All issues
Volume 131 (2002)
- Issue 6 Pages 767-
- Issue 5 Pages 629-
- Issue 4 Pages 495-
- Issue 3 Pages 283-
- Issue 2 Pages 167-
- Issue 1 Pages 1-
Volume 131, Issue 5
Displaying 1-19 of 19 articles from this issue
- |<
- <
- 1
- >
- >|
-
Hideo Kanoh, Keiko Yamada, Fumio Sakane2002 Volume 131 Issue 5 Pages 629-633
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDiacylglycerol kinase (DGK) regulates signal transduction by modulating the balance between the two signaling lipids, diacylglycerol and phosphatidic acid. DGK and its homologs occur in a wide range of multicellular organisms and the mammalian DGK is known to consist of nine members with a considerable incidence of alternative splicing. Recent work has established that DGK serves as a key attenuator of diacylglycerol of signaling functions and that the mammalian isozymes are equipped with molecular machineries which enable them to act in specific intracellular sites and/or in signaling protein complexes.View full abstractDownload PDF (1257K) -
Hiroyuki Arai, Hiroyuki Koizumi, Junken Aoki, Keizo Inoue2002 Volume 131 Issue 5 Pages 635-640
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPlatelet-activating factor (PAF) is one of the most potent lipid messengers involved in a variety of physiological events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity, and its deacetylation induces loss of activity. The deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH). A series of biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Type I PAF-AH is a G-protein-like complex consisting of two catalytic subunits (α1 and α2) and a regulatory β subunit. The β subunit is a product of the LIS1 gene, mutations of which cause type I lissencephaly. Recent studies indicate that LIS1/β is important in cellular functions such as induction of nuclear movement and control of microtubule organization. Although substantial evidence is accumulating supporting the idea that the catalytic subunits are also involved in microtubule function, it is still unknown what role PAF plays in the process and whether PAF is an endogenous substrate of this enzyme. Type II PAF-AH is a single polypeptide and shows significant sequence homology with plasma PAF-AH. Type II PAF-AH is myristoylated at the N-terminus and like other N-myristoylated proteins is distributed in both the cytosol and membranes. Plasma PAF-AH is also a single polypeptide and exists in association with plasma lipoproteins. Type II PAF-AH as well as plasma PAF-AH may play a role as a scavenger of oxidized phospholipids which are thought to be involved in diverse pathological processes, including disorganization of membrane structure and PAF-like proinfiammatory action. In this review, we will focus on the structures and possible biological functions of intracellular PAF-AHs.View full abstractDownload PDF (2297K) -
Tsuyoshi Katoh, Kaoru Konishi, Michio Yazawa2002 Volume 131 Issue 5 Pages 641-645
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH4+/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin sub-fragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.View full abstractDownload PDF (2108K) -
The First Molecular Evidence That Autophagy Relates Rimmed Vacuole Formation in Chloroquine MyopathyTakashi Suzuki, Masahiro Nakagawa, Ayumu Yoshikawa, Noboru Sasagawa, T ...2002 Volume 131 Issue 5 Pages 647-651
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSChloroquine myopathy is a drug poisoning disease involving rimmed vacuole formation. By Western blot analysis, we investigated posttranslational modification of LC3 in cultured cells with a high concentration of chloroquine, and found that the autophagosome membrane-bound form of LC3 increased dose-dependently. We also constructed a disease model by excessive chloroquine injection into rats and unusual immunohistochemical alteration was chased using anti-LC3 antibodies. With chloroquine treatment, muscle atrophy occurred predominantly in soleus muscle and unusual autophagosomes were accumulated. Therefore, we concluded that autophagy plays an important role in rimmed vacuole formation in certain muscular atrophies.View full abstractDownload PDF (2928K) -
Tomoko Nishioka, Kunihiko Sakumi, Tomofumi Miura, Kazuki Tahara, Hiden ...2002 Volume 131 Issue 5 Pages 653-661
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn this study, we established rat 3Y1 embryo cell lines expressing FosB and ΔFosB as fusion proteins (ER-FosB, ER-ΔFosB) with the ligand-binding domain of human estrogen receptor (ER). The binding of estrogen to the fusion proteins resulted in their nuclear translocation. After estrogen administration, exponentially growing cells expressing ER-ΔFosB, and to a lesser extent ER-FosB, underwent morphological alteration from the flat fibroblastic shape to an extended bipolar shape, and ceased proliferating. Such morphological alteration was also induced in quiescent cells expressing ER-ΔFosB and ER-FosB after one round of cell division triggered by estrogen administration. The cells expressing ER-ΔFosB changed shape frequently, and the content of F-actin in the cytoplasm detected by binding of Alexa 488-phalloidin significantly decreased after the morphological alteration. By two-dimensional gel electrophoresis analysis of cellular proteins from the cells expressing ER-ΔFosB, we identified several proteins whose expression either increased or decreased after estrogen administration. Two of these proteins were identified from their amino acid sequences as novel processed form of galectin-1.View full abstractDownload PDF (4638K) -
Shunsuke Kawamura, Gen Toshima, Taiji Imoto, Tomohiro Araki, Takao Tor ...2002 Volume 131 Issue 5 Pages 663-670
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)5, revealed remarkably enhanced production of (GlcNAc)2 and reduced production of (GlcNAc)1, as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)1 and (GlcNAc)2 was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)2 was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.View full abstractDownload PDF (1909K) -
Susumu Ichiyama, Tatsuo Kurihara, Masaru Miyagi, Andrey Galkin, Susumu ...2002 Volume 131 Issue 5 Pages 671-677
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSFluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp105 of the enzyme acts as a nucleophile to attack the α-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His272 [Liu, J. Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated con-comitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp105, into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for charac-terizing the active-site environment as well as for identifying the catalytic residue.View full abstractDownload PDF (848K) -
Hisaaki Mihara, Tomomi Fujii, Shin-ichiro Kato, Tatsuo Kurihara, Yasuo ...2002 Volume 131 Issue 5 Pages 679-685
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSEscherichia coli CsdB is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes both cysteine desulfuration and selenocysteine deselenation. The enzyme has a high specific activity for L-selenocysteine relative to L-cysteine. On the other hand, its paralog, IscS, exhibits higher activity for L-cysteine, which acts as a sulfur donor during the biosynthesis of the iron-sulfur cluster and 4-thiouridine. The structure of CsdB com-plexed with L-propargylglycine was determined by X-ray crystallography at 2.8 Å resolution. The overall polypeptide fold of the complex is similar to that of the uncomplexed enzyme, indicating that no significant structural change occurs upon formation of the complex. In the complex, propargylglycine forms a Schiff base with PLP, providing the features of the external aldimine formed in the active site. The Cys364 residue, which is essential for the activity of CsdB toward L-cysteine but not toward L-selenocysteine, is clearly visible on a loop of the extended lobe (Thr362-Arg375) in all enzyme forms studied, in contrast to the corresponding disordered loop (Ser321-Arg332) of the Thernzotoga maritima NifS-like protein, which is closely related to IscS. The extended lobe of CsdB has an 11-residue deletion compared with that of the NifS-like protein. These facts suggest that the restricted flexibility of the Cys364-anchoring extended lobe in CsdB may be responsible for the ability of the enzyme to discriminate between selenium and sulfur.View full abstractDownload PDF (908K) -
Hideo Akashi, Hiroaki Kawasaki, Won Jong Kim, Toshihiro Akaike, Kazuna ...2002 Volume 131 Issue 5 Pages 687-692
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe activity of a hammerhead ribozyme (Rz) in vivo depends on several factors, such as abundance, stability, and accessibility of Rz to its target mRNA. Among these factors, accessibility is believed to be the rate-limiting factor for Rz-mediated cleavage in vivo. As Rz and its substrate RNA are negatively charged, we examined whether cellular RNA-interacting proteins or artificial polycations might improve the accessibility of Rz to its substrate RNA. Specifically, we examined the effects of two kinds of cationic comb-type copolymer, αPLL-g-Dex, and a cellular RNA helicase on the accessibility of Rz to a model structured RNA in vitro. The cleavage activity of Rz was slightly enhanced by αPLL-g-Dex, probably due to an acceleration of the association/dissociation rate. And also, the RNA helicase-bound hybrid-Rz could cleave the target substrate at a signifi-cantly higher rate due to its unwinding activity for the duplex RNA substrate. These approaches should be useful in the development of efficient gene-inactivating reagents in the post-genomic era.View full abstractDownload PDF (4629K) -
Byeong Wook Jeon, Kee Tae Kim, Soo-Ik Chang, Hak Yong Kim2002 Volume 131 Issue 5 Pages 693-699
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSApoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.View full abstractDownload PDF (3644K) -
Tadahiro Ohmura, Hiroyuki Motoshima, Tadashi Ueda, Taiji Imoto2002 Volume 131 Issue 5 Pages 701-704
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA mutant lysozyme in which Arg14 and His15 were deleted together exhibited higher activity toward glycol chitin than the wild-type lysozyme. Moreover, the mutant lysozyme, which is less stable than the wild-type lysozyme by 7°C, showed a shift of temperature dependence of activity to the low temperature side compared with the wild-type lysozyme [Protein Eng. 7, 743-748 (1994)]. In the free enzyme, the internal motion of the mutant lysozyme was similar to that of the wild-type. The internal motions of the wild-type and mutant lysozymes in the enzyme-substrate complex increased more than those in the free enzymes. Moreover, the increased internal motions of the substrate-complexed mutant lysozyme were greater than those of the substrate-complemed wild-type lysozyme in several residues [J. Mol. Biol. 286, 1547-1565 (1999)]. The structure of the mutant lysozyme was very similar to that of the wild-type lysozyme. Both structures were also alike in the complex of the trimer of N-acetyl-D-glucosamine. The mobility from B-factors agreed to some degree with that from order parameters in the regions showing great mobility of the protein, but this was not the case in the regions showing fast motion. However, we came to the same conclusion that the increased activity of the mutant lysozyme is due to the increase in the fluctuation of the lysozyme molecule. B-factor and order parameter do not always exhibit harmony because the time-scale of the analysis of mobility is different. However, they are not incompatible but complementary for detecting precise protein motions.View full abstractDownload PDF (550K) -
Shungo Deshimaru, Yasuo Miyake, Tadamasa Ohmiya, Yoshiro Tatsu, Yasuko ...2002 Volume 131 Issue 5 Pages 705-712
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain con-sisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2•cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine under-went dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.View full abstractDownload PDF (3414K) -
Umechiyo Tokumoto, Shinobu Nomura, Yoshiko Minami, Hisaaki Mihara, Shi ...2002 Volume 131 Issue 5 Pages 713-719
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)6-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Niaffinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.View full abstractDownload PDF (942K) -
Jin Han, Nari Kim, Juhee Park, Dae-Hyun Seog, Hyun Joo, Euiyong Kim2002 Volume 131 Issue 5 Pages 721-727
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe purpose of this study was to determine whether ATP-sensitive potassium channel (KATP channel) activation generates oxygen free radicals in the rabbit heart. We assayed malondialdehyde (MDA) in rabbit heart slices in vitro as an indicator of oxygen free radical generation. The KATP channel openers, pinacidil and cromakalim, significantly in-creased MDA production in a concentration-dependent manner. MDA formation also increased linearly with incubation time in the presence of KATP channel openers. The KATP channel blockers, glibenclamide and 5-hydroxydecanoate (5-HD), decreased KATP channel opener-induced MDA formation in a concentration-dependent manner. When Fe2+ was administered to heart slices that had been pretreated with KATP channel openers, a marked elevation in MDA was observed, compared to heart slices that were treated with Fe2+ alone. A positive linear correlation between Fe2+ and MDA level was observed. The MDA levels of heart slices subjected to anoxia for 15 min remained unchanged until reperfusion. When the heart slices were reoxygenated for 30 min, a marked increase in MDA formation was observed. However, in the presence of gliben-clamide and 5-HD, reperfusion following anoxia did not result in increased MDA. These results suggest that the opening of mitochondrial KATP channels in rabbit heart slices evokes oxygen free radical generation via a Fenton-type reaction.View full abstractDownload PDF (892K) -
Seishiro Nozaki, Yoshihisa Tomioka, Takanori Hishinuma, Masayuki Inoue ...2002 Volume 131 Issue 5 Pages 729-738
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSNeocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 ± 0.3 μM was obtained for recombinant apo-NCS in the fluo-rescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer poly-merase chain reaction, the association of the apo-NCS mutant and EtBr observed on flu-orescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (ΔITmax) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific pro-teolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.View full abstractDownload PDF (6074K) -
Iwao Ohtsuki, Fumie Shiraishi2002 Volume 131 Issue 5 Pages 739-743
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe investigated the distribution of troponin C•I and troponin I along tropomyosin-actin filaments by immunoelectron microscopy and found that anti-troponin I antibody form-ed transverse striations at 38 nm intervals along the bundle of filaments of both tropo-nin C•I-tropomyosin-actin and troponin I-tropomyosin-actin. Since the length of 38 nm corresponds to the repeating period of filamentous tropomyosin along actin double strands, the present study indicates that troponin I is located at a specific region of each tropomyosin, suggesting that a specific interaction between troponin I and tropomyosin is involved in determining the periodic distribution of troponin I along tropomyosin-actin filaments.View full abstractDownload PDF (3908K) -
Katsuyoshi Matsunami, Shuji Miyagawa, Daisuke Fukuta, Ryota Shirakura2002 Volume 131 Issue 5 Pages 745-749
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHerpes simplex virus type 1 (HSV-1) encodes several immuno-regulatory proteins that allow it to escape from the human immune system. The regulatory function of a HSV-1 glycoprotein gC (HSV-gC1) molecule on complement-mediated swine endothelial cell (SEC) lysis was investigated. The HSV-gC1 gene was obtained by the PCR method from the HSV-1 genome. The complement-regulatory function of this molecule was analyzed by cytotoxicity assay, using Chinese hamster ovarian tumor (CHO) cell and SEC trans-fectants and six human serum samples. FACS and Western blot analysis revealed the expression of the HSV-gC1 molecule on the transfectants. The CHO cell transfectants showed significant resistance to cell lysis by the sera that did not contain the anti-HSV-gC1 antibody. The SEC transfectants, however, showed a marked resistance to cell lysis in all cases. The introduction of a viral immune regulator such as HSV-gC1 into the swine cell provides a new approach for successful xenotransplantation.View full abstractDownload PDF (1855K) -
Hiromiki Kuwahara, Takayuki Yamasaki, Tomomitsu Hatakeyama, Haruhiko A ...2002 Volume 131 Issue 5 Pages 751-756
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCEL-III is a Ca2+-dependent lectin purified from a sea cucumber, Cucumaria echinata. This protein exhibits strong hemolytic activity as well as cytotoxicity toward some cul-tured cell lines. Hemolysis is caused by CEL-III oligomers formed in the cell membrane after binding to specific carbohydrate chains on the cell surface. We have found that the oligomerization of CEL-III is also induced by the binding of simple carbohydrates, such as lactose, in aqueous solution under high pH and high ionic strength conditions. From gel filtration analysis of the oligomerization of CEL-III, it was found that the formation of the CEL-III oligomer is effectively induced by the binding of lactose and lactulose, disaccharides containing a β-galactoside structure. Electron micrographs of the resulting oligomers revealed them to exist as particles with a size of approximately 20-30 nm. The oligomerization process required more than 1 h, which is consistent with the in-crease in surface hydrophobicity as measured using a fluorescent probe, 8-anilinonaph-thalene-1-sulfonate. However, a change in the far-UV CD spectra as well as small-angle X-ray scattering occurred within a few minutes, suggesting that a structural change in the protein takes place rapidly, but the following growth of the oligomer is a much slower process.View full abstractDownload PDF (2051K) -
Hiroshi Oyama, Takatoshi Hamada, Shin Ogasawara, Kenichi Uchida, Sawao ...2002 Volume 131 Issue 5 Pages 757-765
Published: 2002
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (Ki = 0.7 ± 0.14 μM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine resi-due and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.View full abstractDownload PDF (3740K)
- |<
- <
- 1
- >
- >|