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Volume 120 (1996)
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- Issue 3 Pages 467-
- Issue 2 Pages 207-
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Volume 120, Issue 3
Displaying 1-32 of 32 articles from this issue
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Mitsuyoshi Nakao, Hiroyuki Sasaki1996 Volume 120 Issue 3 Pages 467-473
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCertain mammalian genes are expressed exclusively from either the paternal or the maternal chromosome because of a differential marking process that occurs during gametogenesis. This epigenetic marking is called genomic imprinting. Monoallelic expression of imprinted genes is responsible for the inability of uniparental mammalian embryos to develop normally and for the abnormal phenotypes observed with particular chromosomal disomies. Many of the imprinted genes identified to date are involved in the regulation of cell proliferation and differentiation and, together with other pieces of evidence, they are suggested to play a role in tumorigenesis. Here we discuss how imprinted genes cause diseases and tumors and summarize the recent advances of studies on the molecular basis of this epigenetic phenomenon. In particular, we focus on two well-characterized imprinted chromosomal regions, namely the human Prader-Willi/Angelman syndrome region and the mouse INS2/IGF2/H19 region. The correlations between the differential gene activity and the changes in DNA methylation, higher order chromatin structure and replication timing, will shed light on gene regulation at the level of the chromosomal domain.View full abstractDownload PDF (888K) -
Haruki Yamaguchi, Makoto Uchida1996 Volume 120 Issue 3 Pages 474-477
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes a chaperone-like function of the intramolecular N-glycans of bovine pancreatic RNase B. We studied air-oxidative regeneration from reductively denatured species of RNase B and its nonglycosylated form, RNase A. RNase B was reactivated much faster than RNase A, while RNase A was liable to aggregate during the regeneration. An Asn-linked oligosaccharide, Man5GlcNA2Asn, which corresponds to the most predominant sugar chain (ca. 60%) of RNase B, enhanced the reactivation of the denatured RNases A and B. The stimulatory effect of this Asn-oligosaccharide revealed that the N-glycans of RNase B facilitate the transformation of bulky intermediates into folded, compact species.View full abstractDownload PDF (1413K) -
Angela Mai, Sang-Kee Jung, Hiroyuki Tachikawa, Daisaburo Fujimoto1996 Volume 120 Issue 3 Pages 478-480
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing affinity chromatography on lactosyl-Sepharose, a β-galactoside-binding protein of 38 kDa was detected in mouse L1210 lymphocytic leukemia cells. Immunoblotting analysis revealed that it is distinct from any known larger molecular weight galectin. The partial amino acid sequences of the 38 kDa protein indicated that it is a novel member of the galectin family. This 38 kDa lectin is expressed in lymphocytic cell lines but not macro-phage-like cell lines.View full abstractDownload PDF (2776K) -
Isafumi Maru, Jun Ohnishi, Yasuhiro Ohta, Wataru Hashimoto, Yoji Tsuka ...1996 Volume 120 Issue 3 Pages 481-482
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-Acyl-D-glucosamine 2-epimerase from porcine kidney, which was cloned and expressed in Escherichia coli, was crystallized by the vapor-diffusion method, using polyethylene glycol and ammonium acetate as precipitants. The crystals were resistant to X-ray radiation damage and diffracted to more than 2.0 Å resolution. The diffraction pattern indicated that the crystals belong to the orthorhombic system, space group P212121, with unit-cell dimensions of a=78.1, b=97.2, and c=100.7 Å. It is supposed that the asymmetric unit consists of two N-acyl-D-glucosamine 2-epimerase molecules. Collection of data on the native crystals indicated that they are suitable for X-ray structural analysis.View full abstractDownload PDF (1214K) -
Michiaki Yamashita, Shiro Konagaya1996 Volume 120 Issue 3 Pages 483-487
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA salmon cystatin cDNA clone was isolated from a chum salmon cDNA library. The clone encoded a full-length extracellular-type cystatin and its signal peptide and included 5'- and 3'-untranslated regions. The deduced amino acid sequence showed a high degree of sequence similarity to mammalian cystatin C, chicken egg cystatin, and chum salmon pituitary cystatin. By Northern blot analysis, the salmon cystatin was found to show apparently non-tissue specific expression. Because platyfish EHS cells transfected with a cystatin expression vector produced a 13 kDa mature cystatin in the culture medium, the salmon cystatin was considered to act as an extracellular type of cystatin in the fish cells. These findings indicate that the salmon cystatin is a homolog of mammalian cystatin C.View full abstractDownload PDF (2934K) -
Kazuhiro Kimura, Miyuki Hamada, Mitsuaki Moriyama, Yukiko Kannan, Masa ...1996 Volume 120 Issue 3 Pages 488-493
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMannan, a ligand for the mannose/N-acetylglucosamine (GlcNAc) receptor, induces suppression of oxygen consumption and increases glucose production in the perfused rat liver, and repeated infusion of mannan causes desensitization of the responses. In this study, we examined whether activation of Kupffer cells by endotoxin and phorbol ester alters the glycogenolytic responses to mannan. Infusion of lipopolysaccharide (LPS, 10 μg/ml) in the perfusate failed to inhibit the responses to mannan. Intravenous administration of LPS (1mg/kg) 6 and 24 h before perfusion did not desensitize the responses to mannan, suggesting that the responses through mannose/GlcNAc receptors in the liver are retained even after activation of Kupffer cells by LPS. In contrast, prior infusion of phorbol 12-myristate 13-acetate (PMA, 100 nM) in vitro abolished the glycogenolytic responses to subsequently infused mannan, but not that to norepinephrine (100 nM), while prior infusions of 4-α-phorbol 12, 13-didecanoate (100 nM), A23187 (50 nM), or forskolin (1 μM) had no effect on the mannan-induced responses. H-7, an inhibitor of protein kinase C, reduced the glycogenolytic responses to mannan, while it failed to restore the desensitization. These results suggest that protein kinase C may be involved in the process of glycogenolysis by mannan, but is unlikely to be involved in the homologous desensitization of the responses.View full abstractDownload PDF (718K) -
Tomoko Yamaguchi, Nam-Soon Kim, Shingo Sekine, Hiroaki Seino, Fumio Os ...1996 Volume 120 Issue 3 Pages 494-497
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESScDNA encoding a novel human ubiquitin-conjugating enzyme has been cloned from an epidermoid carcinoma KB cDNA library. This clone encodes a protein of 152 amino acids with a calculated Mr of 17, 137. The amino acid sequence showed 80% identity with the Drosophila's bendless gene product (ubiquitin-conjugating enzyme E2). The corresponding transcripts are highly expressed in heart, skeletal muscle, and testis. The product expressed in Escherichia coli exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin-activating enzyme El-dependent manner. These results suggest that the obtained eDNA encodes a novel human E2 which may be involved in protein degradation mainly in the muscles and testis.View full abstractDownload PDF (1482K) -
Sabato D'Auria, Francesco La Cara, Filomena Nazzaro, Nunzia Vespa, Mos ...1996 Volume 120 Issue 3 Pages 498-504
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn NAD-dependent alcohol-aldehyde oxidoreductase was purified to homogeneity and characterized from cell extracts of the thermophilic microorganism Bacillus acidocaldarius. The 500-fold purified homogeneous enzyme had a molecular mass of 154 kDa, as shown by gel filtration and glycerol gradient centrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis the protein showed one band of 38 kDa, indicating that the enzyme is a tetramer composed of subunits of identical molecular weight. Ethanol was the best substrate with the highest kcat/Km values, and the enzyme showed a substrate specificity that included linear, secondary and cyclic alcohols, as well as anisaldehyde, but it was not active on ketones. The protein contains eight zinc atoms per tetramer, four of which are removed by chelating agents with a concomitant loss of thermal stability. Circular dichroism spectra and determination of the NH2-terminal sequence allowed structural and homology comparison with other alcohol dehydrogenases from animal and bacterial sources.View full abstractDownload PDF (877K) -
Mitsutaka Kohno, Jiro Funatsu, Bunzo Mikami, Wataru Kugimiya, Takaharu ...1996 Volume 120 Issue 3 Pages 505-510
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal and molecular structure of Lipase II from Rhizopus niveus was analyzed using X-ray single crystal diffraction data at a resolution of 2.2 Å. The structure was refined to an R-factor of 0.19 for all available data. This lipase was purified and crystallized as Lipase I, which contains two polypeptide chains combined through non-covalent interaction. However, during crystal growth, Lipase I was converted to Lipase II, which consists of a single polypeptide chain of 269 amino acid residues, by limited proteolysis. The structure of Lipase II shows a typical α/β hydrolase fold containing the so-called nucleophilic elbow. The catalytic center of this enzyme is analogous to those of other neutral lipases and serine proteases. This catalytic center is sheltered by an α-helix lid, which appears in neutral lipases, opening the active site at the oil-water interface.View full abstractDownload PDF (709K) -
Torn Mizuguchi, Toshihiro Mitaka, Takashi Kojima, Koichi Hirata, Toshi ...1996 Volume 120 Issue 3 Pages 511-517
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of tryptophan 2, 3-dioxygenase (TO) and serine dehydratase (SDH) has not previously been maintained or re-induced in long-term cultured hepatocytes. In the present study, we succeeded in inducing expression of TO and SDH mRNAs in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide (DMSO). After the start of culture, the expression of TO mRNA rapidly disappeared and at 96 h it was less than 10% of that at isolation. However, after the addition of 2% DMSO from 96 h, the transcript level gradually increased and reached about 40% of that of the isolated cells at day 14. In addition, the expression of TO mRNA was enhanced in cells treated with both 10-5M dexamethasone and 10-7M glucagon. In contrast, the expression of SDH mRNA decreased very rapidly and we could not detect it after 24 h of culture. Furthermore, 2% DMSO failed to induce it. However, when both 10-5M dexamethasone and 10-7M glucagon were added to the culture medium at day 9, we observed dramatic induction of SDH mRNA 24 h later. Primary hepatocytes cultured by this method could express and maintain highly differentiated hepatic functions for a long time. Thus, this in vitro system is suitable for the investigation of hepatic functions.View full abstractDownload PDF (4336K) -
Tsutomu Nakamura, Shigeru Minoguchi, Katsura Izuit1996 Volume 120 Issue 3 Pages 518-524
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) of an extreme thermophile, Thermus sp., which was expressed in Escherichia coli cells, was purified and its enzymological properties were investigated and compared with native Thermus PEPC. The enzyme activity was strongly dependent on acetyl-CoA, an allosteric activator, and inhibited by malate or aspartate. Contrary to the other known PEPCs, Thermus PEPC was not activated but rather inhibited by phosphorylated compounds such as fructose 1, 6-bisphosphate and GTP. The specific activity in the presence of 0.3mM acetyl-CoA and 2mM phosphoenolpyruvate was highest at 70°C. The half-saturation concentrations for both substrates at 70°C were about twice those at 30°C. Half-lives of the enzyme at 85, 90, and 95°C were 220, 110, and 50 min, respectively. Thermus PEPC was highly tolerant also to guanidine hydrochloride (Gdn-HCl): the concentrations required for complete inactivation of Thermus and E. coli PEPCs after incubation at 30°C for 20 h were 3.5 and 0.6M, respectively. The properties of recombinant and native enzyme were similar to each other except for the catalytic activity after incubation with 1-2M Gdn-HCI.View full abstractDownload PDF (861K) -
Shun-ichi Nakamura, Masaaki Matsushima, Haiwei Song, Masakazu Kikuchi1996 Volume 120 Issue 3 Pages 525-530
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that protein disulfide isomerase (PDI) can dissociate the glutathione molecule in vitro from the mutant human lysozyme (hLZM) C77A-a, which is modified with glutathione at Cys95; however, it seems structurally difficult for PDI to attack either the disulfide bond or the side chain of the cysteine residue of a mixed disulfide. To investigate the function of PDI, we introduced several glutathione and cysteine derivatives at Cys95, instead of the glutathione of C77A-a. Using thiol compounds modified by 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), we could easily modify the free thiol group of C77A-b (C77A with no glutathionylation), without denaturation. For all of the modifications we tested, a negative correlation was found between the initial rate and the acceleration ratio of the reductive cleavage of mixed disulfides with PDI. A mutant PDI (hPDIM), which has no thiol-disulfide exchange activity, suppressed the reductive cleavage of the mixed disulfide of C77A-a with hPDI, suggesting that hPDI non-covalently interacted with the substrates. Taking account of the results of the structural analysis, we conclude that one of the functions of PDI in vivo lies in relaxing the structure around the disulfide bond, as well as in exchanging the thiol-disulfide bonds.View full abstractDownload PDF (839K) -
Yasuo Kato, Yasuhisa Asano, Tapas K. Makar, Arthur J. L. Coopert1996 Volume 120 Issue 3 Pages 531-539
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIncubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5'-phosphate form) with 10mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25°C (pH 7.0) results in slight inactivation (_??_15%). However, incubation of the enzyme with glutamate, or prior conversion of the enzyme to the pyridoxamine 5'-phosphate form, results in more extensive inactivation. The inactivation of the enzyme by 2-oxoglutaconic acid dimethyl ester is most pronounced in the presence of both glutamate and α-ketoglutarate. N-Ethylmaleimide was previously shown to alkylate two surface cysteine residues (I and II) and to react syncatalytically with a third cysteine residue (III) of cytosolic pig heart aspartate aminotransferase [Birchmeier et al. (1973) J. Biol. Chem. 248, 1751-1759]. Alkylation of cysteine III results in inactivation of the enzyme, despite the fact that this residue is not essential for catalysis. The present results suggest that 2-oxoglutaconic acid dimethyl ester reacts with the enzyme in a similar fashion to that exhibited by N-ethylmaleimide. Some inactivation by alkylation of a susceptible group at the active site cannot be ruled out. However, the rate of inactivation of cytosolic pig heart aspartate aminotransferase is proportional to the concentration of 2-oxoglutaconic acid dimethyl ester up to a concentration of at least 40mM, suggesting that the compound binds very poorly to the active site or that alkylation at the active site is slow compared with syncatalytic alkylation of cysteine III. The t1/2 for inactivation of pig heart cytosolic aspartate aminotransferase by 40mM 2-oxoglutaconic acid dimethyl ester (in the presence of 10mM L-glutamate, pH 7.2, 25°C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10mM 2-oxoglutaconate for 2 h (25°C, pH 7.2) results in significant inactivation (_??_30%). The enzyme is protected against inactivation by the presence of α-ketoglutarate, but glutamate enhances the inactivation. These findings suggest that 2-oxoglutaconate is an active site-directed inhibitor. The binding of 2-oxoglutaconate to the enzyme exhibits saturation kinetics (K1_??_2mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L-glutamate _??_0.01 min-1; pH 6.0, 25°C; t1/2max _??_70min). This finding suggests that 2-oxo-glutaconate does not readily react in a syncatalytic fashion with cysteine III. Possibly, the two negative charges of 2-oxoglutaconate do not allow ready approach to cysteine III. Rather, the findings suggest that 2-oxoglutaconate binds at the active site of the pyridoxal 5'-phosphate form of the enzyme as an affinity labeling reagent. However, the increased rate of 2-oxoglutaconate-induced inactivation in the presence of glutamate suggests that this unsaturated α-keto acid also exhibits the properties of a kcat inhibitor. 2-Oxoglutaconate inactivates aspartate aminotransferase in cytosolic and mitochondrial fractions of rat kidney and purified pig heart alanine aminotransferase. Injection of 2-oxoglutaconate into mice results in inhibition of kidney aspartate aminotransferase. 2-Oxoglutaconate is a substrate of glutamate dehydrogenase. The kinetic constants are similar to those obtained with α-ketoglutarate. The results suggest that unsaturated α-keto acids and their esters may be useful probes for the study of α-keto acid-utilizing enzymes.View full abstractDownload PDF (1144K) -
Takumi Satoh, Naoto Sakai, Yasushi Enokido, Yasuo Uchiyama, Hiroshi Ha ...1996 Volume 120 Issue 3 Pages 540-546
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the mechanism of oxidative stress-induced death of PC12 cells, we performed confocal and flow cytometric analysis with a reactive oxygen species (ROS)-specific fluorogen, 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Hydrogen peroxide significantly decreased the number of viable PC12 cells after 24 h. Hydrogen peroxide caused membrane blebbing, nuclear condensation and DNA fragmentation, indicating that the PC12 cells died due to apoptosis. The hydrogen peroxidetriggered apoptosis of PC12 cells was associated with enhanced ROS production in a dose-dependent manner by measuring with C-DCDHF-DA. Nerve growth factor (NGF) and Bcl-2 inhibited the hydrogen peroxide-induced apoptosis of PC12 cells. Neither of them, however, reduced the ROS production in PC12 cells. These data suggest that NGF or Bcl-2 protects PC12 cells from hydrogen peroxide-triggered apoptosis independently from ROS production.View full abstractDownload PDF (4173K) -
Masakuni Degawa, Ken-ichi Matsuda, Hiroshi Arai, Yoshiyuki Hashimoto1996 Volume 120 Issue 3 Pages 547-551
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of lead nitrate on the induction of hepatic cytochrome P450IA (CYP1A) isoforms, mainly CYP1A2, by aromatic amines (2-methoxy-4-aminoazobenzene, 2-amino-3-methyl-9H-pyrido[2, 3-b]indole and 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine) and aryl hydrocarbons (3-methylcholanthrene, benzo [a] pyrene and β-naphthoflavone) in male F344 rats were examined at the levels of mRNA, protein and activity of the enzymes. Pretreatment of rats with lead nitrate suppressed the expression of hepatic CYP1A enzyme(s), especially CYP1A2, at both levels of protein and activity of the enzyme(s) by treatment with an aromatic amine or an aryl hydrocarbon. On the other hand, the lead nitrate pretreatment suppressed the induction of CYP1A mRNA(s) by an aromatic amine but not by an aryl hydrocarbon. These findings indicate that lead nitrate suppresses the expression of CYP1A enzymes at both stages of post-translation of mRNAs and transcriptional activation of the genes, and further suggest that the pathway for the transcriptional activation of the CYP1A genes by the aromatic amines is different from that by the aryl hydrocarbonsView full abstractDownload PDF (3109K) -
Shin-ya Saito, Shugo Watabe, Hiroshi Ozaki, Hideo Kigoshi, Kiyoyuki Ya ...1996 Volume 120 Issue 3 Pages 552-555
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depoly-merizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.View full abstractDownload PDF (391K) -
Takashi Hibino, Byung Hyun Lee, Tatsuo Yajima, Akira Odani, Osamu Yama ...1996 Volume 120 Issue 3 Pages 556-563
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe site-directed mutants of negative patches on silene plastocyanin (PC) were used to investigate the change of interactions between photosystem I (PSI) and PC during the course of evolution from cyanobacteria to plants. The net charges of two highly conserved negative patches (#42-45 and #59-61) on silene PC were systematically modified from -4 to +1. PSI complexes from cucumber and Chlamydomonas reinhardtii were efficient electron acceptors for silene PC. The increase of net charge on the negative patch (#42-45) of silene PC decreased the reduction rates of PSI from cucumber and Chlamydomonas, while the modification of the other negative patch (#59-61) had no effect. Though the addition of MgCl2 decreased the reduction rate of cucumber PSI, the decrease was severely diminished in the case of Chlamydomonas PSI, and the reduction rate increased with increasing concentration of MgCl2, when the net charge of the negative patch (#42-45) was modified to + 1. The PSI complexes from Anabaena variabilis and Synechosystis sp. PCC 6803 were inefficient electron acceptors for silene PC and their rates were almost independent of the net charge of the negative patches, as well as the ionic strength of the reaction mixtures. Silene PC specifically cross-linked to the PsaF subunit of PSI complexes from cucumber, Chlamydomonas, Anabaena, and Synechosystis sp. PCC 6803. Modification of the negative patch (#42-45) inhibited the formation of cross-linked adducts in all the cases examined, whereas modification of the other negative patch (#59-61) had essentially no effect. Based on these results, the changes of electrostatic interactions between PC and PSI during the course of evolution from cyanobacteria to plants are discussed.View full abstractDownload PDF (2034K) -
Kohei Oda, Masaaki Ito, Ken-ichi Uchida, Yuji Shibano, Ken-ichi Fukuha ...1996 Volume 120 Issue 3 Pages 564-572
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSXanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, is the second example of the unique carboxyl proteinases [EC 3. 4. 23. 33] which are insensitive to the classical aspartic proteinase inhibitor. The gene coding for XCP was cloned, sequenced, and expressed in Escherichia coli. The XCP gene contains an open reading frame of 2, 481 base pairs encoding a protein of 827 amino acid residues with a Mr of 83, 677. The XCP was synthesized as a large precursor consisting of three regions: NH2-terminal prepro (N-Prepro) (237 amino acid residues); mature XCP (398 a. a. residues); and COOH-terminal pro (C-Pro) (192 a. a. residues). The N-Prepro and mature XCP regions had no sequence similarity to any other proteins reported so far, except the carboxyl proteinase from Pseudomonas sp. 101 [Oda, K., Takahashi, T., Tokuda, Y., Shibano, Y., and Takahashi, S. (1994) J. Biol. Chem. 269, 26518-26524]. The C-Pro region showed high similarity to COOH-terminal regions of other microbial proteinase precursors. E. coli carrying a plasmid containing the cloned wild-type XCP gene produced an 84-kDa protein. This protein was processed into a mature, active form under acidic conditions. This process was completely blocked by tyrostatin, an XCP-specific inhibitor from Kitasatosporia sp. 55, indicating an autocatalytic processing. The purified recombinant XCP had the same characteristics as authentic XCP except for the NH2-terminal amino acid sequence. When the mutant XCP gene truncated in the C-Pro region was expressed in E. coli, an expected 64-kDa protein was detected in the cells, and also processed into the 42-kDa active form under the acidic conditions. Thus, the C-Pro region was not essential for the formation of active mature XCP.View full abstractDownload PDF (3549K) -
Tamotsu Taketomi, Atsushi Hara, Kei-ichi Uemura, Eiko Sugiyama1996 Volume 120 Issue 3 Pages 573-579
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have recently developed a rapid method for the preparation of lysoglycosphingolipids on a small scale, in high yield. This procedure of microwave-mediated saponification of about 1mg of glycosphingolipid with 0.5ml of 0.1M NaOH in methanol for two minutes can be easily repeated if larger amounts of lyso-compounds are needed. We have also found that the new methodology of delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the reflector mode is extremely effective for the confirmation of different lysoglycosphingolipids together with their long chain base components. The combined method of preparation and confirmation of lysoglycosphingolipids is also important for the identification of long chain bases of various sphingolipids, because the usual analytical method of long-chain bases of sphingolipids depends on acidic methanolysis, which results in the formation of by-products such as O-methylsphingosines and threo-sphingosines.View full abstractDownload PDF (646K) -
Tsuyoshi Katoh, Fumi Morita1996 Volume 120 Issue 3 Pages 580-586
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 2-kDa peptide (2K peptide) which was derived from the neck region of porcine aorta smooth muscle myosin heavy chain binds to actin competitively with skeletal myosin subfragment 1 (S1) in the absence of ATP and inhibits acto-S1 ATPase activity [Katoh, T. and Morita, F. (1993) J. Biol. Chem. 268, 2380-2388]. Using this and other peptides, myosin-binding sites on actin were mapped and their functions were studied. The 2K peptide inhibited the acto-S1 ATPase activity without inhibiting the binding of S1 to actin in the presence of ATP. On the other hand, the dansylated 2K peptide (DNS-2K peptide) inhibited not only the acto-S1 ATPase activity but also the binding of S1 to actin in the presence of ATP. Then, DNS-2K peptide was crosslinked to actin with 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide. Amino acid composition and sequencing analyses of the fluorescent lysylendopeptidase-peptides of the crosslinked product indicated that DNS-2K peptide was crosslinked to acidic residues within residues 1-18 (Asp1, Glu2, Asp3, Glu4, and/or Asp11), 19-50 (Asp25), and 85-113 (Glu99 or Glu100) of actin. A competition experiment for the crosslinking with unlabeled 2K peptide showed that the crosslinking to residues 85-113 of actin was specific for DNS-2K peptide. In addition, isolated actin peptide 85-113 was found to show the competitive inhibition of actin-activated ATPase activity of S1 with respect to actin. These results suggest that the site within residues 1-28 of actin participates in the actin-activation of myosin ATPase activity, and the site within residues 85-113 of actin participates in the weak binding of myosin to actin in the presence of ATP.View full abstractDownload PDF (1981K) -
Qian Zhang, Toshio Iwasaki, Takayoshi Wakagi, Tairo Oshima1996 Volume 120 Issue 3 Pages 587-599
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe purified 2-oxoacid:ferredoxin oxidoreductase of a thermoacidophilic and aerobic crenarchaeote, Sulfolobus sp. strain 7, consists of 70-kDa α and 37-kDa β subunits, and contains one thiamine pyrophosphate (TPP), one [4Fe-4S]2+, 1+ cluster, and two magnesium atoms per αβ structure. It exhibits a broad substrate specificity toward 2-oxoacids such as 2-oxoglutarate, 2-oxobutyrate, and pyruvate. The gene encoding the archaeal oxidoreductase was cloned, and the two open reading frames encoding the α (632 amino acids) and β subunits (305 amino acids), respectively, were sequenced. Careful sequence alignment revealed several consensus motifs of this enzyme family, as well as possible cofactor binding residues of the Sulfolobus enzyme. This new structural information also indicates that (i) several genetic fusions and reorganization of the early, possibly αβγδ-type enzyme similar to those from hyperthermophiles have taken place during evolution of the 2-oxoacid:ferredoxin (flavodoxin) oxidoreductase superfamily, which might have occurred in different ways in early aerobic archaea and early anaerobic bacteria, and that (ii) enzymes with different subunit compositions should have an essentially similar catalytic mechanism with one TPP and at least one [4Fe-4S] cluster as the minimal set of redox centers.View full abstractDownload PDF (1709K) -
Sadamitsu Asoh, Takashi Mori, Jun-Ichi Hayashi, Shigeo Ohta1996 Volume 120 Issue 3 Pages 600-607
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe applied an antibody against an apoptosis mediator, Fas/APO-1/CD95, to HeLa-derived cells that completely lack mitochondrial DNA (mtDNA) or have mutant mtDNAs. The anti-Fas antibody killed the cells completely lacking mtDNA (EB8), at concentrations as low as 1 ng/ml, but not control cells harboring wild-type mtDNA (Ft2-11). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling) and analysis of fragmented DNA indicated that the cell death of E138 was due to apoptosis. The antibody was cytotoxic to other two cell lines harboring mutant mtDNA with a point mutation or a large-scale deletion. RT-PCR (reverse transcriptase-polymerase chain reaction) showed that the mRNA content of the Fas gene was 2 to 19-fold higher in the cells with deficient mtDNA than in the control cells. In addition, the expressed Fas protein was detected by immunohistochemical staining in the cells without mtDNA but not in the control cells. Incubating the cells containing wild-type mtDNA with the respiratory inhibitors rotenone and antimycin A enhanced the content of mRNA of the Fas gene 2 to 4-fold and sensitized cells to the antibody. Thus, defects in mitochondria caused apoptotic cell death by anti-Fas antibody and enhanced Fas gene expression.View full abstractDownload PDF (4066K) -
Masayuki Okamoto, Norikazu Hiratani, Kunizo Arai, Shoji Ohkuma1996 Volume 120 Issue 3 Pages 608-615
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of H+-ATPase from rat liver lysosomes were analyzed by reconstituting proton pump activity from solubilized enzyme and Escherichia coli phospholipids in proteoliposomes devoid of anion-channels. The reconstitution procedure involved solubilization of the ATPase with n-octyl-β-D-thioglucoside in the presence of asolectin, and incorporation of the solubilized enzyme into E. coli phospholipid liposomes by dilution, freeze-thawing, and sonication. Proton pump activity of reconstituted H+-ATPase as detected by the ATP-dependent quenching of acridine orange fluorescence indicated that ATP can be replaced with dATP and to a lesser extent with GTP, but not with any other nucleotide, that Mg2+ can be replaced with Mn2+, but not with Ca2+, Sr2+, or Ba2+, that Zn2+, Pd2+, Cd2+, and Hg2+ were inhibitory, and that the enzyme was sensitive to inhibitors of v-type H+-ATPase, including bafilomycin A1, N-ethylmaleimide, DCCD, DIDS, and tri-n-butyltin. The enzyme showed unique sensitivity to anions and was activated by chloride, fluoride, and bromide from inside, but not from outside the vesicles. It was inhibited by sulfate, sulfite, and thiocyanate from outside the vesicles, and by nitrate from both inside and outside the vesicles.View full abstractDownload PDF (1011K) -
Tsuyoshi Ishimoto, Satoshi Akiba, Takashi Sato1996 Volume 120 Issue 3 Pages 616-623
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe association of prostaglandin D2 (PGD2) production as well as arachidonic acid release with the phospholipase D (PLD)-linked mechanism was studied in rat peritoneal mast cells. Stimulation of mast cells with cross-linking of the high-affinity Fc receptor for IgE caused increases in the release of arachidonic acid and PGD2, which are suppressed almost completely by ethanol or RHC 80267, a diacylglycerol lipase inhibitor. Ethanol did not influence inositol phosphate release in response to an antigen. An increase in diacylglycerol, that is inhibited by propranolol, was observed, with a peak within 1min. Antigen stimulation induced little production of lysophosphatidyleholine, while ionomycin as a control markedly induced the production. However, the phospholipase A2 (PLA2) activity in the cytosol of antigen-stimulated cells increased to the level in ionomycin-stimulated cells. The addition of the ADP-ribosylation factor-containing fraction prepared from bovine brain, that is known to specifically activate PLD, to permeabilized mast cells in the presence of GTPγS, apparently increased arachidonic acid and PGD2 release, but not in the presence of ethanol. Furthermore, arachidonic acid release by an antigen was enhanced by melittin, that activates PLA2, but PGD2 production was not. These results suggest that antigen-stimulated PGD2 production as well as arachidonic acid release are strongly associated with the sequential PLD-linked pathway.View full abstractDownload PDF (1055K) -
Ling Ling Jiang, Akio Kobayashi, Hitomi Matsuura, Hirofumi Fukushima, ...1996 Volume 120 Issue 3 Pages 624-632
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman medium-chain enoyl-CoA hydratase was purified from liver, because we noticed the presence of a high medium-chain enoyl-CoA hydratase activity in human skin fibroblasts catalyzed by an enzyme different from the known enzymes catalyzing the enoyl-CoA hydratase reaction. Two enzyme preparations were obtained. One of them, preparation I, consisted of 46-kDa polypeptide, and its molecular mass was estimated to be 86 kDa. The other, preparation II, consisted of a major 77-kDa polypeptide and minor smaller polypeptides including 46-kDa polypeptide. The molecular mass of preparation II was 154 kDa. Both enzyme preparations catalyzed reversible dehydration of medium-chain D-3-hydroxyacyl-CoA to 2-trans-enoyl-CoA, but did not react with L-3-hydroxyacyl-CoA. Catalytic properties and immunochemical reactivities of these enzyme preparations were nearly the same. The cross-reactive material to the antibody was confirmed to be in peroxisomes by immunohistochemical study of cultured human skin fibroblasts.View full abstractDownload PDF (5250K) -
Ling Ling Jiang, Shoko Miyazawa, Takashi Hashimoto1996 Volume 120 Issue 3 Pages 633-641
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously purified two D-3-hydroxyacyl-CoA dehydratase preparations from human liver. One preparation contained a 77-kDa polypeptide and smaller polypeptides, and the other was a homodimer of a 46-kDa polypeptide. Three different purified rat peroxisomal D-3-hydroxyacyl-CoA dehydratase preparations have been reported. There-fore, rat enzyme was purified in this study to confirm the enzyme structure. Two preparations with similar molecular structures to the human enzyme preparations were obtained, and these were similar to each other in immunochemical and catalytic properties. It was suggested that the native enzyme was a homodimer of the 77-kDa polypeptide, and this enzyme was modified to a homodimer of the 46-kDa polypeptide, because conversion of the 77-kDa polypeptide to smaller polypeptides including the 46-kDa polypeptide was clearly observed during purification. Rat liver subcellular fractionation study indicates that this enzyme is located in peroxisomes. The enzyme preparation containing the 77-kDa polypeptide catalyzed the D-3-hydroxyacyl-CoA dehydrogenase reaction as well as the dehydratase reaction. Thus, it is proposed that this enzyme is D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.View full abstractDownload PDF (3469K) -
Akihiko Nakano1996 Volume 120 Issue 3 Pages 642-646
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe product of the yeast SEC12 gene is the most upstream player as far as we know in the events of vesicle budding from the endoplasmic reticulum membrane. To understand how the Sec12p function could be regulated, I identified three extragenic suppressor mutations of the sec12 is mutation. RST1DS is a dominant mutation and causes elevated expression of Sec12p. rst2 and rst3 are recessive and give pleiotropic phenotypes including slow growth at low temperatures and heterogeneous modification of Sec12p. Their possible roles are discussed.View full abstractDownload PDF (3045K) -
Masao Tanji, Etsuko Yakabe, Takashi Kageyama, Kenji Takahashi1996 Volume 120 Issue 3 Pages 647-656
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete primary structure of the major component of tuna pepsinogens was determined by conventional protein chemistry methods. It was composed of a prosegment of 37 residues and a pepsin moiety of 323 residues, having a relative molecular mass of 39, 364. The essential aspartyl residues in the active site and the three disulfide bonds common to other pepsinogens were conserved; however, several unique substitutions and/or deletions characteristic of tuna pepsinogen were found at various positions, especially in the prosegment and subsite regions, as compared with the sequences of other pepsinogens, which may affect the rate of activation of the zymogen, and/or the catalytic function and substrate specificity of the enzyme. Tuna pepsinogen is the least acidic among pepsinogens. The sequence identity between tuna pepsinogen and other pepsinogens ranged from 45 to 52%. A phylogenetic tree based on the primary structures suggested that tuna pepsinogen diverged from the pepsinogen A and prochymosin groups in an early period of pepsinogen evolution.View full abstractDownload PDF (1154K) -
Fumiko Arakawa, Masahide Kuroki, Motohisa Kuwahara, Tarumi Senba, Hiro ...1996 Volume 120 Issue 3 Pages 657-662
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and Vκ) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and Vκ genes to the human heavy and light chain constant region genes (Cγ1 and Cκ) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.View full abstractDownload PDF (709K) -
Isamu Matsunaga, Shiro Oka, Nagatoshi Fujiwara, Ikuya Yano1996 Volume 120 Issue 3 Pages 663-670
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMycoloyl glycolipids cause granulomas in the lungs, liver, and spleen of mice, but the mechanism is not fully understood. To understand the role of macrophage chemotactic factors (MCFs) in granuloma formation, we prepared various mycoloyl glycolipids with different carbohydrate moieties: trehalose dimycolate (TDM), glucose mycolate (GM), mannose mycolate (MM), and fructose mycolate (FM) from Rhodococcus ruber, and examined the relationship between their MCF induction in peritoneal macrophages and the extent of granuloma formation. The molecular mass of each glycolipid was confirmed by fast-atom-bombardment mass-spectrometry. TDM or GM caused granulomas in the lungs, spleen, and liver of ICR mice, but MM and FM did not. The culture supernatant of peritoneal macrophages stimulated with TDM or GM increased macrophage migration, whereas MM and FM had no chemotactic activity. The activity of interleukin-1 (IL-1) in the supernatant was increased equally by each glycolipid and was therefore not related to chemotaxis. Tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were not detected in the four supernatants. The TDM-induced MCF was heat-stable, trypsin-labile, and undialyzable. Furthermore, we separated two MCF active fractions from the supernatant of TDM-stimulated macrophages by gel filtration. These factors acted on macrophages but not on neutrophils. Our results suggested that macrophages recognize the sugar moieties of mycoloyl glycolipids and may, in response, generate a MCF that may play an important role in the macrophage or monocyte recruitment which is essential prior to granuloma formation.View full abstractDownload PDF (2064K) -
Toshio Kojima, Mitsunori Fukuda, Jun Aruga, Katsuhiko Mikoshiba1996 Volume 120 Issue 3 Pages 671-676
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabphilin 3A and Doc2α are synaptic vesicle-associated proteins, and are thought to function as Ca2+ sensors in neurotransmitter release. If either rabphilin 3A or Doc2α plays a role in membrane trafficking, like the synaptotagmins, then non-neural forms should be present. Here we describe the isolation of a mouse cDNA which encodes a novel Doc2 homologue (Doc2β) that is present in all tissues. The encoded protein, which is highly homologous to human Doc2α (70% identity), is composed of 412 amino acids with a calculated relative molecular mass (Mr) of 45, 837. The sequence identity is especially high in two C2 domains (74% in C2A and 84% in C2B). Northern and Western blot analyses have shown that Doc2β is expressed in all cell lines and tissues tested. Ca2+-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but not the C2B domain, of Doc2β binds phosphatidycholine and phosphatidylserine (2.5:1, w/w) liposomes. The binding is Ca2+-dependent, with an EC50 value of approximately 1 μM and a Hill coefficient of approximately 3, which are comparable to those of synaptotagmins, rabphilin 3A and Doc2α. Our results suggest that Doc2β is involved in constitutive membrane trafficking.View full abstractDownload PDF (1949K) -
Yuzi Takahashi, Tokio Tani, Yasumi Ohshima1996 Volume 120 Issue 3 Pages 677-683
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSU1, U2, U4, U5, and U6 small nuclear RNAs are essential for precursor mRNA splicing. We previously found one and four mRNA-type, or spliceosomal introns, in the U2 and U6 genes, respectively, of yeast Rhodotorula hasegawae (Erythrobasidium hasegawianum). We proposed that U2 and U6 RNAs form a catalytic core for precursor mRNA splicing and that the introns in those small nuclear RNA genes may have been acquired through reverse splicing of an intron from a precursor mRNA into a catalytic site in small nuclear RNAs. In the present study, we analyzed U1, U4, and U5 genes in R. hasegawae. One spliceosomal intron was found in the U1 region forming base-pairs with a 5' splice site of a precursor mRNA. The U5 gene has two spliceosomal introns in the region that interacts with 5' and 3' splice sites. In contrast, the gene for U4 RNA, which is released from the spliceosome prior to the first step of the splicing reaction, has no intron. These results lend a further support to the proposed relation between presence and position of an intron in an small nuclear RNA gene and the function of the encoded small nuclear RNA.View full abstractDownload PDF (1782K)
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