巻号一覧
126 巻, 6 号
選択された号の論文の26件中1～26を表示しています
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Tatsuro Irimura, Kaori Denda, Shin-ichiro Iida, Hideyuki Takeuchi, Ken ...1999 年 126 巻 6 号 p. 975-985
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーMucins are major epithelial luminal surface proteins and function as a physical and biological barrier protecting mucous epithelia. Diverse glycosylation of mucins potentially provides a basis for tissue-specific interaction with the milieu. When mucins are associated with malignant epithelial cells, they not only protect these cells from a host environment during metastatic dissemination but also generate immunogenic epitopes which are used by the host in the detection and immunological elimination of carcinoma cells potentially depending upon their status of glycosylation. Diverse mucin structures are generated by the combination of different core peptides, of which 10 have been reported so far, multiple types of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts), and the consequences of stepwise glycosylation events. For example, the mucin 1 (MUC1) associated with malignant cells was previously believed to exhibit unique features with a lower percentage of threonine and serine residues attached to N-acetylgalactosamine and/or without extension through core 2 structures. Some of MUCI-specific monoclonal antibodies and cytotoxic lymphocytes recognize the peptide sequences PDTR within the tandem repeat portion exposed by decreased degree of glycosylation. The specific arrangement of N-acetylgalactosamine residues is shown to be generated by a combination of pp-GalNAc-Ts with different specificities. The role of core 2 branching is somewhat confusing because well-known carcinoma-associated carbohydrate epitopes such as sialyl-Leλ, sialyl-Lea, LeY, and others are often expressed when O-glycans are extended through core 2 branching. The other series of well-known carcinoma-associated carbohydrate structures are truncated O-glycans, conventionally called Tn and sialyl-Tn antigens. Interestingly, these are often found to be aligned on core polypeptides, resulting in three or more consecutive truncated O-glycans. MUC2 and other mucins, but not MUCI, have unique tandem repeats containing three or more consecutive serine or threonine residues, which potentially serve as a scaffold for trimeric Tn and sialyl-Tn epitopes. We recently found, using the MUC2 tandem repeat, that trimeric To is a high-affinity receptor for a calciumtype lectin expressed on the surface of histiocytic macrophages. The biosynthesis of trimeric Tn was strictly regulated by the acceptor specificity of pp-GaINAc-Ts. These results strongly suggest that variation in both glycan structures and distribution of glycans on the core polypeptides give mucins unique and diverse biological functions that play essential roles in carcinoma-host and other cellular interactions.抄録全体を表示PDF形式でダウンロード (942K) -
Noriko Nakagawa, Mitsuaki Sugahara, Ryoji Masui, Ryuichi Kato, Keiichi ...1999 年 126 巻 6 号 p. 986-990
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn the nucleotide excision repair system, UvrB plays a central role in damage recognition and DNA incision by interacting with UvrA and UvrC. We have determined the crystal structure of Thermus thermophilus HB8 UvrB at 1.9 Å resolution. UvrB comprises four domains, two of which have an α/β structure resembling the core domains of DNA and RNA helicases. Additionally, UvrB has an α-helical domain and a domain consisting of antiparallel β-sheets (β-domain). The sequence similarity suggests that the β-domain interacts with UvrA. Based on the distribution of the conserved regions and the structure of the PcrA-DNA complex, a model for the UvrB-DNA complex is proposed.抄録全体を表示PDF形式でダウンロード (1786K) -
Isamu Kameshita, Shigeru Taketani, Atsuhiko Ishida, Hitoshi Fujisawa1999 年 126 巻 6 号 p. 991-995
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA novel peptide with multiple phosphorylation sites, which we designated as multide, was developed to detect a wide variety of protein kinases in crude cell extracts. Multide, KKRKSSLRRWSPLTPRQMSFDC, has been designed to contain consensus sequences for various Ser/Thr protein kinases including cAMP-dependent protein kinase, protein kinase C, MAP kinases, and Ca2+/calmodulin-dependent protein kinases in a single peptide. In-gel protein kinase assay using multide was found to be very useful for analyzing the activities of protein kinases that are altered in response to various extracellular stimuli. The substrate specificities of the protein kinases thus detected were further determined by using five multide analogs with different phosphorylation sites.抄録全体を表示PDF形式でダウンロード (1031K) -
Sung-Joon Park, Young-Dong Cho1999 年 126 巻 6 号 p. 996-1003
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) eDNA (GenBankTM U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The Km value for S-adenosylmethionine (AdoMet) is 23.1 μpM and the K1 value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 μM. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys50, Cys83, and Cys230) and lysine81 residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys50 and Cys230 to alanine had minimal effects on the catalytic activity. Changing Lys81 to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The Km value for AdoMet is 11-fold higher than that of the wild type, but the Vmax value is more than 60%. Taken together, the results suggest that the lysine81 residue is critical for substrate binding.抄録全体を表示PDF形式でダウンロード (1969K) -
Haruna Naito, Atsushi Ikeda, Keiji Hasegawa, Shogo Oka, Kazuhide Uemur ...1999 年 126 巻 6 号 p. 1004-1012
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーSerum mannan binding protein (MBP), a mannose/N-acetylglusosamine-specific lectin, is important in innate immunity. In order to elucidate the mechanism underlying the wide intra- and interracial variety in the MBP serum level, we have studied the transcriptional regulation of human MBP. Rapid amplification of cDNA ends (5' RACE) analysis of Hep G2 RNA indicated the presence of a novel exon, designated as “exon O, ” upstream of previously identified exon 1 [Taylor, M. E. et al. (1989) Biochem. J. 262, 763-771]. Two MBP mRNAs with different sized 5'-noncoding regions were detected: the longer transcript starts at exon O and the shorter one at exon 1. Promoter analysis involving a luciferase assay vector revealed that the transcript starting from exon 1 predominates over that starting from exon O. In addition, a hepatocyto-specific nuclear factor, (HNF)-3, which is known to control the expression of hepatocyto-specific genes, up-regulates the transcription of human MBP from exon 1, while a glucocorticoid, which is known to up-regulate acute phase proteins, markedly suppresses MBP transcription. Recently, polymorphisms were found to occur in the promoter region at two positions [Madsen, H. O. et al. (1995) J. Immunol. 155, 3013-3020]. Functional promoter analysis indicated that three haplotype variants as to these positions, HY, LY, and LX, exhibit high, medium and low promoter activity, respectively, in accordance with the results of a previous population study.抄録全体を表示PDF形式でダウンロード (2213K) -
Junji Yamada, Akihiro Kurata, Michiko Hirata, Tomomi Taniguchi, Hirota ...1999 年 126 巻 6 号 p. 1013-1019
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーAn acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e. g. a maximal velocity of 295 μmol/min/mg and Km of 6.4 μM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C8-18 were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The eDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH eDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.抄録全体を表示PDF形式でダウンロード (2095K) -
Fumie Shiraishi, Sachio Morimoto, Kiyoyoshi Nishita, Takao Ojima, Iwao ...1999 年 126 巻 6 号 p. 1020-1024
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn order to examine the involvement of troponin-linked Ca2+-regulation, in addition to well-known myosin-linked Ca2+-regulation, in the contraction of molluscan striated muscle, myofibrils from Ezo-giant scallop striated muscle were desensitized to Ca2+ by removing both myosin regulatory light chain and troponin C by treatment with a strong divalent cation chelator, CDTA. The ATPase level in the desensitized myofibrils was about half the maximum level in intact myofibrils regardless of the Ca2+-concentration at 25 and 15 C. In the absence of Ca2+, the ATPase of the desensitized myofibrils was suppressed by myosin regulatory light chain but not affected by troponin C at either temperature. The ATPase was activated at higher Ca2+-concentrations by both myosin regulatory light chain and troponin C, but the activating effects of these two proteins were affected differently by temperature. The activation of ATPase by myosin regulatory light chain was much greater than that by troponin C at 25_??_C, whereas the activation by troponin C was much greater than that by myosin regulatory light chain at 15 C. The maximum activation was only obtained in the presence of both myosin regulatory light chain and troponin C at these temperatures. These findings strongly suggest that the contraction of scallop striated muscle is regulated through both myosin-linked and troponin-linked Ca2+-regulation, and that the troponinlinked Ca2+-regulation is more significant at lower temperature.抄録全体を表示PDF形式でダウンロード (1719K) -
Hong Ro Kim, Kwang Il Kang, Ho Sung Kang, Han Do Kim1999 年 126 巻 6 号 p. 1025-1032
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn eukaryotic cells, HSP 90 is associated with several protein kinases and regulates their activities. HSP 90 was also reported to possess an autophosphorylase activity. In this study, we examined in vitro autophosphorylation of HSP 90, which was purified from chick muscle. We show that HSP 90 was not phosphorylated in vitro, but an 84-kDa protein (p 84) was highly phosphorylated. P 84 was neither HSP 90 nor its degradative product, as it was not detected by an antibody (BF 4) specific to HSP 90 in denaturing immunoprecipitation and Western blot analysis. Phosphorylation of a protein similar to p 84 was also detected with purified human brain and HeLa HSP 90, indicating that p 84 is present in many different types of cells. P 84 appeared to exist as large complexes, as determined by HPLC and native gel electrophoresis. Native immunoprecipitation using anti-HSP90 (BF 4)-conjugated Affigel revealed that this phosphoprotein is specifically associated with HSP 90. The interaction of p 84 and HSP 90 was not affected by p 84 phosphorylation. In addition, p 84 phosphorylation was prevented by the presence of divalent cations such as Mg2+ and Mn2+. In contrast, p 84 phosphorylation was significantly activated by addition of exogenous Ca2+- between 100 and 500 μM, although it was blocked by higher concentrations (>1mM) of Ca2+. HSP 90, but not p 84, could be phosphorylated by casein kinase II. Finally, p 84 phosphorylation was specifically prevented by heroin, but not by other kinase inhibitors, indicating that p 84 phosphorylation may be regulated by heme-regulated protein kinase.抄録全体を表示PDF形式でダウンロード (3605K) -
Midori Shimada, Masashi Saito, Tomoya Katakai, Akira Shimizu, Tohru Ic ...1999 年 126 巻 6 号 p. 1033-1042
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe previously characterized p144 bearing N-acetylglucosamine residues in a rat liver nuclear matrix fraction. Based on partial amino acid sequences of rat p144, mouse p144 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence revealed that p144 is a rat homologue of CA150, which is a transcription factor involved in Tat-activated human immunodeficiency virus type 1 transcription. The reported human CA150 consists of 1098 amino acids and has a leucine zipper-like motif in its carboxylregion. However, a clone of mouse p144 cDNA encoded a CA150 consisting of 1, 034 amino acids. The mouse CA150 was shorter by 64 amino acids than hitherto known human CA150 and lacked the leucine zipper-like motif. We designated the longer and shorter CA150 species as CA150a and CA150b, respectively. The partial nucleotide sequences of other mouse p144 cDNA clones were examined and it was found that some clones encode CA150a having a leucine zipper like motif. It was suggested that CA150a and CA150b are splicing isoforms. All rat and mouse tissues examined contained transcripts for both CA150a and CA150b. Both transcripts were detected in human blood and Jurkat cells as well as mouse CD4+ T-cells, which are the HIV-1-sensitive counterpart in humans.抄録全体を表示PDF形式でダウンロード (3401K) -
Nobuo Fuse, Ken-ichi Yasumoto, Kazuhisa Takeda, Shintaro Amae, Miki Yo ...1999 年 126 巻 6 号 p. 1043-1051
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーMicrophthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein, and plays an important role in the development of various cell types, such as neural-crest-derived melanocytes and optic-cup-derived retinal pigment epithelium. Three isoforms of MITF with distinct amino-termini have been described. These include melanocyte lineage-specific MITF-M, heart-type MITF-H, and the recently identified MITF-A. Here we identify a fourth isoform, MITF-C, with a unique amino-terminus of 34 amino acid residues, which shares about 43% sequence identity with putative transactivation segments of two previously identified leukemogenic factors, ENL and AF-9. Reverse transcription-polymerase chain reaction analysis revealed that MITF-C mRNA is expressed in many cell types, including retinal pigment epithelium, but is undetectable in melanocyte-lineage cells. In contrast, MITF-A and MITF-H mRNAs are coexpressed in all cell types examined. Transient cotransfection assays suggested that MITF-C, like other MITF isoforms, functions as a transcriptional activator of certain target genes, but its transactivation specificity for the target promoters is different from those of other MITF isoforms. Therefore, isoform multiplicity provides MITF with differential expression patterns as well as functional diversity.抄録全体を表示PDF形式でダウンロード (3314K) -
Eriko Michishita, Kazuhiko Nakabayashi, Toshikazu Suzuki, Sunil C. Kau ...1999 年 126 巻 6 号 p. 1052-1059
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリー5-Bromodeoxyuridine was found to induce flat and enlarged cell shape, characteristics of senescent cells, and senescence-associated β-galactosidase in mammalian cells regardless of cell type or species. In immortal human cells, fibronectin, collagenase I, and p21wafl sdi-1 mRNAs were immediately and very strongly induced, and the mortality marker mortalin changed to the mortal type from the immortal type. Human cell lines lacking functional p21wafl sdi-1, p16_??_, or p53 behaved similarly. The protein levels of p16_??_ and p53 did not change uniformly, while the level of p21wafl sdi-1 was increased by varying degrees in positive cell lines. Telomerase activity was suppressed in positive cell lines, but accelerated telomere shortening was not observed in tumor cell lines. These results suggest that 5-bromodeoxyuridine activates a common senescence pathway present in both mortal and immortal mammalian cells.抄録全体を表示PDF形式でダウンロード (3293K) -
Ricardo R. Madoery, Clelia González Gattone, Gerardo D. Fidelio1999 年 126 巻 6 号 p. 1060-1066
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn this work we studied the effect of Ca2+ on the ability of immobilized PLA2 to hydrolyze phospholipid substrates either in aggregate or monomeric forms. We use a kinetic methodology for the determination of dissociation constants of soluble and immobilized PLA2-Ca2+, complexes. This approach allows us to obtain the values of the dissociation constants ofenzyme-Ca2+ (Kx) and enzyme-Ca2+-substrate (K'x) complexes from the kinetic data obtained at different substrate and Ca2+ concentrations. Results using mixed micelles of phospholipid-Triton X-100 showed that, in most cases, productive complexes were destabilized by immobilization of PLA2. However, a correct analysis of the interaction must be independent of the classical modes of PLA2 action toward lipid surfaces. Thus, a substrate in monomeric form was also employed to analyze the effect of immobilization on hydrolysis rate in the absence of interfacial activation. Kinetic data showed that the immobilization affected severely the mode of PLA2 action. The kinetic data also suggested that immobilization promoted conformational alterations in the Ca2+-binding site, destabilizing the productive complex enzyme-Ca2+-phospholipid.抄録全体を表示PDF形式でダウンロード (3065K) -
Shuji Miyagawa, Rie Nakai, Mako Yamada, Masahiro Tanemura, Yoshitaka I ...1999 年 126 巻 6 号 p. 1067-1073
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe effect of remodeling of a glycoantigen such as the α-Gal epitope, Galα1, 3Ga1β1, 4GlcNAc-R, by the introduction of glycosyltransferase genes on natural killer (NK) cell-mediated direct cytotoxicity was investigated using human peripheral blood mononuclear cells (PBMC) or an NK-like cell line, YT cells, as an effector, and swine endothelial cells (SEC) as a target. Several SEC transfectants were established by transfection with the genes for β1, 4-N-acetylglucosaminyltransferase III, α2, 3-sialyltransferase and α1, 2-fucosyl-transferase. These transfections led to dramatic reductions in both direct and indirect NK cell-mediated cytotoxicity, by 72-94% in the case of PBMC and 27-72% in that of YT cells, in addition to an effective reduction in xenoantigenicity, which is substantially caused by the α-Gal epitope, to human natural antibodies. The NK cell-mediated direct cytotoxicity was remarkably blocked by an anti-α-Gal epitope monoclonal antibody or GSI lectin which preferentially binds to the epitope. Furthermore, treatment of the parental cells with α-galactosidase resulted in a significant reduction in cytotoxicity. These results suggest that the α-Gal epitope is involved not only in hyperacute rejection and acute vascular rejection, but also in NK cell-mediated direct cytotoxicity. Thus, the genetic remodeling of the α-Gal epitope and probably other glycoantigens as well can be expected to represent a new approach for overcoming not only indirect but also direct immunity to xenografts.抄録全体を表示PDF形式でダウンロード (2241K) -
Takahiro Tsujita, Maho Sumiyoshi, Hiromichi Okuda1999 年 126 巻 6 号 p. 1074-1079
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーThe fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.抄録全体を表示PDF形式でダウンロード (2368K) -
Thomas Zimmer, Atsushi Ogura, Akinori Ohta, Masamichi Takagi1999 年 126 巻 6 号 p. 1080-1089
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーUsing the mRNA differential display technique and Western blot analysis, the present study demonstrates that induction of KAR2 occurs when misfolded membrane-bound cytochrome P 450, mutated in its cytosolically exposed domain, is expressed in Saccharonzyces cerevisiae. Using various KAR2 promoter constructs in front of the Escherichia coli β-galactosidase reporter gene, we found a fast and strong induction through the heat shock element (HSE), which was enhanced several fold by its adjacent GC-rich region. Additionally, a less pronounced induction was detected for the UPR element (UPRE). As expected, this response was absent in the irel disruptant strain. However, the HSE-mediated induction was enhanced upon disruption of IRE1 suggesting that the HSE pathway can compensate for the lack of a functional UPR pathway. Western blotting confirmed that Kar2p levels were increased to the same extent in the irel disruptant and in the non-disruptant strain. Removal of the P 450 membrane-spanning region also abolished the UPRE-mediated induction of KAR2 transcription, but the HSE-mediated response remained. The data show for the first time that the transcription of KAR2 is significantly induced in response to a misfolded membrane-bound endoplasmic reticulum protein, and identifies the HSE and UPRE regions as KAR2 promoter elements responding to the misfolded cytosolic P 450 domain and to the membrane-integrated mutant P 450, respectively.抄録全体を表示PDF形式でダウンロード (3147K) -
Yasuhiro Tomita, Tetsuro Yamashita, Hironori Sato, Hideharu Taira1999 年 126 巻 6 号 p. 1090-1100
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーSendai virus envelope glycoproteins, F and HN, mature during their transport through the endoplasmic reticulum (ER) and Golgi complex. To better understand their maturation processes in the ER, we investigated the time course of their interactions with three ER-resident molecular chaperones, BiP, calnexin (CNX), and calreticulin (CRT), in Sendai virus-infected HeLa cells. Pulse-chase and immunoprecipitation analyses using antibodies against each virus glycoprotein or ER chaperone revealed that F precursor interacted with CNX transiently (t1/2=8min), while HN protein displayed longer and sequential interactions with BiP (t1/2=8min), CNX (t1/2=15min), and CRT (t1/2=20min). HN interacted with the three ER chaperones not only as a monomer but also as a tetramer for several hours, suggesting mechanism(s) to undergo chaperone-mediated quality control of an assembled HN oligomer in the ER. The kinetics of dissociation of the HN-chaperone complexes exhibited a marked delay in the presence of proteasome inhibitors, suggesting that a part of HN associated with BiP, CNX, and CRT is destined to be degraded in the proteasome-dependent pathway. Further, the associations between virus glycoproteins and CNX or CRT were impaired by castanospermine, an inhibitor of ER glucosidase I and II, confirming that these interactions require monoglucosylated oligosaccharide on F_??_ and HN peptides. These findings together suggest that newly synthesized F protein undergoes rapid maturation in the ER through a transient interaction with CNX, whereas HN protein requires more complex processes involving prolonged association with BiP, CNX, and CRT for its quality control in the ER.抄録全体を表示PDF形式でダウンロード (3259K) -
Akiko Shiratsuchi, Yoshinobu Nakanishi1999 年 126 巻 6 号 p. 1101-1106
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーApoptotic cells are rapidly phagocytosed and eliminated from the organism. Although cancer cells apoptose when treated with anticancer drugs, how those cells are recognized by phagocytic cells has remained unclear. The human leukemia cell line Jurkat was cultured with doxorubicin or bufalin and induced to undergo apoptosis accompanied by phosphatidylserine externalization. When apoptotic Jurkat cells were mixed with mouse peritoneal macrophages, efficient phagocytosis was observed. Apoptosis and phagocytosis of Jurkat cells were both inhibited by Z-VAD-FMK, and phagocytosis was significantly reduced in the presence of phosphatidylserine-containing liposomes. These results suggest that anticancer drugs induce apoptosis-dependent and phosphatidylserine-mediated phagocytosis in cancer cells.抄録全体を表示PDF形式でダウンロード (2470K) -
Nobuhiro Ishida, Shigemi Yoshioka, Mai Iida, Kozue Sudo, Nobuhiko Miur ...1999 年 126 巻 6 号 p. 1107-1117
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーUDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galacto-syltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.抄録全体を表示PDF形式でダウンロード (3570K) -
Masahiro Tomita, Katsutoshi Yoshizato, Kazuhiro Nagata, Takashi Kitaji ...1999 年 126 巻 6 号 p. 1118-1126
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe previously demonstrated that insect cells were able to synthesize recombinant human procollagen I as triple-helical heterotrimers when transfected with cDNAs of both proα1 (I) and proα2 (I) chains. However, most of the heterotrimers were retained within the cells, unlike in the case of mammalian cells [Tomita, M., Kitajima, T., and Yoshizato, K. (1997) J. Biochem. 1061-1069]. In an attempt to improve the secretion of the heterotrimers, we introduced the putative collagen-specific chaperone HSP47 into this insect expression model. Mouse HSP47 produced by the insect cells bound intracellularly to both human proα1 (I) and proα2 (I) chains and enhanced the secretion of procollagen I heterotrimers. HSP47 was also coexpressed with either proα1 (I) chains or proα2 (I) chains, which showed that it enhanced the secretion of the former but not the latter. This selective effect of HSP47 was similarly observed in the cells treated with inhibitors of procollagen triple helix formation, indicating that HSP47 can also accelerate the secretion of non-helical procollagens. HSP47 did not change the intracellular solubility of proα1 (I) and proα2 (I) chains in 1% NP-40, eliminating the possibility that it prevents proα chains from aggregating into insoluble forms within the insect cells. We concluded that HSP47 can play a role in the secretion of α1 (I)-procollagen chains in the insect cell model. The present study also demonstrated the dissimilarity in the mechanism of folding and secretion of the expressed procollagen I between the insect and mammalian cells.抄録全体を表示PDF形式でダウンロード (4320K) -
Rintaro Inoue, Hirofumi Usui, Osamu Tanabe, Yasumasa Nishito, Masahiro ...1999 年 126 巻 6 号 p. 1127-1135
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーA heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase α, P-H2B histone, and P-H1 histone in the presence or absence of 20mM MnCl2, or 50mM Mg(CH3COO)2but stimulated the PP activity toward P-H1 histone in the presence of 200mM NaCl and the Mn2+-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'a subunit was isolated from a heterotrimeric form, CAB'δ, of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'δ subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'δ subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'δ subunit with CA and the resulting suppression of the PP- and PTP-activities.抄録全体を表示PDF形式でダウンロード (2446K) -
Hiroaki Konishi, Toshihide Fujiyoshi, Yasuhisa Fukui, Hidenori Matsuza ...1999 年 126 巻 6 号 p. 1136-1143
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーProtein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H2O2 and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3, 4-bisphosphate and PI 3, 4, 5-trisphosphate increased in H2O2-treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3, 4-bisphosphate did not change while that of PI 3, 4, 5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H2O2-treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cells.抄録全体を表示PDF形式でダウンロード (3435K) -
Masayo Okumura, Jun Uematsu, Yoshiaki Hirako, Yuji Nishizawa, Hiroshi ...1999 年 126 巻 6 号 p. 1144-1150
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーHD1 is a 500 kDa hemidesmosomal plaque protein recognized by monoclonal antibody mAb-121. Recent research on inherited skin disease has suggested that it might be identical to plectin or an isoform. To cast light on this question, we have prepared several monoclonal antibodies that recognize a 500 kDa protein in the hemidesmosome fraction. Unexpectedly, some staining pattern heterogeneity was observed on immunofluorescence microscopy. Attention was focused on two monoclonal antibodies which gave different localization in bovine skin and retinal pigment epithelial cells. Determination of the amino-terminal sequence of an antigenic 100 kDa polypeptide fragment derived from the 500 kDa component of an insoluble fraction of bovine hepatocytes revealed it was identical to that of plectin. Using the two antibodies, we screened a cDNA library derived from BMGE+H, a bovine mammary gland epithelial cell line. The isolated cDNA clones corresponded to the rod domain of bovine plectin, with two separate epitope regions for each of the antibodies. From these results we conclude that the hemidesmosomal 500 kDa component HD1 is identical to plectin. As judged on rough estimation of molar ratios on this basis, hemidesmosomes are composed of plectin, BP230, the integrin β4 subunit, and α6 in a 1:1:1:1 ratio.抄録全体を表示PDF形式でダウンロード (2700K) -
Shuji Wakatsuki, Manabu Arioka, Naoshi Dohmae, Koji Takio, Makari Yama ...1999 年 126 巻 6 号 p. 1151-1160
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn our previous paper, we reported that a 15 kDa protein (p15) produced by a fungus, genus Helicosporium, enhanced NGF-induced neurite outgrowth from PC12 cells. Here we further characterized the actions of p15. The complete amino acid sequence of p15 was determined and it was shown to be a hydrophilic protein composed of 118 amino acid residues with two intramolecular disulfide bridges. p15-induced neurite outgrowth was blocked by the depletion of extracellular Ca2+ in the culture medium and was significantly inhibited by L-type Ca2+ channel inhibitor nicardipine. p15 stimulated Src kinase and MAPK activities, and neurite outgrowth was not observed in sreDN2, a dominant negative c-src-express-ing cell line, and was significantly reduced in RasN17-expressing cells. These results suggest that p15 stimulates neurite outgrowth through the potentiation of L-type Ca2+-channels, thereby activating the Src-Ras-MAPK cascade.抄録全体を表示PDF形式でダウンロード (2629K) -
Shin-ichi Nakakita, Krishna K. Menon, Shunji Natsuka, Kazuhiro Ikenaka ...1999 年 126 巻 6 号 p. 1161-1169
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーWe previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and α1-6Fuc residues, (GlcNAcβl-2) Manαl-3(GlcNAcβ1-2Man αl-6) (GlcNAcβ1-4)Manβ1-4G1cNAcβ1-4 (Fucαl-6)GlcNAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochern. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and αl-6Fuc residues as acceptor substrates. While the β1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the β1-4galactosyltransferase (s)in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and al-6Fuc residues as substrates. The β1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to he different from β1-4galactosyltrans-ferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and al-6Fuc residues acts as an inhibitor against “brain type” β1-4galactosyltransferase with a K1 value of 0.29mM.抄録全体を表示PDF形式でダウンロード (1443K) -
Kunio Fujiwara, Ikuo Murata, Shiroki Yagisawa, Toshio Tanabe, Masahiko ...1999 年 126 巻 6 号 p. 1170-1174
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーIn our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AIIA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-α-methyl histamine, 1- and 3-methylhistamine, L-his-tidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10- times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-sub-stituted imidazole and a-methine groups of a HA molecule in addition to the conjugation site of GA including the part (s) reduced with NaBH and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.抄録全体を表示PDF形式でダウンロード (427K) -
Hirokuni Miyamoto, Fumiko Mitani, Kuniaki Mukai, Makoto Suematsu, Yuzu ...1999 年 126 巻 6 号 p. 1175-1183
発行日: 1999年
公開日: 2008/11/18
ジャーナル フリーCircadian rhythms and zonal variations in the cell proliferation of adult rat adrenal cortex were studied by following the cells in the DNA-synthesizing stage (S-phase) as assessed by 5-bromo-2'-deoxyuridine incorporation into the cell-nuclei and/or by visualizing prolifer-ating cell nuclear antigen. The S-phase cells were observed throughout the day in two regions of the adrenal cortex: (i) a region from the inner half of the zona glomerulosa to near the outer margin of the zona fasciculata, and (ii) the outer one-fourth portion of the zona fasciculata. Very little change in number was observed in the former region between day and night, while a burst of cell proliferation occurred in early morning at 3-4 a.m. in the latter region. A prominent rise in the plasma adrenocorticotropic hormone (ACTH) concentration preceded the burst of cell proliferation by about 4h. Upon raising the plasma ACTH concentration by administration of ACTH or metyrapone, prominent cell proliferation also occurred in the same portion of the zona fasciculata 4-6h after the provoked ACTH surge. Thus at least two sites in rat adrenal cortex are responsible for cytogenesis in this endocrine organ, and respond differentially to day/night cycles and circulating ACTH levels.抄録全体を表示PDF形式でダウンロード (2965K)
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