide chain are known to exhibit impaired interactions with both thrombin and heparin, coupled with a secretion defect. To examine the mechanism of pleiotropic effecttype antithrombin deficiency, we expressed three mutants, Oslo (Ala404→Thr), Kyoto (Arg406→Met), and Utah (Pro407→Leu), in baby hamster kidney (BHK) cells, and compared their secretion rates, affinities for heparin and abilities to form thrombin-antithrombin (TAT) complexes with those of wild-type (Wt) antithrombin. Pulse-chase experiments showed that the Oslo- and Kyoto-mutants were secreted at rates similar to Wt antithrombin. In contrast, the Utah-mutant underwent partial intracellular degradation. The intracellular degradation of the Utah-mutant was not inhibited by lysosomotropic inhibitors, but by proteasome inhibitors such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL) and lactacystin, indicating that a part of the Utah-mutant was degraded by proteasome through quality control in the endoplasmic reticulum (ER). Crossed immunoelectrophoresis in the presence of heparin showed that only the Oslo-mutant lacks heparin-binding ability. Incubation with thrombin showed that the Kyoto- and Utah-mutants, but not the Oslo-mutant, formed a weak but detectable TAT complex. Furthermore, heparin enhanced the TAT complex formation by the Kyoto- and Utah-mutants, suggesting heparin cofactor activities of these mutants. These results show that each of the Oslo-, Kyoto-, and Utahmutants exhibits different properties as to secretion, intracellular degradation and functional activity, although they are grouped as pleiotropic effect-type mutants.View full abstractDownload PDF (5644K) -
Chiaki Ogino, Yukinari Negi, Toshiko Matsumiya, Koichi Nakaoka, Akihik ...1999 Volume 125 Issue 2 Pages 263-269
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhospholipase D (PLD), secreted into the culture medium of an actinomycete, Streptoverticillium cinnamoneum, has been purified to homogeneity and characterized. The Stv. cinnamoneum PLD efficiently catalyzes both the hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially Al3+. Nucleotide and amino acid sequence determination of the Stv. cinnamoneum PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.View full abstractDownload PDF (2207K) -
Eiro Muneyuki, Chie Shibazaki, Hiroyuki Ohtani, Daichi Okuno, Makoto A ...1999 Volume 125 Issue 2 Pages 270-276
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe constructed a time-resolved photovoltage measurement system and examined the photovoltage kinetics of wild-type bacteriorhodopsin, its D96N mutant, and halorhodopsins from Halobacterium salinarum and Natronobacterium pharaonis. Upon illumination with a laser flash, wild-type bacteriorhodopsin showed photovoltage generation with fast (10-100 μs range) and slow (ms range) components while D96N lacked the latter, as reported previously [Holz, M., Drachev, L. A., Mogi, T., Otto, H., Kaulen, A. D., Heyn, M. P., Skulachev, V. P., and Khorana, H. G. (1989) Proc. Natl. Acad. Sci. USA 86, 2167-2171]. In contrast, photovoltage generation in halorhodopsins from H. salinarum and N. pharaonis was significant only in the ms time range. On the basis of the photovoltage kinetics and photocycle, we conclude that major charge (chloride) movements within halorhodopsin occur during the formation and decay of the N intermediate in the ms range. These observations are discussed in terms of the “Energization-Relaxation Channel Model” [Muneyuki, E., Ikematsu, M., and Yoshida, M. (1996) J. Phys. Chem. 100, 19687-19691].View full abstractDownload PDF (5589K) -
Tomomitsu Hatakeyama, Taiji Sato, Etsuyo Taira, Hiromiki Kuwahara, Tak ...1999 Volume 125 Issue 2 Pages 277-284
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosaminespecific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, GEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10°C). Identification of the receptor for GEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, abinding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.View full abstractDownload PDF (2744K) -
Haruhiko Tamaoki, Yasuzo Nishina, Kiyoshi Shiga, Retsu Miura1999 Volume 125 Issue 2 Pages 285-296
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism underlying the recognition and activation of the substrate for mediumchain acyl-CoA dehydrogenase (MCAD) was spectroscopically investigated using 3-thiaacyl-CoAs as substrate analogs. The complex of MCAD with 3-thiaoctanoyl-CoA (3-thia-C8-CoA) exhibited a charge-transfer (CT) band with a molar extinction coefficient of ε808=9.1mM-1.cm-1. With increasing 3-thiaacyl-chain length, the CT-band intensity of the complex decreased concomitantly with changes in the FAD absorption at 416 and 482 nm, and no CT band was detected in complexes with chain-lengths longer than C15. Detailed analysis of the absorption spectra suggested that the complexed states represent a two-state equilibrium between the CT-inducing form and the CT-non-inducing form. 13C-NMR measurements with 13C-labeled ligand clarified that 3-thia-C8-CoA is complexed to MCAD in an anionic form with signals detected at 163.7 and 101.2 ppm for 13C(1) and 13C(2), respectively. In the MCAD complex with 13C(1)-labeled 3-thia-C12-CoA, two signals for the bound ligand were observed at 163.7 and 198.3 ppm, and assigned to the anionic and neutral forms, respectively. Only the neutral form signal was measured at 200.6 ppm in the complex with 13C(1)-labeled 3-thia-C17-CoA. These results indicate that the CT band can be explained in terms of an internal equilibrium between anionic (CT-inducing) and neutral (CT-non-inducing) forms of the bound ligand. Resonance Raman spectra of the MCAD. 3-thia-C8-CoA complex, with excitation at the CT band, showed enhanced bands, among which the 854- and 1, 368-cm-1 bands were assigned to the S-C(2) stretching mode of the ligand and to flavin band VII, respectively. Since the enhanced bands were observed at the same wave numbers in complexes with C8, C12, and C14-ligands, it appears that the CT-inducing form shares a common alignment relative to oxidized flavin irrespective of differences in the acyl-chain length. However, with longer ligands, the degree of resonance enhancement of the Raman bands decreased in parallel with the CT-band intensity; this is compatible with the increase in the CT-non-inducing form in complexes with longer ligands. Furthermore, the pH dependence of the CT band gave an apparent pKa=5.6-5.7 for ligands with chain-lengths of C8-C12. The NMR measurements revealed that, like chainlength dependence, the pH dependence can be explained by a two-state equilibrium derived from the protonation/deprotonation of the CT-inducing form of the bound ligand. On the basis of these results we have established a novel model to explain the mechanism of recognition and activation of the substrates/ligands by MCAD.View full abstractDownload PDF (2482K) -
Nicola Volpi, Patrizia Tarugi1999 Volume 125 Issue 2 Pages 297-304
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of chondroitin sulfate samples with decreasing charge densities, different 4-sulfate/6-sulfate ratios, and various molecular masses on Cu2+-induced oxidation of human low-density lipoprotein (LDL) were evaluated by monitoring conjugated diene formation and the tryptophan fluorescence kinetics. Low-sulfated chondroitin sulfate (CS) from beef trachea had a very strong protective antioxidant effect. Quite similar behavior was observed for CS from pig trachea, and a fructose-containing polysaccharide with a chondroitin backbone from Escherichia coli was also strongly protective as to LDL oxidation. CS samples with decreasing charge densities proved effective in inhibiting LDL oxidation. A totally desulfated sample still exhibited a great capacity to protect LDL against oxidation. CS-4-sulfate samples (sulfate to carboxyl ratio of 0.62, about 65% 4-sulfate groups and 5% 6-sulfate groups) retained great ability to inhibit the Cu2+-mediated human LDL oxidation. CS fractions with different molecular masses were examined as possible inhibitors of LDL oxidation. Samples with molecular masses lower than about 8, 570 (13-15 disaccharide units) were unable to protect human LDL from Cu2+-induced oxidation. Similar results were obtained on studying the degradation of tryptophan residues of the LDL protein moiety resulting from Cu2+ complexation through amino acid residues. A low-sulfated CS (sulfate to carboxyl ratio of 0.41, a molecular mass of about 15, 600) having effective anti-oxidant properties as to metal-induced LDL oxidation was isolated from normal human plasma. The protective capacity as to Cu2+-mediated LDL oxidation of CS is discussed in relation to its structure, also considering the physiological role of plasma CS in relation to factors that can alter its properties.View full abstractDownload PDF (980K) -
Sung-Min Suhr, Doo-Sik Kim1999 Volume 125 Issue 2 Pages 305-309
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have found that L-amino acid oxidase (LAO) induces apoptosis in several cultured cell lines by generating H2O2 [Suhr, S. M. and Kim, D. S. (1996) Biochem. Biophys. Res. Commun. 224, 134-139]. It is demonstrated in the present work that the LAO-induced apoptotic mechanism is clearly distinguished from the one stimulated directly by exogenous H2O2. MOLT-4 cells undergo somewhat different morphological changes depending on the apoptotic inducer, LAO or H2O2. LAO-induced apoptosis can be protected by the antioxidant N-acetylcysteine or the free radical scavenger melatonin, while H2O2-induced apoptotic cell death is not protected. A caspase inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (ac-YVAD-aldehyde), prevents cell death when the apoptosis is induced by exogenous H2O2.On the other hand, the ac-YVAD-aldehyde tetrapeptide inhibitor that is dominantly effective on interleukin-1β converting enzyme failed to block the apoptotic event initiated by LAO. Several lines of experimental evidence suggest that apoptotic cell death induced by LAO is not due solely to the hydrogen peroxide produced by the enzymatic reaction.View full abstractDownload PDF (1598K) -
V. Nagarajan, Shigehiro Kamitori, Kenji Okuyama1999 Volume 125 Issue 2 Pages 310-318
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of a triple helical peptide (Pro-Hyp-Gly)10 has been determined at 1.9 Å resolution. Single crystals grown by the hanging drop method, diffracted to a resolution of 1.8 Å. The polymer-like structure of the triple helical repeat Pro-Hyp-Gly was in accordance with the 7/2 model proposed for collagen and very similar to the previously determined structure with a Pro-Pro-Gly sequence repeat. The solvent structure was also very similar to that previously observed, showing similar hydration patterns, but different crystal packing. The presence of hydroxyproline did not have any effect on the molecular structure or the hydration structure. This is in accordance with the recent finding that the inductive effect of the hydroxyl group attached to the Cγ atom of hydroxyproline enhances collagen stability rather than the extensive water network.View full abstractDownload PDF (1994K) -
Ichiji Namatame, Hiroshi Tomoda, Hiroyuki Arai, Keizo Inoue, Satoshi O ...1999 Volume 125 Issue 2 Pages 319-327
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrimary mouse peritoneal macrophages effectively take up and metabolize phosphatidylcholine/cholesterol liposomes containing a small amount of phosphatidylserine, which results in the massive accumulation in the cytoplasm of oil red O positive lipid droplets consisting of cholesteryl ester (CE) and triacylglycerol (TG) [Nishikawa et al. (1990) J. Biol. Chem. 265, 5226-5231]. A number of inhibitors of CE synthesis have been reported, but their effects on the lipid droplet formation have not been fully examined. Furthermore, the contribution of TG synthesis to lipid droplet formation has been poorly investigated. We have investigated the relationship between CE and TG syntheses and cytosolic lipid droplet formation in macrophages cultured in the presence of inhibitors with different modes of action. When macrophages were cultured with liposomes and [14C]oleic acid in the presence of triacsin C, a potent inhibitor of long chain acyl-CoA synthetase, both [14C]CE and [14C]TG syntheses were inhibited to similar extents with IC50 values of 0.19 and 0.10 μM, respectively. On the other hand, pregnenolone, a well-known inhibitor of cellular cholesterol transport, and CL-283, 546, a potent inhibitor of acyl-CoA: cholesterol acyltransferase, inhibited [14C]CE synthesis specifically with IC50, values of 5.0 and 0.038 μM, respectively. Microscopic observation revealed that the inhibitors of cholesterol metabolism produce only a partial decrease in cytosolic lipid droplets even at the highest doses which cause almost complete inhibition of [14C]CE synthesis. However, the triacsin C-dose dependent inhibition of lipid droplet formation was almost complete at 0.59 μM, a concentration that inhibits [14C] CE and [14C]TG syntheses by about 90%. These results show that inhibiton of acyl-CoA synthetase by triacsin C causes a decrease in the cellular levels of acyl-CoA, the common substrate for CE and TG syntheses, leading to an inhibiton of neutral lipid synthesis and eventually to the complete disappearance of cytosolic lipid droplets from macrophages. These findings suggest that TG synthesis, as well as CE synthesis, is responsible for macrophage-derived foam cell formation, and is therefore a potential target for new antiatherosclerotic agents.View full abstractDownload PDF (2952K) -
Helmut Merk, Wolfgang Stiege, Kouhei Tsumoto, Izumi Kumagai, Volker A. ...1999 Volume 125 Issue 2 Pages 328-333
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSingle-chain antibodies (scFv), which can be produced in Escherichia coli cells, have been shown in numerous cases to be active in antigen binding. In the case of the two anti-lysozyme single-chain antibodies, scFvLH and scFvHL, which have the reversed arrangement of the variable domains of the heavy and light chains of the corresponding monoclonal antibodies, the expression level differs greatly when they are produced in Escherichia coli [Tsumoto et al. (1995) Biochem. Biophys. Res. Commun. 201, 546-551]. Although the expression level of scFvLH is high in vivo, the single chain antibody with the reversed orientation (scFvHL) was synthesized in a very low yield and no active product could be obtained. We report here the synthesis of these two anti-lysozyme single-chain antibodies in high yields and with high biological activities in a cell-free E. coli expression system in the presence of reduced and oxidized glutathione, protein disulfide isomerase (PDI), and chaperones. In immunological blotting assays, the synthesized scFvs with both arrangements exhibit specific binding activity to the corresponding antigens, hen egg-white lysozyme, and in an activity assay both inhibited the action of lysozyme. scFvLH is synthesized mainly as a product with the expected molecular weight, whereas scFvHL is produced with additional shorter fragments, suggesting that the low yield isolation through the expression in vivo is due to mistranslation or ribonucleolytic cleavage of the transcript. In the cell-free expression of scFv a certain amount of the product is precipitated but in the presence of chaperones the amount of soluble protein increased from 25 to 90% (PDI and chaperones). The overall expression level and the specific biological activity, however, were hardly influenced. The system reported here can provide significant amounts of various scFv fragments regardless of the order of variable regions, including those which are hardly expressed in vivo.View full abstractDownload PDF (1763K) -
Kikuko Takeuchi, Shingo Senba, Ken-ichi Furukawa, Masami Eto, Fumi Mor ...1999 Volume 125 Issue 2 Pages 334-342
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSmooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogenerously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.View full abstractDownload PDF (6635K) -
Daisuke Nohara, Hiroyuki Sugiura, Hiromu Sakakibara, Mamoru Matsubara, ...1999 Volume 125 Issue 2 Pages 343-347
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRefolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50mM Tris-HCl, the refolding yield of 1 μg/ml of SGT did not exceed 15%. This low yield was assumed to be due mainly to autoproteolysis and/or aggregation occurring concurrently with refolding. On the basis of this assumption, SGT was immobilized on agarose gel in order to suppress such intermolecular interactions, and various refolding media were examined for their ability to minimize misfolding. As a result, 1M Tris, 1M diethanolamine, and 1M triethanolamine were chosen, and their application to the solution system increased the refolding yield considerably, to ca. 45%. A further dramatic increase in yield, to 85%, was observed when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S), engineered as a temporary inhibitor of SGT, was added to the solution system to suppress autoproteolysis during refolding. The application of a temporary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.View full abstractDownload PDF (648K) -
Saori Takahashi, Keitaro Takahashi, Takahiro Kaneko, Hironobu Ogasawar ...1999 Volume 125 Issue 2 Pages 348-353
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe existence of human renin-binding protein (RnBP) in the kidney has been shown by the isolation and characterization of a complex of porcine renin-human RnBP [S. Takahashi et al. (1985) J. Biochem. 97, 671-677]. However, the properties of the free form of human RnBP had not been understood, because of the limitation of materials. In the present study, we have expressed human RnBP in Escherichia coli JM 109 cells under the transcriptional control of taq promoter and purified it by conventional column chromatographies. The purified recombinant human RnBP (rhRnBP) exists as a dimer and inhibits porcine renin activity through formation of a complex of porcine renin with rhRnBP, the so-called highmolecular-weight renin. Moreover, the rhRnBP catalyzes the interconversion between N-acetyl-D-glucosamine (GIcNAc) and N-acetyl-D-Mannosamine (ManNAc) with the apparent Km values of 21.3mM for GlcNAc and 12.8mM for ManNAc, and 0.13mM for effector ATP. ATP is essential for the GlcNAc 2-epimerase activity of human RnBP. These results indicate that the human RnBP is a GIcNAc 2-epimerase.View full abstractDownload PDF (2563K) -
Shingo Senba, Masumi Eto, Michio Yazawa1999 Volume 125 Issue 2 Pages 354-362
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCPI17, a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1), is dominantly expressed in smooth muscle, and the inhibitory activity is potentiated by protein kinase C and its related enzymes [Eto, M. et al. (1997) FEBS Lett. 410, 356-360]. In order to identify its physiological target in smooth muscle, the myofibrillar extract from porcine aorta media was analyzed by affinity chromatography on CPI17-conjugated Sepharose. The binding of phosphatases to the resin depended on thiophosphorylation of CPI17, and about 90% of the phosphatase activities toward phosphorylated myosin (p-myosin) and phosphorylase-a were bound to the resin and could be eluted with 0.5M NaCl. The IC50 values of thiophosphorylated CPI17 toward phosphatases bound to the resin were in the range of 0.5-3 nM, as expected for the PP1 holoenzymes sensitive to CPI17. The CPI17-sensitive fraction was further separated into several peaks of phosphatase activity by column chromatography on Mono Q, which suggested multiple functions of CPI17 as a mediator of the protein kinase C-related signal transduction pathway in aorta smooth muscle. The major activity toward p-myosin was identified as the myofibril-bound PP1 (PP1M), and its subunit composition (140, 37, and 20 kDa) was consistent with that of PP1M from chicken gizzard and porcine bladder. The purified PP1M was completely inhibited by phosphorylated and thiophosphorylated CPI17. Kinetic analysis showed mixed inhibition of PP1M by CPI17 (K1=1.9 nM and K'1=5.1 nM). The concentration of CPI17 in aorta smooth muscle cells was estimated to be at least 0.3 μM from the result of Western analysis. This concentration appears to be sufficient to suppress the in situ PP1M in aorta smooth muscle, and PP1M is thus identified as a target of CPI17 in vascular smooth muscle.View full abstractDownload PDF (4046K) -
Akiko Sasaki, Mai Yamagishi, Tomohiro Mega, Shigemi Norioka, Shunji Na ...1999 Volume 125 Issue 2 Pages 363-367
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme catalyzing the hydrolysis of the Man β1-4G1cNAc linkage of N-linked sugar chains was partially purified and characterized. Endo-β-mannosidase activity was detected using pyridylaminated (PA-) Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc as the substrate in a homogenate of lily flowers (Lilium longflorum Thumb). The enzyme was partially purified by ammonium sulfate precipitation, and Q-Sepharose, Superdex 200, hydroxyapatite, Poros PE/M, Mono Q, and Superdex 200 column chromatographies. The optimum pH was 5.0 and the estimated molecular weight of the enzyme was 78, 000, as determined by gel filtration. The Km value found for Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc-PA was 1.4mM. The enzymatic activity was not influenced by the addition of 10mM EDTA or 2mM Ca2+. Experiments on the hydrolysis of several PA-N-linked sugar chains revealed that the enzyme hydrolyzed MannManα1-6Manβ1-4GlcNAcβ1-4GlcNAc-PA (n=0-2) into a mixture of MannManα1-6Man and GlcNAcβ1-4GlcNAc-PA, indicating that it is an endoglycosidase in nature. However, the enzyme did not hydrolyze β1-4mannohexaose or p-nitrophenyl β-mannopyranoside.View full abstractDownload PDF (554K) -
Norihiko Nakano, Shigeki Higashiyama, Seiji Takashima, Nobuo Tsuruoka, ...1999 Volume 125 Issue 2 Pages 368-374
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA human histiocytic lymphoma cell line, U-937 cells, secretes several vascular endothelial cell growth inhibitors including leukemia inhibitory factor, oncostatin M, tumor necrosis factor-α, and transforming growth factor-β1. Characterization of partially purified fractions from the conditioned media of phorbol ester-treated U-937 cells suggested the existence of unknown endothelial growth inhibitors. Using a combination of copper affinity, heparin affinity, cation exchange, and reversed phase liquid chromatographies, a growth inhibitor for endothelial cells was purified to homogeneity from conditioned media. The purified growth inhibitor migrated as a 65 kDa band on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Microsequencing analyses of the tryptic fragments of the growth inhibitor and a BLAST search analysis revealed a unique sequence showing no homology to known proteins. The purified protein inhibited endothelial cell growth in a dose-dependent manner, but had no effect on smooth muscle cell growth. The factor also blocked endothelial cell growth induced by both fibroblast growth factor-2 and vascular endothelial growth factor, and was additively effective in inhibiting the growth of endothelial cell by U-937 cell-derived endothelial cell growth inhibitors. Thus, this factor appears to be a novel inhibitor with specificity for vascular endothelial cells, and is tentatively called endothelial cell inhibitor (ECI) in this study.View full abstractDownload PDF (2626K) -
Naoki Ohkura, Yoshiya Kitahara, Seiji Inoue, Kiyoshi Ikeda, Kyozo Haya ...1999 Volume 125 Issue 2 Pages 375-382
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA phospholipase A2 (PLAZ) inhibitor was purified from the blood plasma of a sea krait, Laticauda semifasciata, by sequential chromatography on Sephadex G-200, Mono Q, and Phenyl Sepharose columns. The purified inhibitor was found to be the same type as the PLA2 inhibitors, named PLIγ, that had been purified from the blood plasma of the Thai cobra Naja naja kaouthia [Ohkura et al. (1994) Biochem. Biophys. Res. Commun. 200, 784-788] and Chinese mamushi Agkistrodon blomhoffli siniticus [Ohkura et al. (1997) Biochem. J. 325, 527-531]. Like other PLIγs, the L. semifasciata inhibitor (LsPLIγ) inhibited equally all of the PLA2s investigated including Elapid venom PLA2s (group I), Crotalid and Viperid venom PLA2s (group II), and honeybee PLA2 (group III). The LsPLIy was a 100-kDa glycoprotein composed of two distinct subunits, LsPLIγ-A and LsPLIγ-B, with an approximate molar ratio of 2:1. The amino acid sequences of the two subunits were determined by alignment of the peptides obtained by lysyl endopeptidase, endoproteinase Asp-N, and staphylococcal V8 protease digestions. LsPLlγ-A and LsPLlγ-B were composed of 182 and 181 amino acid residues, respectively; and the former subunit was a glycoprotein containing one asparagine-linked sugar chain at the position 157. The sequences of LsPLIγ-A and LsPLIγ-B showed 65 and 74% homology, respectively, to those of the corresponding subunits of N. naja kaouthia PLIγ, and had two tandem patterns of cysteine residues, characteristic of the urokinase-type plasminogen activator receptor (uPAR) and members of the Ly-6 superfamily.View full abstractDownload PDF (3968K) -
Mahadev Rao, Mavinekere Marulapppa Kumar, Mysore Ananthanarayana Rao1999 Volume 125 Issue 2 Pages 383-390
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3mg/kg) i. p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10mg/kg i. p. 1h before cisplatin resulted in partial but significant protection against the cisplatininduced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10mg/kg, i. p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in cisplatin chemotherapy and thus warrants further investigation.View full abstractDownload PDF (876K) -
Takenori Takizawa, Chizuru Tatematsu, Yoshinobu Nakanishi1999 Volume 125 Issue 2 Pages 391-398
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPKR is an interferon-inducible, double-stranded (ds) RNA-activated serine/threonine protein kinase, and has been shown to play roles in viral pathogenesis, cell growth and apoptosis. We expressed PKR as a fusion protein with enhanced jellyfish green fluorescence protein (EGFP) in human embryonic kidney 293 cells to visualize the effect of PKR transfection. The EGFP-fusion construct with wild-type PKR showed both auto- and substrate-phosphorylation activities independent of dsRNA, indicating EGFP-PKR is constitutively active. The EGFP-constrict with a mutant PKR with the first RNA binding domain deleted still possessed kinase activities. On the other hand, the EGFP-fusion with a catalytically inactive mutant of PKR with the substitution of K at 296 with R, which has been shown to have tumorigenic properties, did not possess kinase activities. Transfection of the constitutive active forms of EGFP-PKR constructs induced apoptosis in 293 cells without dsRNA, whereas the EGFP-fusion with the catalytically inactive mutant did not cause apoptosis but rather protected cells from Fas-induced cell death. In addition, Fas-stimulation increased endogenous PKRR activities. These results constitute evidence that PKR is sufficient to induce apoptosis, and plays a role in Fas-mediated apoptosis.View full abstractDownload PDF (4840K) -
Kouhei Saito, Takeshi Kikuchi, Hitoshi Shirakawa, Michiteru Yoshida1999 Volume 125 Issue 2 Pages 399-405
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHigh mobility group (HMG) protein 1 contains two DNA binding motifs, called HMGl/2-boxes, linked with a linker region. The functional relationships between the two boxes and the mechanism of involvement of the linker region for effective binding of HMG1 were examined. The binding analyses of truncated HMG1 peptides with DNA indicated that the structural array of two boxes stabilizes the interaction of HMG1 with DNA. The mutation analyses of the linker region suggested that the region is equipped with tolerance for the deletion of a few amino acid residues to allow appropriate binding of the two boxes with DNA, and that the basic cluster in the linker sequence is in a position to interact with DNA. The existence of tolerance for the linker sequence was found to be conserved during the evolution of HMG1 protein homologues. A structural model for array of two boxes associating with DNA minor groove was constructed on the basis of the experimental results and energy minimization. The model proposes that the DNA binding region in HMG1 covers an 18 by DNA region and induces its bending by about 140 degrees. The linker region may function to maintain the structural array of two HMG1/2-boxes by direct interaction with DNA.View full abstractDownload PDF (2909K) -
Shinn Kimura, Jun Cheng, Koh-éi Tovoshima, Kimimitsu Oda, Takas ...1999 Volume 125 Issue 2 Pages 406-413
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biosynthesis of basement membrane heparan sulfate proteoglycan (HSPG), known as perlecan, in ACC3 cells established from a adenoid cystic carcinoma of the human salivary gland was studied using metabolic labeling and immunoprecipitation with discriminative antibodies specific for HSPG core protein. Treatment of immunoprecipitated HSPG with HNO2, heparitinase, and chondroitinase ABC revealed that ACC3 cells synthesized HSPG molecules composed of 470-kDa core protein and heparan sulfate but not of chondroitin sulfate. The core protein was shown to contain complex type N-linked oligosaccharides by digestion with N-glycanase and endoglycosidase H. Pulse-chase experiments showed that the mature form of HSPG was formed in the cells in 30 min and released into the medium thereafter. Degradation of HSPG was also found in the chase period of 3h. In time course experiments, HSPG was found to be synthesized maximally at day 4 after plating, deposited in the cell layer maximally at day 6, and secreted maximally at day 8. This was also confirmed by immunofluorescence, Northern blotting, and in-situ hybridization. The results indicate that ACC3 cells synthesize, secrete and degrade basement membrane type HSPG, which is analogous to those produced by other cell types, and that the biosynthesis and secretion of HSPG in ACC3 cells are strictly regulated by the cell growth, that may be reflected in the characteristic histology of adenoid cystic carcinomas.View full abstractDownload PDF (3511K) -
Takeshi Murata, Kazuma Takase, Ichiro Yamato, Kazuei Igarashi, Yoshimi ...1999 Volume 125 Issue 2 Pages 414-421
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe report here the large-scale purification of vacuolar (V0V1)-type Na+-ATPase from Enterococcus hirae achieved using column anion-exchange and gel filtration chromatographies; 32 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, and K, was obtained from 20 liter culture. This amount is 500-fold larger than that reported in the previous paper [Murata, T., Takase, K., Yamato, I., Igarashi, K., and Kakinuma, Y. (1997) J. Biol. Chem. 272, 24885-24890]. The purified enzyme shows a high specific activity of ATP hydrolysis (35.7 μmol P1 released/min/mg protein). ATP-driven 22Na+ uptake by reconstituted V0V1-proteoliposomes exhibited an apparent Kt value for Na+ of 40 μM, which is near the Km value (20 μM) for Na+ of the ATP hydrolytic activity. Denatured gel electrophoresis revealed that six subunits, A, B, C, D, E, and F, are releasable as the V1 subunit from the V0V1 complex by incubation with ethylenediaminetetraacetic acid; subunit G was not identified. The remaining V0-liposomes containing I and K subunits catalyzed Na+ uptake in response to potassium diffusion potential (Δψ, inside negative); the Kt value for Na+ of this reaction was estimated to be about 2mM. Inhibition by N, N'-dicyclohexylcarbodiimide (DCCD) of the Na+-ATPase activity and Δψ-driven Na+ uptake by the V0-liposomes was prevented by the presence of Na+, suggesting that the Na+ binding site overlaps with the DCCD-reactive site.View full abstractDownload PDF (1980K) -
L. K. Steinrauf, Michael Y. Chiang, David Shiuan1999 Volume 125 Issue 2 Pages 422-429
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 Å resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 Å in diameter with a repeat of 77.56 Å, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.View full abstractDownload PDF (2851K)
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