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Volume 124 (1998)
- Issue 6 Pages 1051-
- Issue 5 Pages 869-
- Issue 4 Pages 687-
- Issue 3 Pages 473-
- Issue 2 Pages 251-
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Volume 124, Issue 6
Displaying 1-25 of 25 articles from this issue
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Hiroaki Sakai, Tomitake Tsukihara1998 Volume 124 Issue 6 Pages 1051-1059
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFollowing determination of the first crystal structure of the reaction center of Rhodopseudomonas viridis, a membrane protein, by X-ray crystal structure analysis at 3.0 Å resolution, 18 X-ray crystal structures and two electron crystal structures of membrane proteins have been obtained at higher than 3.5 Å resolution. Besides these integral membrane protein structures, three crystal structures of water-soluble proteins, which can enter membranes, have been determined by X-ray crystallography at high resolution. The structural features of membrane proteins have been summarized by inspecting these crystal structures. The polypeptide chain crosses the membrane in a helical conformation or a β-strand. The central +10 Å region of the transmembrane α-helix is dominated by hydrophobic residues. On both sides of the central region are concentrated polar aromatic residues. Charged residues are dominant around +15 Å to +20 Å. All the transmembrane β-structures are found in pore-forming proteins. The central region of the transmembrane β-structure is amphipathic with hydrophobic residues on the membrane exposed side. The distribution of amino acid residues on the membrane exposed surface of the transmembrane β-structure is similar to that of the transmembrane α-helix. α-Helices anchoring the membrane surface region are amphipathic with hydrophobic residues inside and hydrophilic residues outside.View full abstractDownload PDF (4333K) -
Susumu Hirose1998 Volume 124 Issue 6 Pages 1060-1064
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chromatin structure is essential not only for the compact packaging of the eukaryotic genome but also for regulation of transcription. This article provides an overview of chromatin modification upon transcriptional activation or repression, and chromatin remodeling. Interestingly, recent data demonstrate that chromatin remodeling in the promoter region is necessary for transcription.View full abstractDownload PDF (1624K) -
Satoshi Ohno, Takashi Yokogawa, Isao Fujii, Haruichi Asahara, Hachiro ...1998 Volume 124 Issue 6 Pages 1065-1068
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn efficient system was developed for the co-expression of a yeast tRNATyr/tyrosyl-tRNA synthetase (TyrRS) pair in Escherichia coli.. Analysis of suppression patterns using several sets of E. coli and λ phage mutants indicated that the expressed yeast suppressor tRNATyr was aminoacylated only with tyrosine by its cognate yeast TyrRS and not by E. coli TyrRS or other aminoacyl-tRNA synthetases. This extra tRNA/TyrRS pair is expected to be a key bridgehead for developing an in vivo system for the site-directed incorporation of unnatural amino acids into proteins.View full abstractDownload PDF (1360K) -
Nono Takeuchi, Takuya Ueda, Kimitsuna Watanabe1998 Volume 124 Issue 6 Pages 1069-1071
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTranslational initiation in bacteria and some organelles such as mitochondria and chloroplasts requires formyl-methionyl-tRNA (fMet-tRNA). Methionyl-tRNA (Met-tRNA) undergoes formylation by methionyl-tRNA transformylase (MTF), and the resulting fMet-tRNA is utilized exclusively in the initiation process. The gene encoding mammalian mitochondrial MTF (MTFmt) was cloned recently. When the cDNA corresponding to mature MTFmt was cloned into an expression vector, no expression of MTFmt was observed. However, if the cDNA was fused with the histidine-tag sequence at the N-terminus, MTFmt could be expressed in Escherichia coli. The recombinant enzyme was purified by a single step on a histidine-binding metal affinity column. We previously found that native MTFmt is able to formylate E. coli elongator Met-tRNA as well as the initiator Met-tRNA. The specific formylation of the initiator Met-tRNA by E. coli MTF is quite important in bacterial translational initiation. The purified recombinant MTFmt with the histidine-tag showed almost identical kinetic parameters to those of native MTFmt. This expression system is suitable for the rapid, efficient production of MTFmt in amounts adequate for further biophysical studies, which will provide another approach for elucidating the formylation mechanism, in addition to studies on E. coli MTF.View full abstractDownload PDF (3241K) -
Dong-Ho Kim, Yousuke Inagaki, Takashi Suzuki, Ryoichi X. Ioka, Shiroh ...1998 Volume 124 Issue 6 Pages 1072-1076
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe isolation and characterization of rabbit and human cDNAs revealed a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1, 616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibited increased binding of apolipoprotein E (apoE)-enriched β-migrating very low density lipoprotein. Northern blotting and in situ hybridization revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA was increased in the liver and accumulated in cholesterol-laden foam cells of atherosclerotic lesions.View full abstractDownload PDF (2722K) -
Tomofumi Fujino, Tomohiro Tada, Masatoshi Beppu, Kiyomi Kikugawa1998 Volume 124 Issue 6 Pages 1077-1085
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA serine protease that preferentially degrades oxidized and glycated proteins was shown to be present in erythrocyte cytosol. Human erythrocyte cytosol was labeled with [3H] diisopropyl fluorophosphate (DFP) and passed through a column of carboxymethyl-Sephadex to obtain radioactive fractions free of hemoglobin. The fractions contained a single radioactive 80-kDa protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)/fluorography. The radioactive 80-kDa protein bound to unoxidized erythrocyte membranes, and more effectively to oxidized membranes. The radioactive protein was purified through a column of diethylaminoethyl-cellulose and by preparative native-PAGE in a purity of 80%. Antibody against the cytosolic 80-kDa protein bound to 80-kDa protein of erythrocyte membranes, indicating the presence of the same protein in the membrane. The antibody bound more effectively to oxidized membranes than to unoxidized membranes. The 80-kDa protein partially purified from unlabeled cytosol degraded more effectively oxidized bovine serum albumin (BSA), oxidized IgG, and glycated BSA more effectively than the corresponding unoxidized or unglycated proteins. Degradation of oxidized or glycated proteins was effectively inhibited by DFP. Hence, the protein is an 80-kDa serine protease that is adherent to oxidized membranes and is responsible for degradation of proteins modified by oxidation and glycation.View full abstractDownload PDF (4774K) -
Seiki Niioka, Makoto Goto, Teru Ishibashi, Motoni Kadowaki1998 Volume 124 Issue 6 Pages 1086-1093
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAutophagy-related vacuoles, i.e., autophagosomes (AVi), autolysosomes (AVd) and dense bodies (DB), are intracellular organelles within which macroautophagy and bulk proteolysis set out and progress. Separation of these particles in freshly isolated rat hepatocytes, monitored by β-hexosaminidase, a lysosomal marker enzyme, was established by density gradient centrifugation. Percoll density gradients were modified and improved by adding free polyvinylpyrrolidone (PVP, 0.75%) to 60% Percoll, which made it possible to separate AVd (buoyant peak, d=1.090) and DB (dense peak, d=1.131) effectively. Addition of graded levels of a regulatory amino acid mixture (Reg AA) to hepatocyte incubation not only suppressed proteolysis, but also lead to a shift of vacuolar profiles on the density gradients from the buoyant to the dense region. Alterations in the vacuolar shift and proteolysis were highly proportional over a full range of regulation by Reg AA. Morphometric analysis of autophagic vacuoles by electron microscopy revealed changes in the aggregate volumes of both AVi and AVd by Reg AA, which enabled us to estimate autophagic subpopulation of the buoyant peak on the gradient profile. All the results demonstrate that AVd shifts on the density gradients in proportion to alterations in proteolysis regulated by amino acids, and thus the gradient profile can be used as a measure of macroautophagy; and in addition that AVd actively involved in proteolysis occupies only a part of the buoyant peak on the gradients.View full abstractDownload PDF (2068K) -
Young-Kee Kim, Jung Sakong, Kwang-Hyun Cho, Chin Ok Lee1998 Volume 124 Issue 6 Pages 1094-1100
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytosolic free Ca2+ plays important roles in the regulation of physiological processes in tracheal epithelial cells and is probably regulated by many ion-transporting ATPases in these cells. Therefore, the effect of vanadate was investigated to characterize microsomal ion-transporting ATPases. Dose response experiments showed that vanadate had a biphasic effect on the microsomal ATPase activity: a decrease at the vanadate concentration below 100 μM, and a steep decrease at the concentration above 100 μM. The dose response data were fitted to two sigmoidal functions, corresponding to a low-affinity vanadate-sensitive (LAVS) ATPase and a high-affinity vanadate-sensitive (HAVS) ATPase. In 45Ca2+ uptake experiments, both LAVS and HAVS ATPases mediated microsomal 45Ca2+ uptake. The LAYS ATPase was selectively sensitive to thapsigargin in both ATPase activity and 45Ca2+ uptake, suggesting that it is an ER/SR-type intracellular Ca2+-ATPase. Although the HAVS ATPase mediated one-fourth of microsomal 45Ca2+ uptake, its activity was not sensitive to thapsigargin. These results indicate that the activities of these two vanadate-sensitive ATPases are mediated by different enzymes, since thapsigargin only blocks the activity of LAYS ATPase. In conclusion, there are two types of vanadate-sensitive microsomal ATPases, and these ATPases mediate microsomal 45Ca2+ uptake in airway epithelial cells.View full abstractDownload PDF (828K) -
Kay Grobe, Birte Sartori, Christina Traving, Roland Schauer, Peter Rog ...1998 Volume 124 Issue 6 Pages 1101-1110
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSClostridium tertium metabolizes sialoglycoconjugates via a secreted sialidase [EC 3. 2. 1. 18] and an intracellular acylneuraminate pyruvate lyase [EC 4. 1. 3. 3]. The sialidase was enriched 1, 900-fold from the culture medium with a specific activity of 0.7 U per mg protein. It exhibits a temperature optimum of 50°C and tolerates mercury ions at relatively high concentrations (50% inhibition at 5.2mM Hg2+). The sialidase gene was detected on two restriction fragments (HincII, HindIII) of chromosomal DNA and their correct recombination resulted in an active enzyme expressed by Eseherichia coli. The structural gene is represented by 2, 319 by encoding a protein of 773 amino acids with a molecular mass of 85.4 kDa. The first 28 amino acids possess the character of a signal peptide. The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases. It has 42.6% identical amino acids when compared with the sialidase of Clostridium septicum and 64.8% with a sialidase gene amplified from Macrobdella decora. A further open reading frame was detected 30 by downstream from the sialidase gene, which exhibits significant homology with acylneuraminate pyruvate lyases. For the first time, both genes were found in close proximity on a bacterial chromosome, probably being part of one operon.View full abstractDownload PDF (1378K) -
Toshiki Fujita, Hiroshi Maegawa, Atsunori Kashiwagi, Hisamaru Hirai, R ...1998 Volume 124 Issue 6 Pages 1111-1116
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the difference in signaling between insulin and insulin-like growth factor I (IGF-I), we studied the effects of these hormones on the phosphorylation state of Crk-associated substrate (Cas) in cells expressing human insulin receptor (HIRc). In the basal state, Cas was heavily tyrosine-phosphorylated, and insulin dephosphorylated Cas in a time- and dose-dependent manner. On the other hand, IGF-I phosphorylated rather than dephosphorylated Cas in HIRe cells. In HIRY/F 2 cells expressing a mutant insulin receptor lacking a binding site of SHP-2, a protein-tyrosine phosphatase containing src homology 2 (SH2) regions, insulin accelerated phosphorylation of Cas, as did IGF-I. In HIRc cells expressing a mutant SHP-2 lacking a PTPase domain (ΔPTP), which interfered with SHP-2 function, insulin failed to dephosphorylate Cas. In whole cell lysate obtained in the basal state, Gas bound to a glutathione-S transferase fusion protein containing SH2 domains of SHP-2 and dissociated from this GST protein in response to insulin. These results indicate that the opposite regulation of Cas phosphorylation by insulin and IGF-I may be mediated through different properties of their receptors, and that the interaction of the insulin receptor with SHP-2 may play an important role in determining the tyrosine-phosphorylation state of Cas.View full abstractDownload PDF (4456K) -
V. Nagarajan, Shigehiro Kamitori, Kenji Okuyama1998 Volume 124 Issue 6 Pages 1117-1123
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSingle crystals of (Pro-Pro-Gly)10 were grown by the hanging drop method. The crystals diffracted to a resolution of 1.8 Å. In the crystals the polypeptides form triple helices that aggregate end-to-end mediated by the solvent molecules, with the basic repeat being 20 Å along the helical axis. Analysis of the 20 Å structure of (Pro-Pro-Gly)10 using data up to a resolution of 1.9 Å revealed that the overall structure is in accordance with the 7/2 model proposed for collagen. The three strands are held together by the (Gly) N-H…O (Pro-X) hydrogen bond interactions, and additional stability is provided by the (Pro-Y) Cα -H…O (Pro-X) hydrogen bonding interactions.View full abstractDownload PDF (1981K) -
Toshihiro Nagao, Yuji Shimada, Akio Sugihara, Yoshio Tominaga1998 Volume 124 Issue 6 Pages 1124-1129
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSaccharomyces cerevisiae bearing a lipase eDNA from Fusarium heterosporum produced two lipases, A and B. Lipase B was significantly more stable to temperature than lipase A, but their optimum temperatures were similar. Lipase B was composed of one polypeptide (301 amino acids), and lipase A was composed of two polypeptides (275 and 26 amino acids) generated by the cleavage between Arg 275 and Asp 276 with a trypsin-like protease. It was suggested that the C-terminal peptide (26 amino acids) tightened the lipase structure when bound to the catalytic domain (275 amino acids) through a peptide bond. The tight structure was loosened by cleavage of the C-terminal peptide, even though the peptide interacted noncovalently with the catalytic domain, possibly through charged amino acids, in which it is rich. Deletion of the C-terminal peptide greatly decreased the lipase production by the recombinant S. cerevisiae, although its transcriptional level was the same as that of cells carrying the wild-type gene. These facts suggested that the C-terminal peptide affected the lipase production in the post-transcriptional step.View full abstractDownload PDF (3343K) -
Keiji Miyazawa, Akio Mori, Hirokazu Okudaira1998 Volume 124 Issue 6 Pages 1130-1137
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInvolvement of interleukin-6 (IL-6) in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated. In the present study, the regulation of IL-6 gene expression by glucocorticoids and IL-1β in fibroblast-like synoviocytes (FLSs) was investigated. Both synthetic and natural glucocorticoids, i.e., dexamethasone (DEX) and hydrocortisone (HC), respectively, concentration-dependently inhibited protein production and gene expression of IL-6 by human FLSs. The effect of DEX was dependent on de novo protein synthesis. DEX significantly reduced the rate of IL-6 gene transcription without affecting the stability of IL-6 mRNA. The IκBα pathway seemed not to be involved in DEX-mediated inhibition of IL-6 gene expression in IL-1β-stimulated human FLSs. These findings suggest that glucocorticoids suppress IL-6 gene transcription by an as yet undefined mechanism.View full abstractDownload PDF (6077K) -
Svante Hugosson, Jonas Ångström, Britt-Marie Olsson, Jö ...1998 Volume 124 Issue 6 Pages 1138-1152
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe glycosphingolipid binding specificities of Haemophilus influenzae and Neisseria meningitidis were investigated as to the binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby, similar binding profiles, for the binding of the two bacteria to lactosylceramide, isoglobotriaosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and sialylneolactohexaosylceramide, were obtained. On a closer view the binding preferences of the bacteria could be differentiated into three groups. The first specificity is recognition of lactosylceramide. The second specificity is binding to gangliotriaosylceramide and gangliotetraosylceramide, since conversion of the acetamido group of the N-acetylgalactosamine of gangliotriaosylceramide and gangliotetraosylceramide to an amine prevented the binding of the bacteria, and thus the binding to these two glycosphingolipids represents a separate specificity from lactosylceramide recognition. Preincubation of H. influenzae with neolactotetraose inhibited the binding to neolactotetraosylceramide, while the binding to lactosylceramide, gangliotetraosylceramide, or lactotetraosylceramide was unaffected. Thus, the third binding specificity is represented by neolactotetraosylceramide, and involves recognition of other neolacto series glycosphingolipids with linear N-acetyllactosamine chains, such as sialyl-neolactohexaosylceramide. The relevance of the detected binding specificities for adhesion to target cells was addressed as to the binding of the bacteria to glycosphingolipids from human granulocytes, epithelial cells of human nasopharyngeal tonsils and human plexus choroideus. Binding-active neolactotetraosylceramide was thereby detected in human granulocytes and the oropharyngeal epithelium.View full abstractDownload PDF (8969K) -
Keiji Miyazawa, Akio Mori, Hirokazu Okudaira1998 Volume 124 Issue 6 Pages 1153-1162
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel immortalized rheumatoid fibroblast-like synoviocyte (FLS) line, MH7A, was established by stably transfecting FLS cells with SV40 T antigen gene. MH7A cells expressed SV40-specific small t and large T antigens as well as an elevated level of p53 protein. They have already reached over 150 population doublings through culture crisis, and have been growing rapidly compared with the parental FLSs. Constitutive activation of p42/p44 mitogen-activated protein (MAP) kinase was detected in MH7A cells. Serum requirements for the growth of MH7A were markedly decreased compared with those for the parental FLSs. MH7A cells were stained positively for interleukin (IL)-1R, intercellular adhesion molecule-1 (ICAM-1), CD16, CD40, CD80, and CD95. IL-1 β enhanced the production of IL-6 and stromelysin-1, and the surface expression of ICAM-1, in a manner similar to that in the parental FLSs. SB203580, a specific inhibitor of p38 MAP kinase, significantly inhibited IL-1 β-induced IL-6 and stromelysin-1 production by both parental FLSs and MH7A cells; although PD098059, an inhibitor of the p42/p44 MAP kinase pathway, did not affect it. Our results clearly indicate the usefulness of MH7A cells for investigating the regulation of rheumatoid FLSs and the IL-1 signal transduction pathway to develop future RA therapy.View full abstractDownload PDF (3324K) -
Yoichi Kurokawa, Akira Watanabe, Tohru Yoshimura, Nobuyoshi Esaki, Ken ...1998 Volume 124 Issue 6 Pages 1163-1169
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate, D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly. The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH. Thus, alanine racemase catalyzes transamination as a side function. In fact, the apo-form of the enzyme abstracted tritium from [4'-3H] pyridoxamine in the presence of pyruvate. A mutant enzyme containing alanine substituted for Lys39, whose ε-amino group forms a Schiff base with the C4' aldehyde of pyridoxal 5'-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination. However, when methylamine was added to the mutant enzyme, it became active in both reactions. These results suggest that the ε-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme.View full abstractDownload PDF (881K) -
Masatoshi Maki, Kyoko Yamaguchi, Yasuyuki Kitaura, Hirokazu Satoh, Kiy ...1998 Volume 124 Issue 6 Pages 1170-1177
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSALG-2 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain. We established a convenient method for the purification of the recombinant mouse ALG-2 expressed in Escherichia coli. The recombinant protein was first pelleted from a lysate in the absence of a Ca2+-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies. Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-2 occurred as a monomeric form. Ca2+-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEF region was found to be more soluble than the wild type in the presence of Ca2+. Analysis using a fluorescent hydrophobicity probe indicated that ALG-2 exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 μM. Mg2+ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.View full abstractDownload PDF (4516K) -
Shinobu Fujii, Bunpei Inoue, Hiroki Yamamoto, Kenji Ogata, Tomohiro Sh ...1998 Volume 124 Issue 6 Pages 1178-1187
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/Km, for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of kcat for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.View full abstractDownload PDF (1194K) -
Nobuko Kawasaki, Mitsuko Satonaka, Masayoshi Imagawa, Haruna Naito, To ...1998 Volume 124 Issue 6 Pages 1188-1197
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine conglutinin is a Ca2+-dependent, serum lectin that is specific for N-acetylglucosamine and a member of the collectin (collagen-like lectin) family. Here we report the identification of the cis-acting elements involved in regulating expression of the conglutinin gene. The 5'-flanking region of the conglutinin gene was cloned and sequenced by gene walking using vector (cassette)-ligation mediated PCR. A genomic fragment encompassing -741 to +50 by had significant promoter activity when linked to the luciferase reporter gene and transfected into the human hepatoma cell line HepG2. Transfection analysis using a series of luciferase vector/5'-stepwise deletion mutants of the promoter constructs indicated that the sequence of 7 base pairs at around -180 by from the transcription initiation site was necessary for the full expression of the conglutinin gene. The site-directed mutagenesis in the AP-1 (Activator Protein-1) sequence, immediately downstream of the positively controlling cis-element at around -180 bp, resulted in a marked loss of the promoter activity. The novel positively controlling cis-element and the AP-1 sequence regulated synergistically the expression of the conglutinin gene. Gel retardation assay and DNase I footprint analysis demonstrated the presence of the nuclear proteins that bind to these two cis-elements.View full abstractDownload PDF (4087K) -
Bidyottam Mittra, Pranab K. Sadhukhan, Hemanta K. Majumder1998 Volume 124 Issue 6 Pages 1198-1205
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA nuclease activity has been purified from the nuclei-kinetoplast fraction of Leishmania. This enzyme, termed endonuclease M (Endo M), is shown by electrophoresis in a denaturing polyacrylamide gel to be associated with a single polypeptide of molecular mass 52 kDa. Physical analysis of the enzyme indicates that it has a sedimentation coefficient S20, w of 4.5 S, a Stoke's radius of 32.5 Å, and a native molecular mass of 53 kDa. The final Mono Q purified Endo M possesses both DNase and RNase activities. It acts as an endonuclease by introducing random single-stranded nicks into the supercoiled DNA molecules, that often leads to its linearization due to nicking at the opposite strands, and subsequent degradation of the DNA with further incubation. Single-stranded DNA is twice preferred to double-stranded DNA as substrate. Single-stranded RNA is also degraded rapidly and is competitive as a substrate with single-stranded DNA. RNA:DNA hybrids, however, are largely resistant to the Endo M digestion.View full abstractDownload PDF (4237K) -
Takahiro Hirata, Erika Fujihira, Tomomi Kimura-Someya, Akihito Yamaguc ...1998 Volume 124 Issue 6 Pages 1206-1211
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the membrane topology of Tet (K), conventional antibiotic resistance genes were used as reporter molecules. A series of fusion genes comprising a resistance gene -β-lactamase (amp) or chloramphenicol acetyl transferase (cat)-and one loop of the 14 putative transmembrane segments of Tet(K) was constructed. Escherichia coli TG1 with the tet(K)-amp fusion gene at the site of the putative periplasmic loop showed resistance to ampicillin, but the bacterium with the tet (K)-cat fusion gene at the site of the putative cytoplasmic loop showed resistance to chloramphenicol. These findings supported a topology of 14 membrane-spanning segments for Te (K). Three exceptional cases were observed, which were apparently due to the presence of an acidic residue, Glu, in the preceding transmembrane segment. Mutants in which these acidic residues was substituted for alanine were also constructed, and the effect of glutamic acid in the transmembrane segment on the topology of the fusion proteins was examined.View full abstractDownload PDF (719K) -
Benoît Graulet, Dominique Gruffat, Denys Durand, Dominique Bauch ...1998 Volume 124 Issue 6 Pages 1212-1219
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe liver of bovine animals possesses a low ability to secrete triglycerides (TG) as part of the very low density lipoproteins (VLDL) compared with rat liver. We compared hepatic fatty acid (FA) metabolism between rat and calf in order to determine the limiting steps of TG-VLDL secretion in bovine animals. Liver slices from young Sprague-Dawley rats and preruminant Holstein×Friesian calves were incubated for 7 h with increasing concentrations (0.1, 0.2, 0.4, and 0.8mM) of [14C] oleate. The oxidation of oleate to CO2 and acidsoluble products was 2- to 3-fold higher in rat than in calf liver slices. Since oleate uptake was 2-fold higher in rat than in calf, the oxidation rate represented 20-29% of oleate uptake in both animal species. Oleate was essentially incorporated into the neutral lipids (75-87% of total lipids) that were stored mainly in the cytosol in both animal species (81-90% of neutral lipids). The accumulation of neutral lipids in the cytosol was 3.4-fold higher while VLDL secretion was 6- to 18-fold more efficient in rat than in calf liver slices. Our results indicate that the slow rate of VLDL secretion by bovine liver is probably due to the limited availability of TG for VLDL packaging rather than to the preferential oxidation of FA.View full abstractDownload PDF (1053K) -
Eiichi Taguchi, Hisako Muramatsu, Qi-Wen Fan, Nobuyuki Kurosawa, Gen S ...1998 Volume 124 Issue 6 Pages 1220-1228
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSZep, a novel 49 kDa zinc finger protein, was found in the brain of day-13 mouse embryos and cloned. Zep contains two C2112-type zinc finger motifs close to the N-terminal region. The majority of the molecule is composed of a proline-rich domain showing similarity to proline-rich domains in transcription factors and a salivary proline-rich protein. In addition to the proline-rich domain, Zep has an acidic domain and a serine/threonine-rich domain, all of which are frequently found in many transcription factors. The overall organization of Zep shows no similarity to any other proteins. There is a nuclear localization signal in Zep, and the Zep-GFP (green fluorescent protein) fusion protein is located predominantly in the nucleus. In the day-13 mouse embryo, Zep is strongly expressed in the nervous system, i.e. brain, spinal cord, and dorsal root ganglia, with strong to weak expression observed in other regions. Zep continues to be strongly expressed in the neonatal brain; however, its expression is weak in the brain and spinal cord of adult mice. In situ hybridization reveals strong signals for Zep mRNA in the cerebellum and olfactory bulb with moderate signals detected in the hippocampus and cortex. Strong Zep expression is observed in adult thymus, lung, spleen, testis, and ovary. Zep may be involved in the formation and remodeling of various tissues including nervous tissue, probably through transcriptional regulation.View full abstractDownload PDF (5679K) -
Tomohiro Yamaguchi, Kazuhisa Nakayama, Kiyotaka Hatsuzawa, Katsuko Tan ...1998 Volume 124 Issue 6 Pages 1229-1236
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe treatment of normal rat kidney cells with N-ethylmaleimide caused the release of β-COP, a component of coatomer, from the Golgi apparatus without causing disassembly of the organelle. The release of β-COP, which was not due to depolymerization of microtubules, was markedly blocked by the activation of GTP-binding proteins by aluminum fluoride or a nonhydrolyzable analogue of GTP. To determine which component is N-ethylmaleimide-sensitive, we reconstituted the recruitment of coatomer from the bovine brain cytosol onto the Golgi apparatus in digitonin-permeabilized cells. In cells treated with N-ethylmaleimide before permeabilization, β-COP was still recruited onto the Golgi apparatus. In contrast, β-COP was not recruited when N-ethylmaleimide-treated bovine brain cytosol was used. These results suggest that the N-ethylmaleimide-sensitive factor(s) are present in the cytosol. It is known that coatomer and ADP-ribosylation factor-1 (ARF1) are the only cytoplasmic proteins needed for the assembly of Golgi-derived coated vesicles. N-Ethylmaleimide treatment of a coatomer-rich fraction did not affect the binding of β-COP to the Golgi apparatus, whereas the same treatment of an ARF-rich fraction abolished β-COP binding. Similar results were obtained using purified recombinant ARF1. Concomitant with inactivation, 0.85 mol of N-ethylmaleimide was incorporated into 1 mol of ARF1. ARF1 contains only one cysteine residue (Cys-159), which is located near the base moiety of the bound guanine nucleotide.View full abstractDownload PDF (6855K) -
Tuyosi Fujita, Keiichiro Suzuki, Takakiyo Tada, Yutaka Yoshihara, Riek ...1998 Volume 124 Issue 6 Pages 1237-1244
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS2, 3-Bisphosphoglycerate mutase (BPGM) [EC 5. 4. 2. 4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2, 3-diphosphoglycerate (2, 3-DPG) and contains three types of activities in that it functions as a 2, 3-DPG synthetase, a phosphoglycerate mutase and a 2, 3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2, 3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2, 3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an antihexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.View full abstractDownload PDF (1042K)
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