巻号一覧
123 巻, 2 号
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Keiji Tanaka1998 年 123 巻 2 号 p. 195-204
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe proteasome is a multisubunit protease complex with an apparent sedimentation coefficient of 20 S. Two types of regulatory complexes, named PA 700 and PA 28, bind to both ends of the cylindrical 20 S proteasome to form the dumbbell-like and football-like proteasomes, respectively. The former complex, named the 26 S proteasome, is a eukaryotic ATP-dependent protease and appears to be well organized as a large complex of 2 MDa, nsisting of approximately 40 polypeptides, to facilitate rapid proteolysis. It is assumed to be a protein “death machine”, destroying a variety of cellular proteins that have acquired a specific degradation signal (s) such as a multiubiquitin chain. Recently data on in vivo substrates for the ubiquitin-proteasome pathway have been accumulating rapidly, implying its involvement in many biologically important processes, such as cell-cycle regulation, signal transduction, protein quality control, and the immune response. The newlyidentified PA 28 family proteins are inducible by interferons, and may cooperate with the 26 S proteasome or play additional roles. Since the proteasome is capable of catalyzing breakdown of proteins not only irreversibly, but also rapidly and timely, it is thought to be a new regulatory system for biological reactions in eukaryotes.抄録全体を表示PDF形式でダウンロード (3729K) -
Masako Mizuno-Kamiya, Yasunaga Kameyama, Koji Yashiro, Atsushi Fujita1998 年 123 巻 2 号 p. 205-212
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe characterized the Ca2+-independent, membrane-associated phospholipase A2 (PLA2) from rat parotid secretory granules. Among four phosphatidylcholine species with different fatty acyl (palmitoyl, oleoyl, linoleoyl, and arachidonoyl) groups at the sn-2 position, 2-arachidonoyl-phosphatidylcholine was the preferred substrate. Such specificity was also apparent even when 2-arachidonoyl-phosphatidylcholine coexisted with another species. The various well-documented inhibitors of PLA2s, bromoenol lactone, arachidonyl trifluoromethyl ketone, methyl arachidonyl fluorophosphate, and diisopropyl fluorophosphate, did not inhibit granular PLA2 activity. The granular PLA2 was activated markedly by ATP, and to a lesser extent by GTP and ATPγS. GTP also partially suppressed the ATP-mediated activation. UTP, CTP, GTPγS, and the hydrolyzed products of ATP and GTP showed little activation of the enzyme. Neither addition of K-252a nor depletion of Mg2+ affected ATP-mediated activation. Although this enzyme was located in the granular membranes, the granular soluble contents or BSA were required for the full activity and full ATP-mediated activation. These results suggested that the PLA2 located in granular membranes may participate in the liberation of arachidonic acid in parotid cells and be regulated through a mechanism mediated by ATP.抄録全体を表示PDF形式でダウンロード (932K) -
Jun Feng Liang, Toshihiro Akaiket1998 年 123 巻 2 号 p. 213-218
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe have previously demonstrated that treatment of hepatocytes with IFN γ results a series of cellular injury processes, including DNA synthesis arrest, membrane breakage and apoptosis. In the present work, we show that IFN γ suppresses cellular respiration and protein synthesis in hepatocytes, and that cellular respiration suppression is an early event in the IFN γ-induced cellular injuries. Polyunsaturated fatty acids (PUFAs) increased cellular respiration of hepatocytes, but only linoleic acid showed some protective effect against IFN γ-induced cellular respiration suppression. Linoleic acid also reduced other IFN γ-mediated cellular injuries, including membrane breakage and protein synthesis inhibition. Like linoleic acid, fetal bovine serum also inhibited IFN γ-induced cellular damage. Increased NAD levels were found in both IFN γ-treated and non-treated hepatocytes following the addition of PUFAs, but clofibrate, a peroxisome proliferator, bromophenacyl bromide (BPB), an inhibitor of phospholipase, nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, and arachidonic acid, a metabolite of linoleic acid, did not inhibit IFN γ-induced cellular injury. In addition, the combination of linoleic acid and IFN γ induced nitric oxide (NO) synthesis in hepatocytes. These results suggest that fatty acid may play an important role in liver homeostasis during chronic inflammatory states and sepsis.抄録全体を表示PDF形式でダウンロード (3957K) -
Hiroki Tsuruta, Sofia T. Tsuneta, Yoshihiro Ishida, Keiichi Watanabe, ...1998 年 123 巻 2 号 p. 219-225
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe phosphatase of a psychrophile was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, butyl-Cellulofine, Sephacryl S-100, and Mono-Q columns. The purified enzyme preparation was found to be electrophoretically homogeneous on native- and SDS-PAGE, and its molecular mass was determined to be 38.4 kDa by MALDI-TOF mass spectrometry. Maximal activity was observed at 30°C and pH 6.0. Furthermore, the activity of this enzyme at 0 and 5°C was 27 and 28%, respectively, of that at 30°C. The enzyme was stable in the pH range of 6.0 to 8.0 and up to 20°C. The enzyme was affected by metal ions; the activity was enhanced by Mg2+ and Ca2+ ions, but depressed by Zn2+ ions. Analysis of the amino acid composition indicated that this phosphatase contains no S-S bond, and only a few prolyl residues necessary to retain the rigid structure of a protein molecule. The phosphatase shows typical features of a cold enzyme; high catalytic activity at low temperature and rapid inactivation at an intermediate temperature.抄録全体を表示PDF形式でダウンロード (1352K) -
Hisayoshi Akagawa, Ayako Ishii, Satoshi Mizuno1998 年 123 巻 2 号 p. 226-232
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーExpression of a luciferase reporter gene by Chinese hamster ovary cells under the control of the human heat shock protein (hsp) 70 gene promoter was suppressed by incubation at 37°C after treatment with cycloheximide (CHX) during 42°C heat shock exposure. The CHX-induced suppression of hsp gene expression induced no development of thermotolerance. However, 42°C heat shock treatment without CHX followed by CHX inhibition of protein synthesis during recovery incubation at 37°C induced thermotolerance expression by inducing enhanced synthesis of hsps including hsp70 in subsequent heat challenge incubation at 43°C. The results demonstrated a direct correlation between suppression (induction) of stress protein gene expression and non-expression (expression) of thermotolerance. Kinetic analysis showed that the CHX suppression of hsp gene induction was greater than the CHX inhibition of protein synthesis, and that it depended on the severity of heat stress: it decreased with increasing heat stress doses. Moreover, prior feeding of the proline analog L-azetidine 2-carboxylic acid abrogated the CHX-induced suppression of hsp gene expression. In addition, CHX treatment during heat cell-killing at 43°C induced protection of cells. These results were well explained by the proposed model of negative or positive control of stress protein gene expression depending on the level of free hsp70, which may be modulated by both the rate of protein synthesis and the severity of heat stress.抄録全体を表示PDF形式でダウンロード (3496K) -
F. J. Alvarez, J. A. Jordán, A. Herráez, J. C. Dí ...1998 年 123 巻 2 号 p. 233-239
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーPrevious work has shown increased uptake of hypotonically loaded rat RBCs by the spleen and liver “in vivo, ” suggesting that the cells of MPS are involved in their elimination from the circulation. In order to elucidate the mechanism of such elimination, we have undertaken studies on the interaction of such loaded RBCs, in comparison with native RBCs, with peritoneal macrophages. Erythrophagocytosis assays were performed in well plates to which thioglycollate-induced peritoneal macrophages had adhered. Native or loaded 51Cr-RBCs were added under different opsonization conditions to monolayer adherent macrophages, and then the amount of RBCs that were recognized was determined, with separation into adhesion and phagocytosis fractions. Native RBCs are slightly recognized by peritoneal macrophages, about one RBC per macrophage (MΦ). Osmotic treatment of rat RBCs used for encapsulation (independently of the encapsulated substance, 125I-CA or FITC-dextran) produces some modification in the erythrocyte membrane that induces higher recognition of these cells, about three loaded RBCs per macrophage. Consequently, both fluorescent (FITC-Dx) and radioactive (125I-CA) substances previously encapsulated in RBCs were transferred to MΦs. The fluorescence microscopic observations confirmed these results. Moreover, in the case of carrier 51Cr-cells loaded with 125I-CA, the amount of 125I-radioactivity delivered into MΦs was relatively higher than that of 51Cr. The highest ratio, 125I-CA (encapsulated substance)/51Cr-RBCs (carrier cells), present in Mcs means there was a stronger interaction with macrophages of RBCs that carry a higher amount of encapsulated CA, as a function of the heterogeneity of the loaded rat RBCs population previously reported. Finally, the adhesion and phagocytosis of loaded RBCs seem not to involve complement receptors or Fe receptors on the macrophages.抄録全体を表示PDF形式でダウンロード (1917K) -
Yutaka Kariya, Jörg Herrmann, Kiyoshi Suzuki, Takako Isomura, Mas ...1998 年 123 巻 2 号 p. 240-246
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーCompositional analyses of heparin (Hep) and heparan sulfate (HS) have been undertaken with disaccharide units obtained by either enzymatic digestion with heparitinases or hydrazinolysis/deamination reaction of polysaccharides. Unsaturated disaccharide units generated by the enzymatic method are detectable on HPLC with a uv detector recording absorbance at 230 nm. On the other hand, disaccharide units generated by the chemical method possess a component of 2, 5-anhydromannose (AnMan) bearing aldehyde groups in addition to intact iduronic acid (IdoA) or glucuronic acid (GIcA). The aldehyde groups of the disaccharide units are usually reduced with sodium borotritide, and detected by radiochromatography. Both of them, however, involve inevitable experimental problems, such as the use of costly enzymes and radioisotopes. In the present study, we have established a novel composition analysis system for Hep and HS essentially based on the chemical method. After hydrazinolysis/deamination treatment of Hep and HS, the aldehyde groups of AnMan in the disaccharide units generated were coupled with paranitrophenyl (PNP-) hydrazine instead of reduction with sodium borotritide, AnMan-CH=N-NH-PNP (AnMan-PNP) being formed. Then, the PNP-labeled disaccharides were pre-treated on a SepPak C-18 cartridge column, and subsequently separated and detected on ion-pairing reversed-phase HPLC with a detector recording absorbance at 390 nm. With the present system, the order of elution was G1cA-AnMan-PNP (GM), IdoA-AnMan-PNP (IM), IdoA (2S)-AnMan-PNP (ISM), IdoA-AnMan (6S)-PNP (IMS), and IdoA (2S)-AnMan (6S)-PNP (ISMS). As an application, the disaccharide compositions of heparin from bovine intestine and heparan sulfate from bovine kidney were analyzed by the present method, and the results were comparable to those obtained by a well-established enzymatic method. The present compositional analysis was demonstrated to be reliable and economical.抄録全体を表示PDF形式でダウンロード (791K) -
Motohiro Nishimura, Hiroaki Matsuo, Akiko Nakamura, Masanori Sugiyama1998 年 123 巻 2 号 p. 247-252
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーBlasticidin S-producing Streptomyces morookaensis JCM 4673 produces an enzyme which inactivates puromycin (PM) by hydrolyzing an amide linkage between its aminonucleoside and O-methyl-L-tyrosine moieties [Nishimura et al. (1995) FEMS Microbiol. Lett. 132, 95-100]. In this study, we purified to homogeneity the enzyme from the cell-free extracts of S. morookaensis. The molecular weight of PM-hydrolyzing enzyme, estimated by SDS-PAGE and gel filtration, was 68 and 66 kDa, respectively, suggesting that this protein is monomeric. The PM-hydrolyzing activity was strongly inhibited by Zn2+, Fe2+, Cu2+, Hg2+, and N-bromosuccinimide, but was stimulated by DTT. The optimum pH and temperature for PM-hydrolyzing activity were 8.0 and 45°C, respectively. Several L-aminoacyl-β-naph-thylamides were good substrates for the enzyme, suggesting that the PM-inactivating enzyme has an aminopeptidase activity. The N-terminal sequence of the first 14 amino acids (Val-Ser-Thr-Ala-Pro-Tyr-Gly-Ala-Trp-Gln-Ser-Pro-Ile-Asp) of the enzyme showed no significant homology with any published hydrolase sequences.抄録全体を表示PDF形式でダウンロード (1517K) -
Tomihiko Uemura, Kumiko Hirai1998 年 123 巻 2 号 p. 253-262
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーWe have purified L-kynurenine 3-monooxygenase from pig liver mitochondria using a procedure involving seven steps composed of (1) preparation of mitochondrial outer membrane, (2) preparation of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio] -1-propane sulfonate (Chaps)-insoluble outer membrane material, (3) extraction of the enzyme with β-octylglucoside, (4) ammonium sulfate fractionation, (5) DEAE-Sepharose CL-6B chromatography, (6) Matrex gel orange A affinity chromatography, and (7) high-performance liquid chromatography (HPLC) gel filtration. The final preparation had an about 160-fold purified enzyme activity with a yield of 0.8%. The apparent molecular mass of the aggregated form of the native enzyme was determined to be close to 300 kDa by HPLC gel filtration in the presence of 0.005% Triton X-100. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) showed a main protein band with an apparent molecular mass of about 49 kDa. The enzyme was found to be about 86% pure by the criterion of SDS-PAGE. The dissociated form of the enzyme contains 1 mol of non-covalently bound FAD/mol of protein monomer. The UV/visible spectrum had absorption peaks at 275, 384, and 450 nm, typical of a simple flavoprotein. Five inhibitory monoclonal antibodies against the enzyme were obtained. They could stain moderately a single protein band (49 kDa) in a Western blot.抄録全体を表示PDF形式でダウンロード (5127K) -
Nobuo Hisano, Yutaka Yatomi, Masayuki A. Fujino, Yasuyuki Igarashi, Sh ...1998 年 123 巻 2 号 p. 263-268
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーTo elucidate the physiologic role of sphingolipid-derived products as signaling molecules, we analyzed the levels of endogenous sphingosine (Sph) derivatives in human platelets. When the platelets were stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate, neither ceramide formation nor sphingomyelin hydrolysis was observed, which suggests that the sphingomyelin cycle may not be an essential part of the signaling pathway under these conditions. In contrast, Sph was found to increase in platelets upon stimulation. The level of Sph 1-phosphate, which is formed from Sph by the action of Sph kinase, was not affected under our conditions. Although it has been established that Sph inhibits protein kinase C, which regulates the functional responses of the platelets, Sph levels which exert an inhibitory effect on protein kinase C cannot be attained under physiological conditions (without exogenous Sph). Considering the stimulation of the synthesis of Sph by the physiological agonist thrombin, we speculate that Sph is a signaling molecule of physiological importance in platelets, but protein kinase C may not be its target.抄録全体を表示PDF形式でダウンロード (2652K) -
C. Fini, M. Coli, A. Floridi, S. D'Auria, M. Staiano, R. Nucci, M. Ros ...1998 年 123 巻 2 号 p. 269-274
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe effects of temperature on the three-dimensional organization and on the secondary structure of GPI-anchored 5'-nucleotidase from bull seminal plasma and of its anchor-less form (solubilized ecto-5'-nucleotidase), obtained after GPI anchor removal by phosphatidylinositol-specific phospholipase C were investigated in parallel by circular dichroism and fluorescence spectroscopy. The structural features of the two enzymes were correlated to their functional properties in the temperature range of 25-90°C. The kinetic data indicated that the enzyme activities were temperature dependent, showing the maximal values at 60°C. The relevant Arrhenius plots were linear in the temperature range of 20-60°C and the activation energies were 44.4 and 51.8 kJ/mol for the solubilized and GPI-anchored 5'-nucleotidase, respectively. The time-course measurements of enzyme activity, in the temperature range of 25-55°C, revealed that the two enzymes were of different thermal stability, the solubilized ectoenzyme showing lower thermal deactivation constants and longer half lives. Fluorescence and near UV circular dichroism spectroscopy showed that temperature increases induced remarkable changes in the protein tertiary structure of the two enzymes, whereas far-UV circular dichroism analysis revealed only a small temperature effect on the protein secondary structure content.抄録全体を表示PDF形式でダウンロード (648K) -
Akiko Ibuka, Takashi Tonozuka, Hiroshi Matsuzawa, Hiroshi Sakai1998 年 123 巻 2 号 p. 275-282
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーTVA I, an α-amylase from Thermoactinomyces vulgaris R-47, is a versatile enzyme which hydrolyzes the α-(1→4)-glucosidic linkages of pullulan to produce panose, known as neopullulanase activity, and the α-(1→6)-glucosidic linkages of certain oligosaccharides. We modified the Ala-357, Gln-359, and Tyr-360 residues located in region II, one of the four regions conserved in a-amylase family enzymes, and deleted 11 consecutive amino acid residues located after the C-terminus of region II of the TVA I sequence by means of site-directed mutagenesis. The action pattern of the mutated enzyme for pullulan was greatly altered and it hydrolyzed mainly the α-(1→6)-glucosidic linkages of pullulan to produce maltotriose, while the action patterns for starch and maltooligosaccharides were almost identical to those of the wild-type enzyme. This means that the mutated TVA I has lost the neopullulanase activity, and thus can be designated as an amylopullulanase-typnzyme. The kcat/Km value of the mutated enzyme for α-(1→)-glucosidic linkages was virtually unaltered, while that for α-(1→4)-glucosidic linkages was about 100 times smaller than that of the wild-type enzyme.抄録全体を表示PDF形式でダウンロード (5519K) -
Miki Watanabe, Hiromi Tsuda, Shuhei Yamada, Yukinaga Shibata, Takeshi ...1998 年 123 巻 2 号 p. 283-288
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーHeparinase was purified to homogeneity from the cell extract of an oral bacterium, Prevotella heparinolytica, by a combination of anion exchange chromatography, gel filtration chromatography, and hydroxyapatite chromatography. Properties of the purified P. heparinolytica heparinase (P. heparinase) were investigated. The enzyme exhibited a maximum activity in 50mM Tris-HCl buffer, pH 7.5-8.0, containing 75mM sodium acetate, 0.1M NaCl, and 1mM CaCl2. Optimum conditions for the maximum activity of P. heparin-ase were similar to those of the heparinase from Flavobacterium heparinum (F. heparinase). The two enzymes also yielded similar digestion profiles of various glycosaminoglycans and heparin tetrasaccharides, suggesting that they have a similar substrate specificity. Kinetic study of the P. heparinase reaction using porcine intestinal heparin as substrate gave a Km, value of 3.8×10-5M and a Vmas value of 11.4 μmol/min•mg protein. The Michaelis constant of P. heparinase was slightly larger than but not significantly different from that of F. heparinase. The amino acid composition of P. heparinase was also similar to that of F. heparinase, but its N-terminal sequence of 20 amino acid residues was different and hitherto unreported. These results together indicate that these heparinases are different proteins with closely similar enzymatic properties. Since F. heparinum produces not only heparinase but also heparitinase II, which has a broad substrate specificity, F. heparinase may be contaminated with this enzyme. In contrast, P. heparinolytica does not produce heparitinase II, and P. heparinase should prove a useful tool for degrading heparin without the risk of contamination with heparitinase II.抄録全体を表示PDF形式でダウンロード (633K) -
Akiyoshi Tanaka1998 年 123 巻 2 号 p. 289-293
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThermal unfolding of Pseudomonas cepacia lipase (PCL) was studied by differential scanning calorimetry (DSC) at pH 7. The peak temperature tp of the DSC trace increased with increasing concentration of the protein. The DSC traces could be successfully analyzed on the basis of the following mechanism, assuming the dissociation of a calcium ion upon denaturation; N Ca2+_??_D+Ca2+, where N and D represent native and denatured states of PCL, respectively. In the presence of 1-5% alcohols (methanol, ethanol, n-propanol, and n-butanol), tp decreased with increasing alcohol concentration and longer alkyl chain. In contrast to the case of tp, the denaturation enthalpy dh did not depend on the protein concentration or alcohol concentration used. The change in heat capacity on denaturation, Δcpd, evaluated directly from the DSC traces, was close to zero both in the absence or presence of alcohol, which could be due to the open conformation of the enzyme exposing a large hydrophobic surface to the solvent.抄録全体を表示PDF形式でダウンロード (469K) -
Kikuo Ogata, Rie Ohno, Kazuo Terao, Kentaro Iwasaki, Yaeta Endo1998 年 123 巻 2 号 p. 294-304
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe ATPase activity of rat liver 30S-5SRNP particles prepared by EDTA treatment of 80 S ribosomes, and that of 40S subunits were investigated in correlation with polypeptide elongation. The ATPase activity of 30S-5SRNP particles was higher than that of 40S subunits. Poly(U) and TMV RNA stimulated the ATPase activity of 30S-5SRNP particles more markedly than that of 40S subunits. These two kinds of particles also showed intrinsic GTPase. Poly (U) enhanced the GTPase activity of 30S-5SRNP particles but not that of 40S subunits. An elongation factor (EF-1α, EF-2, or EF-1αβγ) alone or in combination with poly (U) and/or other elongation factors stimulated the ATPase activities of both particles. The extent of stimulation of the ATPase activity by a combination of these components was usually somewhat higher than or similar to the sum of those with the individual components. The extents of stimulation by these components were higher in the case of 30S-5SRNP particles than that of 40S subunits, indicating the importance of the 5 SRNP moiety in the former particles. The intactness of 18 SrRNA was required for promotion of the ATPase activity of 30S-5SRNP particles by phe (+), (-) tRNAphe. The ATPase activities of the two kinds of particles by themselves or those observed with the combinations of the components mentioned above were inhibited by several kinds of translation inhibitors. The degrees of inhibition were generally higher for 30S-5SRNP particles. The ATPase activity of 40S subunits was enhanced by spermidine, suggesting the importance of the conformational change induced by it. These results imply the participation of the intrinsic ATPase of 30S-5SRNP particles and 40S subunits in polypeptide elongation, and the important role of the 5 SRNP moiety of 30S-5SRNP particles in the ATPase activity.抄録全体を表示PDF形式でダウンロード (1084K) -
Naoko Abe, Tomoko Kadowaki, Kuniaki Okamoto, Koji Nakayama, Masamichi ...1998 年 123 巻 2 号 p. 305-312
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed “Lys-gingipain, ” was purified from the culture supernatant of the Arggingipain-deficient mutant of P. gingivalis (KDP 112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of Mr=51, 000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and α1-anti-chymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.抄録全体を表示PDF形式でダウンロード (3032K) -
Yoshito Abe, Tadashi Ueda, Keiichi Kawano, Yoshitsugu Tanaka, Taiji Im ...1998 年 123 巻 2 号 p. 313-317
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーUsing hen lysozyme in which the ε-carbons of two methionine residues are enriched with 13C nuclei, we found that there is a subtle difference in the chemical shift of the ε-carbon resonances between Met 12 and Met 105 in thermally denatured lysozyme without any reduction of disulfide bonds at pD 3.8, and also in reduced S-alkylated lysozyme at pD 3.8 and 35°C. The difference in the chemical shift was abolished on digestion with TPCK-trypsin and the chemical shifts of both resonances converged to that of Met 12, whose chemical shift is identical to that in the randomly coiled state. Therefore, it is suggested that the chemical shift in the ε-carbon resonance of Met 105 is different from that in the randomly coiled state due to an interaction involving Met 105. In order to locate the interaction involving Met 105, fragmentation of the reduced S-alkylated lysozyme into the peptides was carried out by means of chemical cleavage or specific endoprotease digestion. As a result, the local interaction of Met 105 or the residues around Met 105 with eleven residues at the C-terminus of lysozyme is suggested to occur.抄録全体を表示PDF形式でダウンロード (3298K) -
Jason T. C. Tzen, Rachel L. C. Chuang, Jeff C. F. Chen, Lawrence S. H. ...1998 年 123 巻 2 号 p. 318-323
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosin isoforms with molecular masses of 18 and 16 kDa are present in rice oil bodies. Chicken antibodies raised against oleosin 18 kDa and rabbit antibodies raised against oleosin 16 kDa did not cross-recognize these two homologous isoforms. This peculiar non-cross recognition was used to locate the two oleosin isoforms on the surface of oil bodies via immunofluorescence detection using anti-chicken IgG conjugated with FITC (fluorescein isothiocyanate) and anti-rabbit IgG conjugated with Texas-Red. The results revealed that both oleosin isoforms resided on each oil body in vivo and in vitro. Artificial oil bodies were reconstituted via sonication using triacylglycerol, phospholipid, and oleosins. The results indicated that the two rice oleosin isoforms could stabilize artificial oil bodies individually whereas oleosin 16 kDa provided better stability to the organelles than oleosin 18 kDa.抄録全体を表示PDF形式でダウンロード (4057K) -
Masao Miki, Tomoyoshi Kobayashi, Hiroyuki Kimura, Akihiko Hagiwara, Ho ...1998 年 123 巻 2 号 p. 324-331
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーFluorescence resonance energy transfer spectroscopy has been used to study the spatial relationships between probes attached to actin and troponin in the reconstituted skeletal muscle thin filament in the presence and absence of Ca2+ ions. Gln-41 and the nucleotidebinding site of actin were selectively labeled with the acceptor probe: fluorescein cadaverine and 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine 5'-diphosphate (TNP-ADP), respectively. Troponin was selectively labeled at positions 9 or 133 of troponin-I and 98 of troponin-C with a donor probe; 5-(2-iodoacetylaminoethyl) aminonaphthalene 1-sulfonic acid (IAEDANS). The distances between probes attached to position 133 of TnI and Gln-41 or the nucleotide site of actin were determined to be 51.6±1.2 and 42.7±0.9 Å respectively in the presence of Ca2+, and these distances decreased by 11.5 and 9.3 Å respectively in the absence of Ca2+ ions. The distances between the probes attached to position 9 of TnI and Gln-41 or the nucleotide site of actin were determined to be 59.1±2.0 or 49.3±1.5 Å respectively in the presence of Ca2+, and the distances decreased by 5.3 or 3.7 Å in the absence of Ca2+. The distances between probes attached to position 98 of TnC and Gln-41 or the nucleotide site of actin were determined to be 55.1±1.7 and 57±5 Å in the presence of Ca2+ and the distances increased slightly by _??_1 Å in the absence of Ca2+. The results suggest that the C-terminal domain of troponin I moves to the outer domain of actin during inhibition, while the C-terminal domain of TnC does not move much.抄録全体を表示PDF形式でダウンロード (1980K) -
Shih-Ching Ho, Shuji Wakatsuki, Manabu Arioka, Makari Yamasaki, Katsuh ...1998 年 123 巻 2 号 p. 332-338
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーTo identify and analyze the function of proteins expressed in the growth cones, we have screened monoclonal antibodies raised against the preparation of the growth cone particles derived from fetal porcine brains and found a novel neuronal antigen, termed NP-190. Biochemical characterization of NP-190 demonstrated that it was an integral membrane protein with an apparent molecular weight of 190 kDa and that it was mainly expressed in fetal brains. Homologous antigens with molecular weights of 200 and 170 kDa were also identified in the fetal brain extracts of chickens and rats, respectively. Immunoblot experiments of brain extracts from chickens and rats in various stages of development indicated that the expression of NP-190 homologs was developmentally regulated; it began to appear and increased in the embryonic stage, then decreased to very low level in the adult brains. Immunostaining of cultured primary of neurons from the embryonic day 18 rat cerebral cortex demonstrated that rat NP-190 homolog localized in the cell bodies, axons and growth cones, but not in dendrites. Partial amino acid sequence analysis of affinity-purified NP-190 from fetal porcine brains demonstrated that it was a novel protein. These results suggest that NP-190 plays a distinct role in brain development.抄録全体を表示PDF形式でダウンロード (4318K) -
Chika Yokota, Shuji Takahashi, Akira Eisaki, Makoto Asashima, Shamima ...1998 年 123 巻 2 号 p. 339-346
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーMidkine (MK) is a heparin-binding growth factor that has been implicated in neural survival and differentiation, fibrinolysis, and carcinogenesis. It is expressed in the nervous system during early Xenopus development. In the present study, we demonstrated that injection of vegetal blastomeres with Xenopus MK at the 8-cell stage results in incomplete invagination. In the case of dorsal vegetal injection, hypertrophic neural tissue is produced. Animal caps isolated from embryos that have been injected with Xenopus MK and cultured with activin do not elongate, and all mesoderm markers examined, including both head and trunk/tail ones, are greatly diminished. In contrast, head-specific neural markers, XANF-1 and Xotx 2, are induced, while trunk/tail neural markers, XlHbox 6 and F-spondin, are decreased. Moreover, MK showes the same effects in animal caps injected with Xenopus Smad 2 mRNA.抄録全体を表示PDF形式でダウンロード (4817K) -
Masanori Bun-ya, Motohiro Maebuchi, Tatsuyuki Kamiryo, Takao Kurosawa, ...1998 年 123 巻 2 号 p. 347-352
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oic acid and 3α, 7α-dihydroxy-5β-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydro-genase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3α, 7α, 12α-trihydroxy-5β-cholest-24-en-26-oyl-CoA and 3α, 7α-dihydroxy-5β-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.抄録全体を表示PDF形式でダウンロード (567K) -
Keiichiro Suzuki, Young Ho Koh, Hisako Mizuno, Rieko Hamaoka, Naoyuki ...1998 年 123 巻 2 号 p. 353-357
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーThe glycation reaction (Maillard reaction) plays a major role in diabetic complications, since some reaction intermediates are responsible for the modification and cross-linking of long-lived proteins, resulting, in turn, in a deterioration of normal cell function. The reaction intermediates include methylglyoxal (MG) and 3-deoxyglucosone (3-DG), both of which are cytotoxic dicarbonyl compounds and are elevated during hyperglycemia. Aldehyde reductase (ALR) catalyzes the reduction of both compounds. To examine the intracellular role of ALR in the diabetic complications of neural cells, its gene was overexpressed in rat pheochromocytoma PC 12 cells, which normally express a low level of ALR. Western blot analysis showed that ALR protein in the ALR gene-transfected cells was more than twice as much as in the control cells. In the parental cells, cytotoxicity, including apoptotic cell death, which was determined by fluorescent microscopy using the fluorescent DNA binding dye Hoechst 33258, was observed at 100 μM MG. In the ALR gene-transfected cells, the cytotoxicity of both MG and 3-DG and apoptotic cell death were decreased. This suggests that intracellular ALR protects neural cells from the cytotoxicity of 3-DG or MG, and that neural cells, which normally express a low level of ALR, might be susceptible to diabetic complications caused by intermediate products of the Maillard reaction, such as 3-DG and MG.抄録全体を表示PDF形式でダウンロード (2347K) -
Yumi Yamashita, Daisuke Fukuta, Atsushi Tsuji, Akira Nagabukuro, Yoich ...1998 年 123 巻 2 号 p. 358-368
発行日: 1998年
公開日: 2008/11/18
ジャーナル フリーRecently, we found a novel murine cell-surface glycoprotein, designated as p 91, expressed mainly in myeloid cells such as macrophages and mast cells. The molecule has six immunoglobulin-like extracellular domains, a transmembrane segment, and a cytoplasmic tail containing four immunoreceptor tyrosine-based inhibition motif (ITIM) or ITIM-like sequences, resembling the structural features of human killer-cell inhibitory receptors (KIR). Here we show that p 91 comprises a polymorphic gene family, harboring one potent inhibitory-type p 91 and at least two other p 91 genes. Tyrosine-phosphorylated, but not nonphosphorylated, synthetic peptides matching the third ITIM and the fourth ITIM-like sequences, respectively, found in the cytoplasmic portion of p91A, the sole inhibitory-type p 91, were associated with the tyrosine phosphatases, SHP-1 and SHP-2. In addition, the phosphotyrosyl peptide matching the third ITIM sequence also bound the inositol 5-phosphatase, SHIP. These results support the notion that p91A may function as an inhibitory cell-surface molecule against cell activation. The p91 genes were shown to be clustered in the proximal region of mouse chromosome 7, a syntenic position of human chromosome 19 where the genes for the KIR family are found. A human cDNA clone cross-hybridizing to a murine p 91 probe was isolated from a human spleen cDNA library, and was found to code for a molecule quite similar to members of the immunoglobulin-like transcript (or ILT) family. The gene was found to be located on human chromosome 19q 13.3-13.4. These results establish the existence of a novel set of potent regulatory receptors in mouse and man, similar but different from the KIR family.抄録全体を表示PDF形式でダウンロード (5316K)
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