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Volume 120, Issue 1
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Shuichi Tsuji1996 Volume 120 Issue 1 Pages 1-13
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the regulatory mechanism for carbohydrate expression and to understand the meaning of the carbohydrate-structural diversity, we started to clone sialyltransferase (ST) genes based on two different strategies, i.e. expression and homology cloning. So far, 13 STs have been cloned in our laboratory, 7 of which turned out to be new ones. The primary enzyme structures deduced from all the cloned ST genes suggest a putative domain structure with a type II transmembrane topology. There are no significant amino acid sequence similarities among these cloned STs, except for in two sialyl motifs, L and S, which are proposed to be the CMP-sialic acid recognition and/or catalytic sites. Northern blot analysis revealed the developmental stage-dependent and/or tissue-specific expression of most of the cloned STs. The cloned STs are classified into four families according to the carbohydrate linkages they synthesize, i.e. the ST3Gal-, ST6Gal-, ST6Ga1NAc-, and ST8Sia-families. Generally, enzymes in these families exhibit strong activity toward certain acceptor groups but show very weak activity toward other acceptor groups, and the substrate specificities of the enzymes overlap one another, as indicated by in vitro experiments. Enzymes in the ST3Ga1-family are expressed mainly in a tissue-specific manner. However, those in the ST6Ga1NAc- and STSSia-families are expressed in a tissueas well as developmental stage-specific manner. In vivo conditions are supposed to be more complex. Therefore, it is quite important to examine their substrate specificities in vivo and the mechanism of their expression to elucidate the physiological role of each enzyme and the meaning of the diversity in carbohydrate structure. Using cloned cDNAs and expressed enzymes, we have been studying how sialylcarbohydrate expression is regulated and what the functions of sialylcarbohydrate chains are. Recently, we found that transfection of the GD 3 synthase, an α2, 8-ST (ST8Sia I), gene triggers cholinergic neuritogenesis in Neuro2a cells through the de novo expression of GD 3, suggesting that the GD 3 synthase gene behaves as a neural differentiation inducer.View full abstractDownload PDF (4264K) -
Hisashi Mizutani, Ikuko Miyahara, Ken Hirotsu, Yasuzo Nishina, Kiyoshi ...1996 Volume 120 Issue 1 Pages 14-17
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (1196K) -
Katsuhito Takahashi, Tetsushi Tazunoki, Tsutomu Okada, Kinya Ohgami, T ...1996 Volume 120 Issue 1 Pages 18-21
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe human smooth muscle cell (SMC) calponin gene, which is composed of seven exons and six introns, spanning an approximately 11.2 kilobase (kb) genomic DNA, has been isolated and characterized by sequence analysis. As determined by primer extension mapping and rapid amplification of mRNA transcripts, a major transcription start site of the calponin gene is located at 101 base-pairs (bp) upstream of the ATG start codon. A striking feature of the 5'-flanking, 5'-untranslated, and amino-terminal protein coding regions is the presence of an Alu repetitive sequence and a stretch of DNA sequence identified using a methyl CpG-binding protein affinity column [Cross, S. H., Charlton, J. A., Nan, X., and Bird, A. P. (1994) Nature Genet. 6, 236-244]. The results of this study provide a new insight into the molecular mechanism underlying regulation of SMC-lineage specific gene expression.View full abstractDownload PDF (1938K) -
Sang Eun Lee, Jae Myoung Suh, Scott Scheffter, Jean L. Patterson, In K ...1996 Volume 120 Issue 1 Pages 22-25
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDouble-stranded Leishmania RNA virus 1-4 (LRV 1-4) has at least four open reading frames (ORFs). The two small ORFs located near its 5' terminus, ORF 1 and ORFx, could encode 34- and 60-amino acid polypeptides, respectively. ORF 2 encodes an 82-kDa major capsid protein, and ORF3 encodes a 98-kDa polypeptide which contains the consensus sequence for RNA-dependent RNA polymerases of plus-strand and double-stranded RNA viruses. The complete sequence of LRV 1-4 shows that ORF 2 and ORF 3 overlap by 71 nucleotides, and that ORF3 lacks a potential translation initiation site, suggesting that the viral polymerase may be synthesized as a 180-kDa fusion protein with the virus capsid. In this report, we present evidence for the synthesis of a fusion protein through a ribosomal frameshift. In vitro-translation experiments and immunostudies involving antiserum against the viral capsid protein demonstrated that the overlapping 71 nucleotides of ORF 2 and ORF 3 are contained in a region which promotes translational frameshifting. Computer analysis of the putative frameshift region revealed a potential pseudoknot structure located within the overlapping 71 nucleotide sequence.View full abstractDownload PDF (1754K) -
Hiroaki Sakai, Noriyoshi Sakabe, Kyoyu Sasaki, Wataru Hashimoto, Hidey ...1996 Volume 120 Issue 1 Pages 26-28
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structure of GGT [EC 2. 3. 2. 2] from E. coli K-12 was studied at 3 Å resolution by X-ray crystallography. Initial protein phases were calculated using two kinds of Pb2+ derivatives. The phases were refined by non-crystallographic 2-fold symmetry electron density averaging combined with solvent flattening and histogram matching. The GGT molecule has overall dimensions of 60×50×40 Å There are two antiparallel β-pleated sheets consisting of 6 and 7 β-strands. The two β-sheets form a wall-like structure. Twelve short α-helices were detected, of which the maximum length appears to be four helix turns.View full abstractDownload PDF (2863K) -
Akihiro Iwamatsu, Nobuko Yoshida-Kubomura1996 Volume 120 Issue 1 Pages 29-34
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sequential in situ digestion of proteins immobilized on polyvinylidene difluoride (PVDF) has been systematically designed and optimized. The method consists of immobilization of the proteins on PVDF, S-carboxymethylation, and then successive in situ digestions using specific proteases. In order to obtain high yields of the peptide fragments, from which specific amino acid residues connected to the N- or C-terminal of the resulting digestion fragments can be deduced, the cleavages are performed in the following order: (1) Achromobacter protease I (API), (2) endoproteinase Asp-N, and (3) trypsin. Procedures for recovering the numerous fragments remaining on the PVDF membrane after the third digestion with trypsin are also discussed. Application of sequential in situ digestion for the acquisition of fragments suitable for sequencing from digests of large-molecular-weight proteins is also presented.View full abstractDownload PDF (2842K) -
Taishin Takuma, Tokuro Ichida, Noriko Yokoyama, Shinri Tamur, Takashi ...1996 Volume 120 Issue 1 Pages 35-41
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCofilin is an actin-depolymerizing protein, whose depolymerizing activity is supposed to be regulated in part by phosphorylation and dephosphorylation. Thus, we studied the phosphorylation states of cofilin in rat parotid acinar cells during stimulation for amylase exocytosis. Isoproterenol and carbachol induced rapid and extensive dephosphorylation of cofilin; 60-70% dephosphorylation was clearly detectable within 1min. Membranepermeable cyclic AMP (CPS-cAMP), phorbol ester (PMA), and Ca ionophore A 23187 mimicked the effect of isoproterenol and carbachol. Protein phosphatase inhibitors (calyculin A or FK 506 plus cyclosporin A) did not block the dephosphorylation in response to isoproterenol or carbachol. Furthermore, calyculin A alone strongly dephosphorylated cofilin. Although no exogenous protein phosphatases tested dephosphorylated cofilin in the homogenate, the cofilin that was isolated by immunoprecipitation was clearly dephosphorylated by protein phosphatases 1, 2 A, and 2 C.View full abstractDownload PDF (5953K) -
Miyuki Shimoji, Susumu Itoh, Kenji Toide, Kazuo Nakayama, Shinichi Aiz ...1996 Volume 120 Issue 1 Pages 42-48
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCYP3A rapidly disappears in primary hepatocytes, although the primary cells are suitable for studies of the regulation of n CYP3A genes. In the present study, we found that Cyp3a mRNA could be expressed in the primary hepatocytes from p53-knockout mice for at least 2 weeks when the cells were cultured in the presence of dexamethasone. Propoxycoumarin O-depropylase activity, which is known to be mainly catalyzed by CYP3A, was maintained at a level of 50% of the initial activity even after 5 days of culture, and the activity correlated with the expression level of Cyp3a mRNA in the primary hepatocytes from p53-knockout mice. The cells remained morphologically intact during 4 weeks. These results suggest that hepatocytes from p53-knockout mice are a useful tool for studies of the expression of Cyp3a.View full abstractDownload PDF (2921K) -
Shun-ichi Kurata, Midori Matsumoto, Uki Yamashita1996 Volume 120 Issue 1 Pages 49-52
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDuring in vitro activation of mouse peritoneal macrophages with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), their synthesis of peroxynitrite and their cytostatic activity against mouse lymphocytic leukemia (L 1210) cells were examined. The activation of the genes for nitric oxide synthase (iNOS) and heme oxygenase (HO-1) was also determined during the activation of the macrophages. Results showed that activation of peroxynitrite synthesis in macrophages was accompanied by the transcriptional activation of iNOS and HO-1 genes. Both genes seem to be activated simultaneously upon activation of the macrophages. Simultaneous activation of iNOS and HO-1 genes may be important because degradation of heme by HO-1 is one of the most important reaction that produces CO in higher organisms, and nitric oxide (NO) and carbon monoxide (CO) can react with heme-containing guanylate cyclase.View full abstractDownload PDF (3100K) -
Tilmann Achsel, Yoshiro Shimura1996 Volume 120 Issue 1 Pages 53-60
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe excision of introns with weak polypyrimidine tracts at their 3' splice sites can be enhanced by sequence elements in the downstream exon or by a downstream 5' splice site. The enhancers inside the exon do not conform to a strict consensus, but they are generally rich in purines. Here, we show that members of the family of SR proteins recognize these elements. Not only does SF2/ASF activate many different polypurine enhancers, but also at least one other SR protein, most likely SC 35, is active as well. The degree of splicing activation varies with the polypurine enhancers and the SR proteins. Further, we show that the similar activation by downstream 5' splice sites requires U 1 snRNP, which is not the case with purine-rich enhancers. These results are consistent with a model showing that U 1 snRNP binds to the 5' splice site and SR proteins to exonic sequences upstream of the 5' splice site. Both interact with U2AF at the 3' splice site. This represents a molecular explanation for the exon recognition which is important for splice site selection in mammals.View full abstractDownload PDF (4892K) -
Tsutomu Hashizume, Masahiko Nakao, Takashi Satn1996 Volume 120 Issue 1 Pages 61-65
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe modulating effect of sphingosine on the metabolism of inositol phospholipids was investigated using rabbit platelets. When [3H] arachidonic acid- or [3H] inositol -labeled platelets were incubated at 37°C with sphingosine, the radioactivity of the phosphatidylinositol (PI) fraction obtained on TLC decreased time-dependently up to 5min, and phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) increased concomitantly, though neither arachidonic acid nor 1, 2-diacylglycerol was formed. The effect of sphingosine was dose-dependent, the maximum effect being observed at 20 μM. Treatment with a sphingosine derivative, sphingosine-l-phosphate (Sph-1-P) or N-hexanoyl-sphingosine (C6-ceramide), did not result in an increase in PIP. The increased radioactivity of PIP with sphingosine was attributable to an increase in phosphatidyl-inositol 4-phosphate, but not phosphatidylinositol 3-phosphate. Furthermore, wortmannin, an inhibitor of PI 3-kinase, did not affect the modulating effect of sphingosine at 100 nM, at which the enzyme is known to be completely inhibited. The activity of PI 4-kinase in the platelet lysate was increased by sphingosine but not by Sph-1-P. These results suggest that sphingosine enhances the activity of PI 4-kinase and thereby contributes to the regulation of inositol phospholipid metabolism.View full abstractDownload PDF (726K) -
Hidevoshi Hiaashi, Sachivo Yoshida, Kazuki Sato, Tatsuva Yamagatat1996 Volume 120 Issue 1 Pages 66-73
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the interaction between gangliosides and synthetic peptides of calmodulin (CaM)-dependent enzymes to confirm the hypothesis that interaction between gangliosides and the CaM-like site (CLS) of the enzyme is a mechanism for the modulation of the enzyme activity by gangliosides. Gangliosides, GD1b, GT1b, and GD1a, inhibited the activity of CaM-independently activated cAMP-phosphodiesterase and their inhibition was cancelled by a peptide consisting of 17 amino acid residues of a plasma membrane Ca2+-pump CLS, suggesting the involvement of the interaction between the peptide and the gangliosides. The peptide of an assumed CLS of phosphodiesterase also cancelled the inhibition. On the other hand, the gangliosides interacted with synthetic CaM-binding site (CBS) peptides of phosphodiesterase, calcineurin, Ca2+-pump, and Ca2+/calmodulin-dependent protein kinase II. Moreover, gangliosides GM 3 and LM 1, that activate but do not inhibit phosphoiesterase, interacted with the CBS peptides, whereas they did not bind to CLS peptides. On the basis of these new findings, we propose a revised model for the ganglioside-mediated modulation of CaM-dependent enzymes, i.e. without CaM, gangliosides bind to CBS and thus stimulate the enzyme activity, acting like CaM. At higher concentrations, they bind to CLS of the enzymes as well and inhibit the activity, acting like the CBS of the enzyme.View full abstractDownload PDF (983K) -
Rachel L. C. Chuang, Jeff C. F. Chen, Jessie Chu, Jason T. C. Tzen1996 Volume 120 Issue 1 Pages 74-81
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body stores a matrix of triacylglycerols surrounded by phospholipids and alkaline proteins termed oleosins. Oil bodies in rice seeds are present in embryos and aleurone layers. They do not coalesce in crowded environments, as observed on electron microscopy. The detected isoelectric point of purified rice oil bodies is pH 6.2. This implies that rice oil bodies possess a negatively charged surface at neutral pH. The suspension of rice oil bodies in pH 6.5 buffer induces aggregation. Presumably, the negatively charged surface causes electrostatic repulsion that maintains rice oil bodies as discrete organelles. Rice oil bodies lose their integrity on trypsin treatment. Undoubtedly, oleosins play an important role in the stability of oil bodies. There are two oleosin isoforms in rice oil bodies. Antibodies raised against these two homologous isoforms do not cross-recognize each other. Both isoforms are restricted to oil bodies, as detected on immuno-assaying. Partial amino acid sequences of these two isoforms were obtained, and compared with the deduced sequences of two maize and two rice oleosin genes. The comparison confirmed that the two major proteins in rice oil bodies are the two oleosin isoforms.View full abstractDownload PDF (5908K) -
Yoshiaki Azuma, Hiroaki Seino, Takashi Seki, Satoru Uzawa, Christian K ...1996 Volume 120 Issue 1 Pages 82-91
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCharged amino acid residues of human RCC 1 were converted to alanine and mutants which were unable to complement tsBN 2 cells (a temperature-sensitive reel- mutant of the hamster BHK 21 cell line) were selected. These RCC 1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN 2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC 1 mutants were unstable in tsBN 2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC 1 repeat showed an increased Km, while their kcat values were comparable to that of wild-type RCC 1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC 1 repeat showed a value of Km similar to that of wild-type RCC 1, while the kcat values of these mutants were reduced, depending upon the RCC 1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC 1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcal among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.View full abstractDownload PDF (4315K) -
Hitoo Iwase, Atsushi Tanaka, Yoshiyuki Hiki, Tohru Kokubo, Ikuko Ishii ...1996 Volume 120 Issue 1 Pages 92-97
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA 1. In our previous report, only one glycoform was obtained from the IgA1 of healthy individuals. However, it was found to be composed of heterogeneous IgAl components having mutually different glycoforms. First, the IgA 1 was separated into two subfractions having different affinities toward jacalin. Among them, the high-affinity subfraction was mainly composed of polymerized IgA1. Comparative study of the carbohydrate chain showed a relative abundance of Galβ1, 3GalNAc in the polymerized form. A simultaneous analysis of the N-glycan of these subfractions was also carried out. Three major components, two biantennary and one triantennary oligosaccharides, were obtained from both subfractions and the relative contents of these components were almost the same. On the other hand, IgA 1 was artificially polymerized by heating at 63°C for 2h. The heat-stable IgA 1 was separated from the heat-aggregated material on a Sephacryl S-300 column. The obtained heat-stable IgA 1 (approximately 20%) was not further aggregated by more heating under the same conditions. The heat-stable IgA 1 contained a much higher amount of the sialylated Galβ1, 3GalNAc. Thus, it was shown that the degree of completeness of the hinge O-linked oligosaccharide might be correlated with the stability and polymerization process of the IgAl molecule.View full abstractDownload PDF (664K) -
Koji Ishiguro, Kazunori Sugimoto1996 Volume 120 Issue 1 Pages 98-103
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInduction of the expression of the β-lactamase gene, blaB, is regulated by the blaA gene located just upstream of blaB in the opposite direction in Proteus vulgaris. The expression of the blaA gene is negatively autoregulated by its own product BlaA, the activator of the blaB gene. The P. vulgaris BlaA protein shares high amino acid homology with the LysR family members, which are prokaryotic transcriptional activators that possess a putative helix-turn-helix DNA binding motif. To characterize its function, we purified the BlaA protein to homogeneity from Escherichia coli carrying the expression plasmid of the blaA gene driven by the tac promoter. The gel shift assay and DNasel footprinting showed that purified BlaA specifically bound to the blaA promoter region, which resides immediately upstream of that of blaB. The binding region contained an inverted repeat, including a T-N11-T sequence which is similar to the LysR motif (T-N11-A) that is conserved in some LysR family members [Goethals et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1646-1650]. We also showed that the BlaA protein forms a dimer in solution, using glycerol gradient centrifugation and glutaraldehyde crosslinking.View full abstractDownload PDF (2795K) -
Takeshi Yamamoto, Motoko Nakashizuka, Hisashi Kodama, Yasuhiro Kajihar ...1996 Volume 120 Issue 1 Pages 104-110
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA bacterial sialyltransferase, named sialyltransferase 0160, was purified from a marine bacterium that had been isolated from seawater from Sagami Bay, Kanagawa. This strain has been identified as Photobacterium damsela, and named P. damsela JT0160. Sialyltransferase 0160 was purified 688-fold to homogeneity from the crude extract of the cells with a yield of 19% using a combination of anion exchange chromatography, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography. The purified enzyme migrated as a single band (61 kDa) on sodium dodecyl sulfate-polyacryl-amide gel. This sialyltransferase was found to be a β-galactoside α2, 6-sialyltransferase [EC 2. 4. 99. 1] which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6 on the basis of an analysis of the enzymatic reaction products with HPLC, 1H-, 13C-NMR spectroscopy, and fast atom bombardment mass spectroscopy.View full abstractDownload PDF (839K) -
Lee-Ming Chuang, Tong-Yuan Tai, Ronald C. Kahn, Huey-Peir Wu, Sheng-Ch ...1996 Volume 120 Issue 1 Pages 111-116
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIRS-1 has been found to relay the signals from the receptors for insulin, insulin-like growth factor-1, growth hormone, and many cytokines for the downstream effects in the various cell types tested. For interleukin 4 signaling, most studies were performed on hematopoietic cells and cell lines transfected with rat liver IRS-1 cDNA. In a liver cell lineage, IRS-1 expression has been found to be increased in hepatoma cells and hepatocytes in regenerating liver. To elucidate the possible function and the signal transduction pathway for interleukin 4, in comparison with insulin, in liver cells, we used the Hep 3B hepatoma cell line as a model system. Following insulin and interleukin 4 stimulation, rapid tyrosyl phosphorylation of IRS-1 occurred. Interleukin 4, but not insulin, stimulated the tyrosine phosphorylation of JAK1 and, to a lesser extent, JAK2. In contrast to the other cell types, the association of IRS-1 and Grb2 through the SH2 of Grb2 was demonstrated after IL-4 and insulin stimulation of the Hep3B hepatoma cells. Both insulin and interleukin 4 stimulated tyrosine phosphorylation and the enzyme activity of Erk1 kinase. Our results indicate that interleukin 4 and insulin might modulate hepatic cell growth and differentiation through many different or common pathways for the activation of JAK kinases and the usage of IRS-1 as a docking protein. The binding of IRS-1 with Grb2 after IL-4 as well as insulin stimulation may lead to MAP kinase activation, probably through the Grb2/sos/p21ras pathway.View full abstractDownload PDF (4233K) -
Kiyoshi Nakazawa, Ayako Morita, Hiroe Nakano, Chizuko Mano, Nahoko Toz ...1996 Volume 120 Issue 1 Pages 117-125
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe corneal stromal cells from 2-day-old chicks were cultured on plastic dishes or within three-dimensional collagen gel in the presence or absence of growth factor (EGF, bFGF, PDGF, TGF-β1, or their combinations). The cells were labeled with [35S]sulfate and [3H]-glucosamine, and the radio-labeled proteoglycans were examined. Keratan sulfate was synthesized to some extent (15.4-16.9% of total synthesis for medium fraction; 8.0% for cell layer fraction) in a primary culture even when the cells were cultured on plastic dishes, although the values were very much lower than that (42.7%) in the stromal fraction of organ culture of corneal explants. The primary culture in collagen gel showed some increase in the proportion of keratan sulfate synthesis as compared with the culture on plastic. Among growth factors, addition of EGF to the culture in gel caused a further increase in the proportion of keratan sulfate synthesis. bFGF and TGF-β1 increased proteoglycan synthesis as a whole to some extent, but chondroitin sulfate/dermatan sulfate synthesis was increased preferentially and, consequently, the proportion of keratan sulfate synthesis to total synthesis was decreased. PDGF also caused some decrease in the proportion. In the culture after one passage (secondary culture), the keratan sulfate synthesis decreased markedly (8.6-8.3% of total synthesis for medium fraction; 2.7% for cell layer or gel fraction) and a large chondroitin sulfate/dermatan sulfate proteoglycan appeared whether the cells were cultured on plastic or in collagen gel. But, when the medium was changed to CG medium (serum-free medium) in the middle of either primary or secondary cultures, the keratan sulfate synthesis (27.8% for medium fraction; 15.6% for gel fraction) was maintained at the level of that of the primary culture in gel. EGF and bFGF were not additive to the effect of CG medium on the keratan sulfate synthesis in the secondary culture. Instead, EGF and bFGF stimulated hyaluronic acid synthesis in the culture. The mechanism of these changes in the expression type of proteoglycan and their significance remain to be clarified.View full abstractDownload PDF (1177K) -
Yasuhiko Yamamoto1996 Volume 120 Issue 1 Pages 126-132
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNMR signals arising from the HisB5 NδH and HisEF5 NεH protons in sperm whale skeletal and horse heart myoglobins have been located for the first time in the downfield shifted portion of the spectra. The shifts and hydrogen exchange rates indicate that these His imidazole ring NH protons are involved in the inter-segmental hydrogen bonds of the protein in solution, as demonstrated by a crystallographic study [Takano, T. (1977) J. Mol. Biol. 220, 381-399]. The assigned His imidazole ring NH proton resonances can serve as new sensitive structural probes in the study of the local conformation of myoglobin. The applicability of the NMR spectral parameters in the study of the tertiary structure of apomyoglobin, the denaturation of the protein, and the protein stability of sperm whale and horse myoglobins is presented in some detail.View full abstractDownload PDF (914K) -
Masao Iwata, Yoshikazu Sado, Tasuku Sasaki, Yasutada Imamura, Yoshifum ...1996 Volume 120 Issue 1 Pages 133-137
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe recently reported that the bovine lens capsule contained a shorter α1(IV) chain (160 k) as a major polypeptide in addition to the 180 k α1(IV) chain [J. Biochem. (1995) 117, 1298-1304]. Two experiments were performed to examine whether or not the 160 k polypeptide retained the carboxyl-terminal NC1 domain. On immunoblotting analysis with a monoclonal antibody (H11) raised against the NC1 domain of the human α1(IV) chain [positions 1643-1650; near the carboxyl-terminal end of the human α1(IV) chain], the 180 k and 160 k polypeptides showed identical immunoreactivity, suggesting that the two chains had the same human α1(IV) collagen NC1 domain sequence. Another monoclonal antibody (H21) specific for the NC1 domain of human α2(IV) did not react with these polypeptides, but with the bands corresponding to 175 k and 155 k. The 160 k polypeptide was selectively solubilized from bovine lens capsules, leaving the other major polypeptides, 180 k and 175 k, insoluble. The 160 k polypeptide was separated by preparative electrophoresis. Bacterial collagenase digestion of the separated 160 k polypeptide produced collagenase-resistant segments of about 29 k and 30 k in size based on globular standards. These sizes corresponded well with those of the NC1 domains of type IV collagen α chains (25-30 k). The results indicated that the 160 k polypeptide retained the carboxyl-terminal NC1 domain of the α1(IV) chain. In turn, the 20 k polypeptide of the amino-terminal region or the 7 S domain of 180 k α1(IV) would have been excised to yield 160 k α1(IV), assuming that the 160 k α1(IV) chain is a processed form of the 180 k α1(IV) one and not an alternatively spliced chain of the α1(IV) gene.View full abstractDownload PDF (1843K) -
Koji Yamada, Pham Hung, Kazunari Yoshimura, Saburo Taniguchi, Beong Ou ...1996 Volume 120 Issue 1 Pages 138-144
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of UFA on Ig production by rat MLN lymphocytes was examined to clarify their influence on allergic reactions. A 4-h treatment at 1mM inhibited the production of IgA, IgG, and IgM by the lymphocytes, but stimulated IgE production. The IgE production-stimulating activity became stronger with increasing number of carbon atoms and/or double bonds. However, no saturated fatty acid with carbon numbers from 12 to 18 affected IgE production by the lymphocytes. Hydrogen peroxide exerted Ig production-regulating activity similar to that of UFA, suggesting that the effect of UFA is at least partly due to oxidation products. Thus, the effect of antioxidants on the Ig production-regulating activity of arachidonic acid was examined. α-Tocopherol and BHT annulled the stimulation of IgE production by arachidonic acid, but ascorbic acid was not effective. The IgE production-enhancing activity of UFA was closely related to their oxidation rate in culture medium. These results suggest that UFA enhance the allergic reaction through the stimulation of IgE production and the inhibition of IgA production, and that hydrophobic antioxidants are partially effective to annul the adverse effect of UFA.View full abstractDownload PDF (744K) -
Takashi Yamamoto, Kazuhiko Yamamoto, Hyogo Sinohara1996 Volume 120 Issue 1 Pages 145-152
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUrinary excretion of trypsin inhibitor increased after injection of a carcinogen, N-nitrosobis(2-oxopropyl)amine, into Syrian hamsters. Two inhibitors were purified to apparent homogeneity from urine collected during the course of the carcinogenesis experiment. Their complete amino acid sequences were determined by Edman degradation of the intact proteins and partially degraded fragments. One corresponded to a hamster liver cDNA clone that hybridized with human bikunin probe [Ide et al. (1994) Biochim. Biophys. Acta 1209, 286-292], except that the protein sequence lacked C-terminal serine and the other was trypstatin, the C-terminal half of the bikunin molecule. Three proteins containing covalently linked bikunin were also identified in pooled blood plasma. They were all dissociated into heavy and light chains by treatment with chondroitinase ABC or 50mM NaOH, but not by heating at 100°C in the presence of sodium dodecyl sulfate and dithiothreitol. N-terminal amino acid sequence analyses of the native chains and partially degraded fragments thereof revealed that these proteins are (i) human-type inter-α-trypsin inhibitor, consisting of heavy chains 1 and 2 and bikunin, (ii) bovine-type inter-α-trypsin inhibitor, consisting of heavy chains 2 and 3 and bikunin, and (iii) pre-α-trypsin inhibitor, consisting of heavy chain 3 and bikunin. Heterodimer of bikunin/heavy chain 1 or bikunin/heavy chain 2 was not detected. These results suggest that the composition, and hence function, of the inter-α-trypsin inhibitor family differs considerably from species to species.View full abstractDownload PDF (3854K) -
Kenji Sugimoto, Taku Yamada, Yoshinao Muro, Michio Himeno1996 Volume 120 Issue 1 Pages 153-159
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal component tightly associated with the pericentromeric heterochromatic region of fruit fly, mouse, and human throughout the cell cycle. Drosophila HP1 has been shown to be involved in position effect variegation and to be required for the correct chromosome segregation in vivo, while the biological activity of human homolog (HP1Hsα) has not yet been characterized. We previously reported that human CENP-B and CENP-C, two major centromere heterochromatin autoantigens often recognized by autosera in scleroderma patients, possess DNA-binding activity in vitro. Here, we show that human HP1, which is also an autoantigen targeted by some types of anticentromere autosera, is a DNA-binding protein. Human HP1 was expressed as a GST-fusion in Escherichia coli and purified with glutathione-Sepharose. The DNA-binding activity of the recombinant HP1 was demonstrated by gel mobility shift assay and South-Western-type blotting. The minimum DNA-binding region was further limited to the internal 64-amino acid stretch that is less-conserved between human and fruit fly but retains a helix-enriched motif with weak similarity to CENP-C. This suggests that HP1 is involved in the pericentromeric heterochromatin formation by directly associating with genomic DNA.View full abstractDownload PDF (4876K) -
Mitsutoshi Yoneyama, Wakako Suhara, Yukiko Fukuhara, Mayumi Sato, Keik ...1996 Volume 120 Issue 1 Pages 160-169
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInterferon regulatory factor (IRF)-1 and IRF-2 have been implicated for the virus-induced expression of the interferon-α and β (type I IFN) genes. However, recent gene disruption studies in mice suggested the presence of other factor(s) interacting with overlapping promoter elements. In the present paper, we describe the characterization of a DNA binding factor which is strongly induced after virus infection and recognizes these promoter elements. After extensive purification, the factor was revealed to be identical to IFN-stimulated gene factor 3 (ISGF3), a transcription factor complex activated by IFN treatment. ISGF3 binds to the promoter element of IFN-β, positive regulatory domain I (PRDI), with significantly higher affinity than IRF-1, 2, and mutational analysis of PRDI showed that the gene expression and binding of ISGF3, but not of IRF-1, 2, are highly correlated. Furthermore, our functional analysis involving a dominant negative inhibitor for ISGF3 activation and an anti-IFN neutralizing antibody clearly demonstrated the presence of a positive feedback pathway for type I IFN genes mediated by ISGF3.View full abstractDownload PDF (7772K) -
Asako Kameyama, Mark S. Shearman, Kazuo Sekiguchi, Masaki Kameyama1996 Volume 120 Issue 1 Pages 170-176
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe voltage-dependent L-type Ca2+ channel in the heart is regulated by cAMP-dependent protein kinase (PKA) and possibly by protein kinase C (PKC). We have investigated the channel modulation through phosphorylation by these protein kinases, using liposomes into which Ca2+ channels from bovine heart were reconstituted. Phosphorylation of the proteoliposomes with PKA increased the dihydropyridine-sensitive Ca2+ efflux from them by about 70%. PKA rapidly phosphorylated membrane proteins of 210 and 170 kDa. A dihydropyridine-class Ca2+ channel blocker, [3H] azidopine, specifically photo-labeled a protein of 210 kDa, suggesting that the 210-kDa phosphoprotein might be the α1 subunit of the Ca2+ channel. In contrast, phosphorylation of the proteoliposomes with PKC failed to modulate the Ca2+ efflux. Although PKC catalyzed the phosphorylation of membrane proteins of 150, 130, 95, 67, and 62 kDa, the 210- and 170-kDa proteins were not phosphorylated by this kinase. These results suggest that phosphorylation of the 210-kDa protein in the cardiac sarcolemma by PKA may be responsible for modulation of the channel function, whereas modulation of the channel by PKC, if it occurs, must be the result of an indirect mechanism, e. g. phosphorylation of a cytoplasmic protein or an associated channel polypeptide, that cannot function in the reconstituted system.View full abstractDownload PDF (3582K) -
Keiji Kasahara, Kaeko Hayashi, Tsutomu Arakawa, John S. Philo, Saburo ...1996 Volume 120 Issue 1 Pages 177-183
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of a white kidney bean (Phaseolus vulgaris) α-amylase inhibitor (PHA-I), which is composed of two kinds of glycopolypeptide subunits, α and β, was established by conventional methods. The polypeptide molecular weight of PHA-I determined by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that PHA-I has the subunit stoichiometry of (αβ)2 complex. Inhibition test of PHA-I with increasing amounts of porcine pancreatic α-amylase (PPA) suggested that an inactive 2:1 complex is formed between PPA and PHA-I. In fact, two complexes differing from each other in the molar ratio of PPA to PHA-I were separated by gel filtration, and molecular weight estimation by the light-scattering technique confirmed that they are complexes of PHA-I with one or two PPA molecules. The binding of PPA to PHA-I appeared to follow simple binomial statistics, suggesting that two binding sites on PHA-I are independent and of high affinity for PPA.View full abstractDownload PDF (858K) -
Tetsuo Adachi, Naoaki Morihara, Naoya Yamazaki, Harutaka Yamada, Arao ...1996 Volume 120 Issue 1 Pages 184-188
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMolecular genetic studies of extracellular-superoxide dismutase (EC-SOD) have shown that individuals with high serum EC-SOD content have a single base substitution generating the exchange of glycine for arginine-213 (R213G) in the heparin-binding domain of this enzyme [Sandstriöm, J. et al. (1994) J. Biol. Chem. 269, 19163-19166], which causes the impairment of its binding ability to endothelial cell surface [Adachi, T. et al. (1996) Biochem. J. 313, 235-239]. Serum EC-SOD in healthy individuals without the above mutation is heterogeneous with regard to heparin affinity and consists of five fractions, forms (I) to (V), of which (IV) and (V) are the main fractions with high affinity for heparin [Adachi, T. et al. (1995) J. Biochem. 117, 586-590], whereas the major fraction in hemodialysis patients was serum EC-SOD form (I), which is thought to be the proteolytic truncated form. On the other hand, serum EC-SOD in both healthy individuals and hemodialysis patients with the R213G mutation consisted mainly of the high heparin-affinity type. This observation suggests that the susceptibility of EC-SOD to proteinases is reduced by the R213G mutation. The affinity of normal EC-SOD (n-EC-SOD) for heparin decreased by the treatment with trypsin, accompanied by a reduction in the molecular mass. The IC50, of trypsin for the heparin affinity of R213G mutant EC-SOD (m-EC-SOD) was 0.15 μg/ml, fivefold that for n-EC-SOD. Heparin affinity of n-EC-SOD was again more susceptible to neutrophils than that of m-EC-SOD. These results suggested that m-EC-SOD is more resistant to trypsin and neutrophil-release trypsin-like proteinases than n-EC-SOD, which causes the heparin affinity of serum EC-SOD to differ in individuals with and without the R213G mutation.View full abstractDownload PDF (2065K) -
Tsuyoshi Katoh, Fumi Morita1996 Volume 120 Issue 1 Pages 189-192
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe dissociation constant for the binding of myosin subfragment 1 (S1) and chromatographed actin in the presence and absence of nucleotide was measured at various ionic strengths and various temperatures. The dissociation constant was of nM order in the absence of nucleotide and increased by _??_100- and _??_100, 000-fold in the presence of ADP and ATP, respectively. The dissociation constant also increased with increasing ionic strength, irrespective of the presence of nucleotide, and its dependence on the ionic strength was increased by the presence of ATP but decreased by the presence of ADP. The standard enthalpy change and entropy change for the binding of S1 to actin were both positive, irrespective of the presence of nucleotide, indicating that the binding was entropy-driven. The standard entropy change was essentially unaffected by the presence of ADP but was greatly decreased by ATP, suggesting that the large increase in the dissociation constant in the presence of ATP was due to the decrease of hydrophobic interactions. On the other hand, the increase in the dissociation constant for acto-S1 in the presence of ADP might be induced by the decrease of electrostatic interactions.View full abstractDownload PDF (453K) -
Ago Rinken1996 Volume 120 Issue 1 Pages 193-200
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSixteen different detergents were studied for solubilization of functional complexes between m2 muscarinic acetylcholine receptors (mAChR) and guanine nucleotide-binding regulatory proteins (G proteins). More than 40% of solubilized mAChR retained their GTP-dependent high affinity for agonist binding after solubilization with sucrose monolaurate, whereas all other detergents studied gave considerably lower solubilization yields or caused the loss of the high affinity for agonist binding. The preformation of mAChR-G protein complexes in membranes revealed that a large excess of G proteins did not increase the portion of high-affinity binding sites, but caused GTP- and Mg2+-dependent inhibition of the binding of radioactive antagonists to mAChR. The optimization of detergent concentration and other experimental conditions revealed that up to 47% of the solubilized receptors indicated the GTP-dependent high affinity for agonist binding after mixing solubilized mAChR with purified G proteins in sucrose monolaurate in the presence of Mg2+ and carbachol. These results give the first clear proof of the formation of functional complexes between mAChR and G proteins in solution and indicate that GTP-dependent high-affinity agonist binding is connected to the direct interactions between mAChR and G proteins and that other membrane components are not necessary.View full abstractDownload PDF (1138K) -
Mikio Tomida, Osamu Gotoh1996 Volume 120 Issue 1 Pages 201-205
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe human gene encoding the differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) receptor was cloned and its structure was analyzed. The gene spans more than 70 kilobases and contains 20 exons. The D-factor/LIF receptor can be subdivided into several regions: cytokine receptor homologous domain 1, an Ig-like domain, cytokine receptor homologous domain 2, three fibronectin type III domains, a transmembrane domain and a cytoplasmic region. Each domain of the receptor is encoded by a set of exons. There is a TATA sequence upstream of the transcription initiation site. One unit of the Alu sequence is present in the 5' flanking region. An NF-IL6 site is located 31 bases downstream of the transcription initiation site.View full abstractDownload PDF (621K)
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