All issues
Volume 124 (1998)
- Issue 6 Pages 1051-
- Issue 5 Pages 869-
- Issue 4 Pages 687-
- Issue 3 Pages 473-
- Issue 2 Pages 251-
- Issue 1 Pages 1-
Volume 124, Issue 5
Displaying 1-27 of 27 articles from this issue
- |<
- <
- 1
- >
- >|
-
Takashi Nagano, Hussam Jourdi, Hiroyuki Nawa1998 Volume 124 Issue 5 Pages 869-875
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA group of proteins found at cell-cell junctions have a common structural domain, called PDZ_??_a stretch of 80-90 amino acid residues initially identified in the three proteins PSD-95, Dlg, and ZO-1. This domain is found in various proteins from bacteria to mammals and is involved in protein-protein interaction. Recently, many proteins containing this domain were identified in the nervous system by molecular cloning and shown to interact with other synaptic proteins, including various transmitter receptors, ion channels, and signal transducers. These PDZ-containing proteins are mostly located near the synaptic membrane and are, therefore, speculated to transport associated proteins to the synapse and/or anchor them at the synaptic sites. Alternatively, as a single molecule often contains multiple PDZ domains that can interact with each other, it may cluster all these synaptic molecules and facilitate their signaling at synaptic sites. This review focuses on the best characterized PDZ-containing proteins that interact with N-methyl-D-aspartate (NMDA)-type glutamate receptors and discusses their functions in synaptic organization.View full abstractDownload PDF (1062K) -
Tsuyoshi Inoue, Masaharu Gotowda, Deligeer, Kunishige Kataoka, Kazuya ...1998 Volume 124 Issue 5 Pages 876-879
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of the blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) has been determined at 2.05 Å resolution. AxgNIR contains both type 1 and 2 Cu sites, the geometry of the former being distorted tetrahedral. The superpositioning of the type 1 Cu sites in the blue enzyme and a green nitrite reductase revealed that the orientation of the Met150 side chain differed. The deviation of the Sδ(Met150) atom from the axial position of the NNS plane formed by two Nδ(His95 and His145) and one Sβ(Cys136) atom caused the difference in the colors of the enzymes, i.e. blue and green.View full abstractDownload PDF (1136K) -
Pasquale Gallo, Filomena Rossi, Michele Saviano, Carlo Pedone, Giovann ...1998 Volume 124 Issue 5 Pages 880-885
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLike cyclosporin A, cyclolinopeptide A binds specifically bovine cyclophilin A, inhibiting its peptidyl-prolyl cis-trans isomerase activity. We describe here the protein interaction with several synthetic analogues of cyclolinopeptide A, which are either homodetic or disulphide bridged heterodetic cyclopeptides characterized by different ring dimensions, in terms of dissociation and inhibition constants evaluated by fluorescence and inhibition of the enzyme activity, respectively. Dissociation constants from fluorescence experiments are practically identical and about 20-fold lower than for cyclosporin A. On the other hand, inhibition constants differ from compound to compound and are higher than for cyclosporin A. This result is therefore difficult to rationalize, but we would suggest decoupling between binding and inhibitory ability of cyclopeptides. The Pro1 residue of cyclolinopeptide A seems to play a fundamental role in determining the inhibition of the rotamase activity of cyclophilin A, as the homodetic analogue lacking this residue does not show any inhibitory ability. Similarly, heterodetic analogues with a ring size smaller than 7 residues do not display inhibition. We presume that the sequence -Pro-Pro-Phe-Phe- and a ring size of 8 residues for homodetic cyclic peptides could be used as starting points in the targeted synthesis of cyclopeptides able to bind both cyclosporin A and calcineurin. The only peptide showing similar values of the dissociation and inhibition constant is cyclolino-peptide A. This compound can be considered a novel model for the molecular design of immunosuppressant drugs.View full abstractDownload PDF (794K) -
Naoki Ichikawa, Miwa Fukuda, Tadao Hashimoto, Kunio Tagawa1998 Volume 124 Issue 5 Pages 886-891
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe role of the carboxyl-terminal region of the yeast mitochondrial ATPase inhibitor was investigated. Three progressive C-terminal deletion mutants of the inhibitor were constructed; (i) Ile58→end; (ii) Ile51→end; and (iii) Gln43→end. The truncated inhibitor was detected in extracts of Ile58→end mutant yeast cells. For the Ile51→send mutant, the truncated inhibitor was only detected when the cells were grown on medium containing the membrane-permeable metal chelator, o-phenanthroline, which inhibits mitochondrial proteases. The most greatly truncated inhibitor protein, Gln43→end, was never detected even in the cells grown in the presence of the metal chelator. The rates of ATP synthesis and hydrolysis in the mutant mitochondria containing the Ile51→end inhibitor were similar to those in wild type control cells, while the Ile51→end inhibitor protein was degraded in the cells unless they were incubated in the presence of the chelator. These results indicate that the carboxyl-terminal region of the ATPase inhibitor is not involved in the its inhibitory action on the F1F0-ATPase, but is required for the stable conformation of the protein which is protected against degradation by proteases.View full abstractDownload PDF (2573K) -
Haoran Wang, Xiangrong Gao, Soji Fukumoto, Sayuri Tademoto, Kenzo Sato ...1998 Volume 124 Issue 5 Pages 892-899
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe traditional two-step EGTA/collagenase method is widely used in studying nitric oxide (NO) production in hepatocytes. The present study first revealed that hepatocytes isolated by this method spontaneously express an iNOS mRNA. Thereafter, based on this novel finding, we characterized the expression and regulation of the gene in primary cultured hepatocytes. Using Northern blot analysis, the iNOS mRNA was observed 4h after isolation, reached peak at 8h, and declined to an undetectable level after 24 h. iNOS gene expression was shown to be serum-independent and not due to lipopolysaccharide contamination. Time-course analysis of the effects of actinomycin D demonstrated that the increase in iNOS transcripts is the result of an accompanying great increase in iNOS gene transcription and lower iNOS mRNA stability; also blockage by cycloheximide suggests that it is dependent on de novo protein synthesis. Inhibition by pyrrolidine dithiocarbamate, a NF-κB/c-rel inhibitor, further implies the involvement of NF-κB/c-rel. To clarify reason (s) for the induction, hepatocytes were isolated with the collagenase buffer perfusion step omitted. As a consequence, iNOS mRNA was undetectable in the hepatocytes. These findings show that the traditional hepatocyte-isolation culture does indeed transiently express a serum-independent but de novo protein synthesis-dependent iNOS mRNA due to collagenase (type IV) buffer perfusion.View full abstractDownload PDF (5061K) -
Laura Riboni, Alessandro Prinetti, Rosaria Bassi, Paola Viani, Guido T ...1998 Volume 124 Issue 5 Pages 900-904
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeuro2a cells were exposed to different doses (1-40 nmol/106 cells) of [C3-3H]sphingosine and the relationship between metabolism and biological effects of sphingosine was investigated. Sphingosine appeared to be rapidly taken up and metabolized. The incorporation of sphingosine was not merely dependent on its concentration but primarily on the dose per cell of administered sphingosine. At low doses, [3H] sphingosine represented a minor portion of the cellular radioactivity, and N-acylated metabolites, particularly ceramide, largely prevailed over degradation products. Concomitantly with ceramide increase, Neuro2a differentiation took place. With increasing exogenous sphingosine/doses, the acylation process reached saturation. From this point on, [3H] sphingosine started accumulating and eventually cell toxicity occurred. In conclusion, the biological effects exerted by exogenous sphingosine on Neuro2a cells are not merely dependent on the long-chain base concentration in the culture medium, but are strictly related to the cellular dose of exogenous sphingosine and to the capacity of cells to metabolize sphingosine.View full abstractDownload PDF (2049K) -
Yoshihiro Fuchikami, Tohru Yoshimura, Aldo Gutierrez, Kenji Soda, Nobu ...1998 Volume 124 Issue 5 Pages 905-910
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSD-Amino acid aminotransferase [EC 2. 6. 1. 21] catalyzes the inter-conversion between various D-amino acids and α-keto acids. The subunit of the homodimeric enzyme from Bacillus sp. YM-1 consists of two domains connected by a single loop, which has no direct contact with the active site residues or the cofactor, pyridoxal 5'-phosphate [Sugio, S., Petsko, G. A., Manning, J. M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669]. We constructed two plasmids, one encoding a polypeptide fragment corresponding to the N-terminal domain, and the other a fragment corresponding to the C-terminal domain. When both polypeptide fragments were expressed together in the same host cell, an active fragmentary enzyme consisting of two sets of the two polypeptide fragments was produced. When the two polypeptide fragments were expressed separately, each of them provided a soluble protein but with no activity. However, D-amino acid aminotransferase activity appeared upon incubation of a mixture of the two fragments. The active fragmentary enzyme was purified to homogeneity and characterized; it was found to be similar to the wild-type enzyme in various enzymological properties except substrate specificity, inhibition by α-ketoglutarate, and thermostability. The fragmentary enzyme showed higher catalytic activity toward several substrates, such as D-lysine and D-arginine, than the wild-type enzyme.View full abstractDownload PDF (1989K) -
Keiko Doi-Kawano, Yoshiaki Kouzuma, Nobuyuki Yamasaki, Makoto Kimura1998 Volume 124 Issue 5 Pages 911-916
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSunflower cystatin Scb differs from other phytocystatins in that it is a highly basic protein with a pI value of 9.6 and includes six additional amino acids (Arg30-Leu-Gln-Arg-Thr34, Thr37) in the middle region as compared with other phytocystatins [Kouzuma et al. (1996) J. Biochem. 119, 1106-1113]. We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different. Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain. The result revealed that the additional sequence (Arg30-Leu-Gln-Arg-Thr34) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile1-Pro2) as well as the N-terminal glycine residues Gly3 and/or Gly4 play an important role in manifesting the inhibitory activity toward papain.View full abstractDownload PDF (2172K) -
Chang-Rak Ha, Ichiro Iuchi1998 Volume 124 Issue 5 Pages 917-926
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom our previous studies, we have suggested that “egg envelope (chorion) hardening-enzyme” (first proposed by Zotin [J. Embriol. Exp. Morph. 6, 546-568 (1958)]) in the rainbow trout, Oncorhynchus mykiss, is transglutaminase (TGase), and that it coexists with its substrate, the unfertilized egg chorion, and forms ε-(γ-glutamyl) lysine cross-links between the chorion proteins. In the present study, we extracted the TGase activity from the isolated chorions by homogenization with isotonic saline (143mM NaCl-10mM Tris•HCl, pH 7.2) and fractionated the extract by Toyopearl HW55S gel filtration with the isotonic saline containing 5mM CaCl2 and 5mM 2-mercaptoethanol (2-ME). One peak of TGase activity (P2) was obtained. When the eluates were dialyzed against 5mM CaCl2-5mM 2-ME-10mM Tris•HCl (pH 7.2), another peak of the activity (P1) appeared. P1 TGase activity, which becomes apparent in a medium of low ionic strength, is involved in acceleration of the chorion hardening after egg activation in fresh water, so-called water activation. We purified the two TGases, P1 and P2, by SP-Sepharose, Q-Sepharose, and TSK-gel column chromatography. The molecular mass of the native form of P1 TGase was estimated as 103 kDa by Toyopearl HW55S gel filtration and as 100 kDa by the TSK-gel filtration. SDS-PAGE analysis showed that it consisted of heterogeneous 86- and 76-kDa proteins. However, these proteins closely resembled each other in amino acid composition, which was characterized by high content of Thr, Gly, and Pro residues as compared with P2 TGase. In contrast, the P2 TGase was isolated as a homogeneous 76-kDa protein and characterized by high content of Glx (Glu/Gln) and His residues. Neither of the chorion TGases of rainbow trout, P1 and P2, was similar to the liver-type TGase of red sea bream or the tissue-type TGase of chum salmon in amino acid composition. Examination of susceptibility to various inhibitors, reactivation by CaCl2, pH dependency, and activity of the polymerization of chorion proteins suggested that the P1 and P2 TGases were essentially similar to each other in enzymatic properties.View full abstractDownload PDF (4232K) -
Misaki Asano-Miyoshi, Yuko Kusakabe, Keiko Abe, Yasufumi Emori1998 Volume 124 Issue 5 Pages 927-933
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo identify genes specifically expressed in taste tissues, we constructed a subtraction cDNA library of epithelium of rat circumvallate and foliate papillae and carried out differential screening of this library. Dot blot analysis showed 46 out of 88 clones obtained by this method to be expressed in the epithelium of papillae. The cDNA inserts in these clones were sequenced and analyzed for similarity to entries the GenBank database. About 54.3% of the clones were known sequences, including the sequences of ebnerin, cytokeratin 18, and Na+, K+-ATPase, that were shown by in situ hybridization to be expressed in the circumvallate papillae. About 41.3% of the papillae-specific clones had no significant similarity to known sequences and are candidates for novel taste bud-specific marker genes.View full abstractDownload PDF (3342K) -
Takehiko Sasaki, Tomohiko Maehama, Toshiyoshi Yamamoto, Shunsuke Takas ...1998 Volume 124 Issue 5 Pages 934-939
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysophosphatidic acid (LPA) induced activation of c-Jun N-terminal kinase (JNK) in Swiss 3T3 fibroblasts. This activation reached the maximum at 20 min and required a high concentration of LPA with an EC50 value of approximately 3 μg/ml. LPA-induced activation of JNK was not suppressed by prior treatment of the cells with pertussis toxin, whereas it was completely blocked by suramin, a non-selective inhibitor of ligand-receptor interactions. The kinetics and concentration-dependency of LPA-induced JNK activation were in sharp contrast with those of LPA-induced extracellular signal-regulated kinase (ERK) activation, which reached the maximum within 3 min and occurred with an EC50 of 0.1 μg/ml. The ERK activation was susceptible to pertussis toxin, whereas it was not inhibited by suramin. These results indicate that the signal transduction pathways of LPA-induced JNK and ERK activations are distinct. Thus, this is the first report showing that LPA induces not only ERK activation but also JNK activation, which may be responsible for the induction of DNA synthesis in LPA-stimulated Swiss 3T3 fibroblasts.View full abstractDownload PDF (2320K) -
Kazuya Matsuura, Hiroaki Shiraishi, Akira Hara, Kumiko Sato, Yoshihiro ...1998 Volume 124 Issue 5 Pages 940-946
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman 3α-hydroxysteroid dehydrogenase exists in four isoforms, which belong to the aldo-keto reductase (AKR) superfamily and are named AKR1Cl-AKR1C4. The properties of AKR1C3 have not been fully characterized compared to the other isoforms. In addition, a cDNA that shows more than 99% homology with AKR1C3 cDNA has been cloned from human myeloblasts. We have here expressed and purified a recombinant enzyme (designated as DBDH) from this cDNA. DBDH oxidized xenobiotic alicyclic alcohols and 3α- or 17β-hydroxy-5β-androstanes, and catalyzed the reversible conversion between prosta-glandin D2 and 9α, 11β-prostaglandin F2 more efficiently than that of 3α- or 17β-hydroxy-steroids: the respective Km, values were 0.6 and 6.81 μM, and kcat/Km values were about 1, 000 min-1•mM-1. Anti-inflammatory drugs highly inhibited the enzyme. The recombinant AKR1C3 prepared by site-directed mutagenesis of DBDH also showed the same properties as the wild-type DBDH. Analyses of expression of mRNAs for DBDH and AKR1C3 by reverse transcription-PCR indicated that only one mRNA species for DBDH is expressed in 33 human specimens of liver, kidney, lung, brain, heart, spleen, adrenal gland, small intestine, placenta, prostate, and testis. These results suggest that AKR1C3 acts as prosta-glandin D2 11-ketoreductase, and that its principal gene in the human has a coding region represented by DBDH cDNA.View full abstractDownload PDF (3541K) -
Kenichi Watanabe, Kazumi Ishidoh, Takashi Ueno, Nobuhiro Sato, Eiki Ko ...1998 Volume 124 Issue 5 Pages 947-956
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDecreased lysosomal proteolysis in regenerating liver after 70% hepatectomy was analyzed. The activities of cathepsins B and L increased transiently 4 h after hepatectomy, began to decrease gradually reaching about 30% of the control level at 24 h, then returned to near control level after 7 days. Immunoblot and RNA blot analyses confirmed that the changes in cathepsin activities coincided with changes in protein levels and mRNA levels. In parallel with the changes in cathepsins, we found that the amounts of LGP120, LGP110, and LGP85, three integral lysosomal membrane proteins, declined significantly after hepatectomy, suggesting that the lysosomal levels are also diminished in regenerating liver. We isolated dextran-loaded lysosomes and found that the protein content and marker enzyme activities of dextran-loaded lysosomes from partially hepatectomized livers are lower by 50 and 40%, respectively, compared with control livers. This indicates that there is a significant reduction in the cellular lysosomal level in regenerating liver. In addition, we used a sensitive biochemical assay to quantify leupeptin-induced autolysosomes and found that the autophagic activity is markedly suppressed in regenerating liver as compared with normal liver. Thus, the suppression of lysosomal proteolysis in regenerating liver is attained through three steps, i.e., decreased biosynthesis of cathepsins, decreased lysosomal biogenesis, and decreased cellular autophagy.View full abstractDownload PDF (4421K) -
Hajime Masumoto, Toshio Yoshizawa, Hiroyuki Sorimachi, Takeshi Nishino ...1998 Volume 124 Issue 5 Pages 957-961
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant human m-calpain was produced in a soluble form at a level of 20 mg/liter of Sf-9 cell culture by the coexpression of recombinant human m-calpain large (m8OK) and small (30K) subunits using a baculovirus expression system. The expressed m-calpain was purified by sequential column chromatographies on DEAE-Toyopearl, gel-filtration, and Mono Q by the same method used to purify native m-calpain. The recombinant m-calpain had a specific activity of 691 U/mg and a Ka value (Ca2+ requirement for 50% caseinolysis activity) of 0.4mM, which are essentially identical to those of native rabbit m-calpain. A mutant m-calpain large subunit, m-C105S-80K, where the active-site cysteine-105 is converted to serine by site-directed mutagenesis, was coexpressed with 30K in Sf-9 cells, purified, and characterized. m-105S-calpain does not degrade casein nor an artificial tetra-peptide substrate, succinyl-Leu-Leu-Val-Tyr-MCA. Further, it shows no autolytic activity with Ca2+. This is the first report of the large-scale production of a fully active m-calpain species in the baculovirus system.View full abstractDownload PDF (2993K) -
Takeshi Shimogiri, Masanobu Kono, Hideyuki Mannen, Makoto Mizutani, So ...1998 Volume 124 Issue 5 Pages 962-971
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOrnithine transcarbamylase (OTC) is one of the urea cycle enzymes. While the chicken is a uricotelic animal, it has measurable OTC activity in its kidney. OTC activity is highly variable within and between chicken breeds. Chicken OTC may have some physiological significance because of its significant activity in the kidney. We cloned the OTC cDNA from chicken kidney and found 77% homology between the deduced amino acid sequence of the mature protein and that of mammals. The chicken OTC gene spans 26 kb, consists of 10 exons and 9 introns, and utilizes the same exon-intron boundaries as the human gene. The 5'-flanking region contains a putative TATA box and two potential regulatory sites, but neither the 5'-flanking region nor the splice sites correlated with variation in OTC activity. In intron 3, two polymorphic sites were found: one comprising a deletion of 401 nucleotides; and the other was a length and sequence polymorphic region located 8 bases upstream from the deletion. The latter polymorphism provides an explanation for phenotypic variation in OTC. Linkage analysis has suggested reassignment of the chicken OTC gene from the suggested Z chromosome to chromosome 1q.View full abstractDownload PDF (2337K) -
Ryuji Hamamoto, Keisuke Yamada, Masamichi Kamihira, Shinji Iijima1998 Volume 124 Issue 5 Pages 972-979
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied spheroid (multicellular aggregate) formation by hepatocytes and the expression of liver-specific functions such as albumin secretion when hepatocytes were cultured with various extracellular matrices. Hepatocytes cultured on Primaria® and poly-D-lysine coated dishes, and in the presence of a polymer, Eudragit, formed spheroids, and they also exhibited higher liver-specific functions and poor growth compared to monolayer cultures. The results indicated that the cell morphological change and cell-cell interaction caused by the spheroid formation were key factors promoting the expression of the liver-specific functions. To elucidate the mechanism underlying the poor growth in spheroids, we examined the HGF signaling pathway. Phosphorylation and down-regulation of the HGF receptor (c-Met proto-oncogene product) were observed for the cells from both monolayer and spheroid cultures, but Ras activation was partly blocked in spheroids. Furthermore, we found that CDK inhibitors, p21 and p27, were highly expressed in spheroids. These results suggested that the reduced Ras signaling and high expression of the CDK inhibitors might cause the lower growth in spheroids. We then examined the relationship between liver-enriched transcription factors (C/EBPα and β) and liver-specific functions. The results revealed that the high expression of C/EBPα was maintained during cultures when hepatocytes formed spheroids. Antisense oligonucleotides of C/EBPα repressed albumin secretion and the expression of p21, suggesting that the transcription factor, C/EBPα, may play a crucial role in the growth and differentiation of hepatocytes in spheroids.View full abstractDownload PDF (5885K) -
Xueyuan Jiang, Minyue Zhang, Yi Ding, Jun Yao, Hao Chen, Dexu Zhu, Mut ...1998 Volume 124 Issue 5 Pages 980-985
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteinase In has previously been described as displaying a trypsin-like proteinase activity that momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication [Kato, M., Irisawa, T., Ohtani, M., and Muramatu, M. (1992) Eur. J. Biochem. 210, 1007-1014]. We purified the proteinase In from E. coli C600 and found that the 15 amino acid residues of its amino-terminal were identical with those of oligopeptidase A (OpdA), the product of the E. coli prlC gene. The purified proteinase had a molecular mass of approximately 67 kDa, which was also the same as that of oligopeptidase A. To further elucidate the relationship between proteinase In and oligopeptidase A, we assembled an expression vector to direct the synthesis of E. coli oligopeptidase A. The protein was expressed at a high level in E. coli BL21(DE3) and was produced mostly in the soluble, active form. Both the recombinant enzyme (rPrlC) and the purified proteinase In could hydrolyze trypsin substrates for proteinase In as well as benzyloxycarbonyl Ala-Ala-Leu p-nitroanilide (Z-AALpNA), described as a synthetic substrate for oligopeptidase A. The effects of various protease inhibitors on rPrlC were also very similar to those on proteinase In. The trypsin inhibitors 4-guanidino benzoic acid 4-tert-butylphenyl ester and antipain strongly inhibited the trypsin-like proteinase activity of the recombinant enzyme, but had no effect on its Z-AALpNA hydrolyzing activity. Cobalt ion, which greatly enhanced the OpdA activity, slightly inhibited the trypsin-like activity of the recombinant enzyme. These results strongly suggest that proteinase In is encoded by the E. coli prlC gene and is a multi-functional proteinase with two separate active sites.View full abstractDownload PDF (2007K) -
Yasuo Komatsu, Miho Shirai, Eiko Ohtsuka1998 Volume 124 Issue 5 Pages 986-991
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-domain hairpin ribozyme was improved by increasing the number of linker bases connecting domain I' with domain II from six to seven, and a new ribozyme was designed to release a trans-acting ribozyme that is able to bind with target RNAs by three hybridizing arms. The trans-cleavage activities of the trimmed ribozyme were used to cleave a long target RNA, with 85 bases transcribed from a synthetic gene encoding induced nitric oxide synthase (iNOS). Although the ribozyme with three-hybridizing arms did not efficiently cleave the long target RNA, the overall activity, from self-trimming to the trans-cleavage reaction of the target RNA, was higher than that of the ribozyme with two hybridizing arms.View full abstractDownload PDF (2223K) -
Hiroyuki Suzuki, Kazuhiro Takahashi, Ken-ichi Yasumoto, Shintaro Amae, ...1998 Volume 124 Issue 5 Pages 992-998
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTyrosinase-related protein 2 (TRP-2)/DOPAchrome tautomerase is an enzyme involved in melanin biosynthesis and plays an important role in cytoprotection by preventing the production of a toxic melanin precursor, 5, 6-dihydroxyindole. Neurofibromin is the protein product of a gene linked to neurofibromatosis type 1 (NF1), which is characterized by multiple neurofibromas and abnormalities in skin pigmentation. To explore the pathogenesis of NF1, we studied the role of neurofibromin in the regulation of TRP-2 gene expression. By means of transient cotransfection assays, we show that the expression of a reporter gene under the control of the TRP-2 gene promoter was increased by a neurofibromin-dependent signal through the 71-bp region (positions -415 to -345). A Lys-to-Glu substitution at position 1425 in neurofibromin abrogated this activating function. A dominant negative Ki-ras inhibitor mimics neurofibromin's function, and additively increases TRP-2 promoter activity when coexpressed with neurofibromin. Therefore, we suggest that neurofibromin is involved in the regulation of TRP-2 gene expression. Moreover, we found a single case of a glioblastoma multiforme that expresses TRP-2 mRNA but not tyrosinase mRNA, suggesting that TRP-2 may function in human neural tissues under certain conditions.View full abstractDownload PDF (2140K) -
Ken Sasai, Jun-ichi Aikawa, Sakura Saburi, Hideaki Tojo, Satoshi Tanak ...1998 Volume 124 Issue 5 Pages 999-1003
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLeukemia inhibitory factor (LIF) is a pluripotent growth factor which acts in various cell systems. LIF is a glycoprotein containing six putative N-glycosylation sites. We established Chinese hamster ovary (CHO) cell lines to evaluate the biological roles of the N-glycosylation in rat LIF (rLIF). The bioactivity of rLIF was evaluated in two different bioassay systems using F9 and DA-1a cells. Employing site directed mutagenesis, six N-glycosylation-deficient LIF mutants were generated by replacing each asparagine residue (N) (at positions N9, N34, N63, N73, N96, and N116) by glutamine (Q). The resultant mutants showed similar activity in the bioassay using F9 cells. However, N34Q was about 3 times more potent than the wild-type rLIF in the assay using DA-1a cells. These findings suggest that the presence of N-glycan at N34 suppresses cell proliferation. In contrast, N63Q was about 2.5 times less potent than the wild-type rLIF indicating the pivotal role of N63 glycosylation for rLIF bioactivity. Taken together, our data suggest that the N-glycans of LIF play different roles depending on the cell line and that glycosylation of each specific residue contributes differently to its bioactivity.View full abstractDownload PDF (1570K) -
Kazuo Kawamura, Daichika Hayata, Shigeki Fujiwara, Toshitsugu Yubisui1998 Volume 124 Issue 5 Pages 1004-1012
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo identify genes expressed during budding of the tunicate Polyandrocarpa misakiensis, we isolated and sequenced 624 clones from a directionally constructed cDNA library to prepare a catalog of expressed sequence tags (ESTs). A total of 233 ESTs matched genes of known sequence in the SwissProt database. About 24% out of them showed high similarity to ribosomal proteins, twice the value (12%) of pre-budding animals. ESTs involved in the respiratory chain also appeared with significant redundancy, suggesting that tunicate budding is accompanied by the enhancement of energy conversion as well as protein synthesis. Serine protease inhibitor (serpin) afforded another striking example of a gene that was highly expressed in the process of budding. The deduced amino acid sequences of five serpin cDNAs all had two consensus signatures of the Kazal's type of secretory protease inhibitor, one of which had an active site for trypsin and the other for elastase. In line with this, recombinant GST-fusion protein showed both trypsin and elastase inhibitor activities. In accordance with the EST analysis, the hemolymph taken from the budding stage showed the highest activity of trypsin inhibitor. We discuss a possible role that Polyandrocarpa serpins may play in bud development by counteracting trypsin-like serine protease, which could facilitate dedifferentiation of formative tissues.View full abstractDownload PDF (2909K) -
Makoto Tsuneoka, Eisuke Mekada1998 Volume 124 Issue 5 Pages 1013-1019
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOncogenic cooperation was found between the N-myc and v-ras oncogenes in a rat fibroblast cell line, 3Y1. To investigate the specific role of N-myc in the transformation, we established transformed cell lines that expressed N-myc under a controllable promoter. Using these cells, we found that constitutive expression of N-myc is necessary to maintain the transformation, and that the expression level of N-myc is closely correlated with the transformation. Since another myc family gene, c-myc, directly activates expression of RCC1, which has important functions for eukaryotic cell proliferation, we focused on the relationship between N-myc and RCC1. Cells transformed by N-myc and v-ras expressed several times more RCC1 mRNA than the parent 3Y1 cells, and the expression of RCC1 changed in a parallel with the expression of N-myc. Gel retardation analysis and experiments with reporter plasmids constructed from a DNA fragment of the RCC1 gene indicated that the N-Myc protein controls expression of RCC1 by binding directly to CACGTG elements in the RCC1 gene. These results suggest that N-myc can directly transactivate expression of RCC1, a c-myc target gene.View full abstractDownload PDF (3761K) -
Yoshiko Saeki, Tsukasa Seya, Kaoru Hazeki, Michio Ui, Osamu Hazeki, Hi ...1998 Volume 124 Issue 5 Pages 1020-1025
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have established hepatoma clones from benzopyrene-treated liver cells, one of which (G-5) shows extensive metastasis to the lung when injected subcutaneously into mice [Tanigaki, Y. et al. (1995) Invasion Metastasis 15, 70-80]. In the present study, we performed in vitro assays suitable for examination of the adhesive and invasive properties of the highly metastatic cells. G-5 cells efficiently entered the pores of fibronectin-coated filters. Treatment of the cells with an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), wortmannin, significantly impaired the invasive activity. A structurally unrelated inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also prevented invasion. Both inhibitors suppressed cell adhesion to fibronectin-coated dishes. G-5 cells were next transfected with a mutant regulatory subunit (Δp85) of PI 3-kinase, which was expected to impair the function of PI 3-kinase. The transfectants showed suppressed adhesion to the dishes and did not efficiently migrate into the filters. The lower adhesive ability of the transfected cells was not further affected by inhibitors of PI 3-kinase. Thus, PI 3-kinase activity contributes significantly to the adhesive and invasive properties of G-5 cells.View full abstractDownload PDF (4115K) -
Shingo Morii, Chifumi Fujii, Toshio Miyoshi, Masafumi Iwami, Eiji Itag ...1998 Volume 124 Issue 5 Pages 1026-1032
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene encoding 3-ketosteroid-Δ1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1, 536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Δ1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Δ1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.View full abstractDownload PDF (2074K) -
Hiroshi Ueda, Rika Morishita, Ritsuko Katoh-Semba, Kanefusa Kato, Tomi ...1998 Volume 124 Issue 5 Pages 1033-1037
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe βγ subunits of G proteins were coimmunoprecipitated with antibodies against various α subunits, and analyzed by silver stain and immunoblotting with conventional transfer procedure and membrane-blocking buffer containing 2% BSA. Multiple isoforms of γ were coimmunoprecipitated with no significant difference in form or ratio among the antibodies against α subunits used, suggesting antibodies against any α subunit could coimmuno-precipitate all forms of γ. Therefore, this method was applicable to analyze γ subunits in various cells, especially to clarify what forms of γ subunits are major components. The major isoforms were: γ5 in C6, NG108-15, HeLa, HEK293, and F9 cells; γ12 in Swiss 3T3 and BRL-3A cells; and γ3 in PC12 cells. In addition to most γ subunits identified, unidentified γ subunits were present in PC12, NG108-15, and BRL-3A cells. Furthermore, the method was applied to examine changes of isoforms of γ during differentiation of HL-60 cells. Undifferentiated cells mainly contained γ5, but retinoic acid treatment of cells replaced most γ5 with γ2. Thus, this method is useful to determine the major isoforms which seem to be the more important in cells.View full abstractDownload PDF (3351K) -
Nobukazu Komatsu, Tatsuya Oda, Tsuyoshi Muramatsu1998 Volume 124 Issue 5 Pages 1038-1044
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the involvement of caspases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in U937 cells. We found that caspase-3- and caspase-6-like activities, but not caspase-1-like activity, increased during toxin-induced apoptosis. Z-D-CH2-DCB, a caspase-like inhibitor, completely inhibited the generation of caspase-3- and caspase-6-like activities and blocked all features of apoptosis induced by toxins: nuclear morphological changes, DNA fragmentation, and cytotoxicity. However, three caspase-specific inhibitors, Ac-YVAD-CHO, Ac-DEVD-CHO, and Ac-VEID-CHO, had no effect, even though Ac-DEVD-CHO and Ac-VEID-CHO inhibited the increased caspase-3- and caspase-6-like activity, respectively. These results suggest that the generation of caspase-3- and caspase-6-like activities is redundant, and other caspases distinct from caspase-3 and -6 may be important in toxin-induced apoptosis. Furthermore, serine protease inhibitor, 3, 4-dichloroisocoumarine (DCI), abolished the apoptotic cell death and DNA fragmentation caused by toxins, without affecting the increased caspase-3- and caspase-6-like activities. Our results suggest that multiple proteases with different preferences for apoptotic substrates participate in toxin-induced apoptotic death of U937 cells.View full abstractDownload PDF (1864K) -
Myeong-Cheol Song, Tadashi Ogishima, Akio Ito1998 Volume 124 Issue 5 Pages 1045-1049
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously identified distal and proximal arginine residues in the N-terminal portion and an aromatic amino acid at position 1(P1' site3) relative to the cleavage site as important recognition signals in substrates of mitochondrial processing peptidase [Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24714-24722; Ogishima, T., Niidome, T., Shimokata, K., Kitada, S., and Ito, A. (1995) J. Biol. Chem. 270, 30322-30326]. To further elucidate the elements required for the specific recognition and cleavage by the enzyme, we synthesized synthetic peptides that possessed only the distal and proximal arginine residues and phenylalanine at the P1' site in a poly alanine sequence, and analyzed the processing reaction toward them. They were not cleaved by the peptidase although they inhibited the peptidase activity. However, when serine was introduced into the C-terminal portions of the sequence, processing was observed. The efficiency of the resultant peptides improved as the number of serine residues was increased. A peptide with serine or histidine at P2' and threonine at P3' was processed most efficiently. These results indicate that the processing reaction catalyzed by the peptidase depends not only on the N-terminal portion but also on the C-terminal portion from the cleavage site in the substrates.View full abstractDownload PDF (616K)
- |<
- <
- 1
- >
- >|