巻号一覧
128 巻, 6 号
選択された号の論文の25件中1～25を表示しています
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Edmar Vaz de Andrade, Flávia Caixeta de Albuquerque, Líd ...2000 年 128 巻 6 号 p. 891-895
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーWe report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22. The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A. The second vector codes for the scFv fused to the Fc fragment of the human IgGl. The fusion partner simplified the detection and purification of the secreted protein. These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.抄録全体を表示PDF形式でダウンロード (1234K) -
Yoshie Takaki, Nobuhisa Iwata, Satoshi Tsubuki, Sayuri Taniguchi, Sato ...2000 年 128 巻 6 号 p. 897-902
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーAmyloid β peptide (Aβ) is a physiological peptide that is constantly catabolized in the brain. We previously demonstrated that an endopeptidase sensitive to phosphoramidon and thiorphan conducts the initial rate-limiting proteolysis of Aβ in vivo, but the exact molecular identity of the peptidase (s) has remained unknown because of the molecular redundancy of such activity. We analyzed the brain-derived enzyme by means of inununo-depletion and gene disruption, and demonstrate here that neprilysin accounts for the majority of the Aβ-degrading activity. Furthermore, kinetic analysis, giving a Km value of 2.8±0.76 μM, indicated that Aβ1-42 is a relevant substrate for neprilysin.抄録全体を表示PDF形式でダウンロード (1630K) -
Maki Murata-Hori, Katsumi Fumoto, Yasuaki Fukuta, Takahiro Iwasaki, As ...2000 年 128 巻 6 号 p. 903-907
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーPrevious studies demonstrated that the phosphorylated myosin II regulatory light chain (MRLC) is localized at the cleavage furrow of dividing cells, suggesting that phosphorylation of MRLC plays an important role in cytokinesis. However, it remains unclear which kinase (s) phosphorylate MRLC during cytokinesis. AIM-1, an Aurora/Ipllprelated kinase from rat, is known as a serine/threonine kinase that is required for cytokinesis. Here we examined the possibility that AIM-1 is a candidate for a kinase that phosphorylates MRLC during cytokinesis. As a result, we showed that AIM-1 monophosphorylated MRLC at Ser 19 using two-dimensional phosphopeptide mapping analysis and several MRLC mutants. Furthermore, AIM-1 was colocalized with monophosphorylated MRLC at the cleavage furrow of dividing cells. We propose here that AIM-1 may participate in monophosphorylation of MRLC during cytokinesis.抄録全体を表示PDF形式でダウンロード (1817K) -
Katsuhiko Fujii, Naoto Urano, Hideki Ushio, Masataka Satomi, Haruka Ii ...2000 年 128 巻 6 号 p. 909-916
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーNonylphenol (NP) is an important intermediate in the production of various commercial and industrial materials, but is also known as a ubiquitous pollutant in urban aquatic environments. We recently studied the NP-degrading activities of microflora in several aquatic environments, and found a notable degrading activity for wastewater from a sewage treatment plant in Tokyo. This result led us to isolate NP-degrading microbes and identify biodegradation products. Using conventional plate culture techniques and molecular biological methods, Pseudomonas and Sphingomonas species, which are known for their degradation activities of many aromatic compounds, have been isolated. But it has also been found that Sphingomonas sp. (S-strain) is necessary and sufficient for the degradation of NP. Although the role of Pseudomonas sp. (P-strain) remains unclear, P-strain seems to provide some co-nutrients for the growth of S-strain. The degradation products were analyzed by GC/MS and NMR. More than 95% of NP was degraded within 10 days and aromatic compounds other than NP were not found, suggesting that the phenolic part of NP was completely degraded. We also examined the potential of S-strain for bioremedial applications. S-strain cells immobilized on chitosan or alginate beads retain their NP-degrading activity in flask-scale experiments. Furthermore, the chitosan-bound cells in a lab-scale bioreactor have been found to be persistent for repeated use, suggesting that S-strain is applicable to the treatment of NP-contaminated wastewater.抄録全体を表示PDF形式でダウンロード (640K) -
Keitaro Fujikura, Yuan-Wei Zhang, Hideki Yoshizaki, Tokuzo Nishino, Ta ...2000 年 128 巻 6 号 p. 917-922
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe primary structures of cis-prenyltransferases are completely different from those of trans-prenyltransferases. To obtain information about amino acid residues relating to catalytic function, random mutation of the undecaprenyl diphosphate synthase gene of Micrococcus luteus B-P 26 was carried out to construct a mutated gene library using an error-prone polymerase chain reaction. From the library, the mutants showing poor enzymatic activity were selected by the colony autoradiography method. Among 31 negative clones selected from 3, 000 mutants, two clones were found to contain only one amino acid substitution at either Asn-77 or Trp-78. To determine the functional roles of these interesting residues, we prepared six mutated enzymes with substitutions at residues Asn-77 or Trp-78 by site-directed mutagenesis. Substitution of Asn-77 with Ala, Asp, or Gln resulted in a dramatic decrease in catalytic activity. but the Km values for both aBylic and homoallylic substrates of these mutant enzymes were comparable to those of the wild-type. On the other hand, three Trp-78 mutants, W781, W78R, and W78D, showed 5-20-fold increased Km values for farnesyl diphosphate but not for Z-geranylgeranyl diphosphate. However, these mutants showed moderate levels of enzymatic activity and comparable Km values for isopentenyl diphosphate to that of the wild-type. These results suggest that the Asn-Trp motif is involved in the binding of farnesyl diphosphate and enzymatic catalysis.抄録全体を表示PDF形式でダウンロード (1403K) -
Hiroaki Kato, Shi Chen, Hiroshi Kiyama, Ken Ikeda, Naoki Kimura, Kinic ...2000 年 128 巻 6 号 p. 923-932
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーIn the present study, we have identified a novel gene, NDRP (for neuronal differentiation-related protein), which is predominantly expressed in developing and regenerating neurons. The predicted NDRP comprises 1, 019 amino acid residues and has 6 WD repeats in the N-terminal half and multiple potential nuclear localization signals (NLSs) at the C-terminal part. This molecule shows no significant structural similarity with any other molecules in available databases. In situ hybridization and immunohistochemistry revealed the highest expression of NRDP in sensory neurons, for instance, olfactory epithelia and neural layer of retina during embryonic development, as well as in perinatal dorsal root ganglions. The expression of this gene in intact motor neurons such as in the hypoglossal nerve was undetectable but became obvious after axotomy. These results suggest that the product of this gene might be involved in the development of sensory neurons as well as the regeneration of motor neurons.抄録全体を表示PDF形式でダウンロード (2691K) -
Michiko Hamatake, Kazuhiro Iguchi, Kazuyuki Hirano, Ryoji Ishida2000 年 128 巻 6 号 p. 933-939
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーTo investigate the mode of zinc-induced cell death, the associated morphological changes, and biological events were examined in zinc-treated Molt-4 cells. Fluorescence microscope observations with double staining of zinc-treated cells with Hoechst 33342 and propidium iodide (PI) indicated that the metal induced both necrosis and apoptosis. To confirm this, cells were stained with both PI and FITC-labeled annexin V, which binds phosphatidylserine, and then analyzed by flow cytometry. The results also confirmed that zinc induces mixed types of cell death, necrosis and apoptosis, and that the former induction occurs earlier and at a greater frequency. Hallmarks of apoptosis such as abnormal chromosome condensation and release of cytochrome c, as well as the appearance of annexin-positive cells, appeared along with the expression of mitochondrial membrane protein 7A6. However, zinc did not induce increases in caspase-3 like protease and caspase-8 activities, and caused slightly hypodiploid cells. Furthermore, the induction of cell death and annexin-positive cells was not blocked by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. These results indicate that zinc induces both necrosis and apoptosis, without caspase-3 activation.抄録全体を表示PDF形式でダウンロード (1504K) -
Nae Kadota, Akiko Suzuki, Yoshiko Nakagami, Toshiaki Izumi, Takeshi En ...2000 年 128 巻 6 号 p. 941-949
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーMeltrin α (ADAM12) is a member of the ADAM (MDC) protein family characterized by the presence of metalloprotease and disintegrin domains. ADAM proteins contain single transmembrane domains, and the processed mature proteins are postulated to span the plasma membrane. It has been reported that transfection of a truncated meltrin α cDNA lacking the prodomain and metalloprotease domain promotes skeletal muscle cell fusion. We show here that meltrin a was constitutively expressed in both undifferentiated and differentiated C2 skeletal muscle cells and also in fibroblasts. Both its precursor and processed mature forms were present in these cells. Thus, meltrin α may play general roles in addition to its roles in myogenesis. Since endogenous meltrin α cannot be detected by immunofluorescence microscopy, we examined the location of the exogenously expressed protein by transfection. Unexpectedly, the exogenously expressed meltrin α was located to a network structure of the endoplasmic reticulum (ER) but not to the plasma membrane. Cell fractionation revealed that the intrinsic mature protein was associated with the plasma membrane. However, the exogenously expressed protein remained unprocessed. These results seem to imply that the exogenously expressed meltrin a is not translocated from the ER to the trans-Golgi network, where a processing enzyme resides, and that it is consequently not converted to the mature form. Thus, the transfected meltrin a is unlikely to exert its physiological functions. Conversely, the ER may serve as a reservoir of the latent form of intrinsic meltrin α.抄録全体を表示PDF形式でダウンロード (4396K) -
Saori Takahashi, Masanori Kumagai, Sho Shindo, Kyuichi Saito, Yukio Ka ...2000 年 128 巻 6 号 p. 951-956
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーRenin-binding protein (RnBP) is a highly specific renin inhibitor first isolated from porcine kidney. Our recent studies demonstrated that the human RnBP is the enzyme Nacetyl-D-glucosamine (GIcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have developed a new assay method for GlcNAc 2-epimerase activity using a system of N-acyl-D-hexosamine oxidase coupled with peroxidase and employed this method to study the effects of renin on GIcNAc 2-epimerase activity. The recombinant human (rh) RnBP existed as a dimer and its GleNAc 2-epimerase activity was strongly inhibited by the purified renin concomitant with the formation of RnBP-renin heterodimer, so-called high molecular weight (HMW) renin. The renin activity was also inhibited by rhRnBP in a dose-dependent manner. These results indicate that renin is an inhibitor of GIcNAc 2-epimerase, and the renin-RnBP heterodimer HMW renin is an inactive form of both renin and GlcNAc 2-epimerase activities.抄録全体を表示PDF形式でダウンロード (994K) -
Caroline Cluzel, Claire Lethias, Robert Garrone, Jean-Yves Exposito2000 年 128 巻 6 号 p. 957-963
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーOne of the mechanisms involved in the regulation of the fibril diameter is the retention of the N-propeptide. In sea urchin embryo, thin collagen fibrils harbor numerous extensions at their surface, which we have suggested correspond to the large N-propeptide of the 2α collagen chain. To investigate the function of the N-propeptide during fibrillogenesis, we engineered constructs coding for the globular region of the 2α N-propeptide. To obtain homotrimeric molecules, the N-telopeptide, the central triple helix and the C-propeptide of the 2α chain were replaced by human domains of the proal(I) chain. A single restriction site allowed insertion of distinct versions of the minor triple helix of the N-propeptide. Several human cell lines were transfected, and with one of them we were able to produce intact homotrimeric procollagen molecules. Rotary shadowing of these purified molecules indicates the presence of three large 2α N-propeptides that are similar to the extensions present at the surface of the sea urchin thin fibrils. This cassette-vector will be useful in determining the respective contributions of the globular and minor triple helical domains of the N-propeptide in the regulation of fibril diameter.抄録全体を表示PDF形式でダウンロード (3146K) -
Takako Hishilki, Hideo Shimada, Shingo Nagano, Tsuyoshi Egawa, Yasukaz ...2000 年 128 巻 6 号 p. 965-974
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252IIe. X-ray crystallographic analyses of the ferric d-camphor-botmd form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H2O. The enzyme showed a low monooxygenase activity of ca. 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric dcamphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerequisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.抄録全体を表示PDF形式でダウンロード (1911K) -
Tadashi Fujii, Takao Narita, Hitosi Agematu, Naoki Agata, Kunio Isshik ...2000 年 128 巻 6 号 p. 975-982
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe pcd gene from Flavobacterium lutescens IFO3084 encoding Δ'-piperideine-6-carboxylate dehydrogenase (PCD) was cloned, sequenced, and expressed in Escherichia coli. The deduced amino acid sequence of PCD from F. lutescens IFO3084 showed strong similarity to that from Streptomyces clavuligerus. The molecular mass of the recombinant PCD was estimated to be approximately 58, 000 Da by SDS-PAGE and native PAGE, which indicated that the enzyme molecule is a monomer. The in vitro analysis of L-α-arnittoadipic acid (L-AAA) production showed that L-AAA is synthesized from L-lysine in two steps catalyzed by L-lysine 6-aminotransferase (LAT) and PCD from F. lutescens IFO3084.抄録全体を表示PDF形式でダウンロード (2347K) -
Yasushi Hasegawa2000 年 128 巻 6 号 p. 983-988
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーIt has been reported that catch and striated muscle myosin heavy chains of scallop are generated through alternative splicing from a single gene [Nyitray et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12686-12690]. They suggested that the catch muscle type myosin was expressed in various tissues of scallop, including the gonad, heart, foot, and mantle. However, there have been no reports of the primary structure of myosin from tissues other than the adductor muscles. In this study, we isolated a cDNA encoding the motor domain of myosin from the mantle tissue of scallop (Patinopecten yessoensis), and determined its nucleotide sequence. Sequence analysis revealed that mantle myosin exhibited 65% identity with Drosophila non muscle myosin, 60% with chicken gizzard smooth muscle myosin, and 44% with scallop striated muscle myosin. The mantle myosin has inserted sequences in the 27 kDa domain of the head region, and has a longer loop 1 structure than those of scallop striated and catch muscle myosins. Phylogenetic analysis suggested that the mantle myosin is classified as a smooth/nonmuscle type myosin. Western blot analysis with antibodies produced against the N-terminal region of the mantle myosin revealed that this myosin was specifically expressed in the mantle pallial cell layer consisting of nonmuscle cells. Our results show that mantle myosin is classified as a nonmuscle type myosin in scallop.抄録全体を表示PDF形式でダウンロード (1281K) -
Li Jun Hao, Dan Yang, Yoshito Fujii, Akira Yamauchi, Norio Suzuki, Hir ...2000 年 128 巻 6 号 p. 989-998
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーIn order to improve transient gene transfer into PLB-985 cells, we treated cells with 12- tetradecanoylphorbol 13-acetate (TPA) 4 h before transfection, and increased by 540-fold the reporter activity of the firefly luciferase gene transfected by TFL-01, a cationic liposome. Dioctanolyglycerol added before TPA addition inhibited the TPA-dependent increase in activity, suggesting it to be a TPA competitor for PKC binding. H 7, staurosporine, GO 6976, and H 8, but not GF 109203X and forskolin, inhibited the TPA-dependent increase in reporter activity when added 8 h after TFL-01/gene addition. Forskolin and GF 109203X also inhibited the increase when added before TPA. Therefore, for the potentiation of transfection by the TPA/TFL-01 method, conventional PKC activity with significant but low protein kinase A (PKA) activity are first required, and then a novel PKC activity with significant PKA activity. TPA enhanced the uptake of FITC-labeled phosphorothioated oligonucleotides and their prolonged maintenance by cells, suggesting increased TFL-01-assisted plasmid uptake and its stabilization in TPA-treated PLB-985 cells. This method was used successfully for the sensitive analysis of the promoter function of the gp91phox gene, implying the method to be generally useful for promoter analyses of various genes expressed in differentiated human monocytic cells.抄録全体を表示PDF形式でダウンロード (1229K) -
James R. Ketudat Cairns, Voraratt Champattanachai, Chantragan Srisomsa ...2000 年 128 巻 6 号 p. 999-1008
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーDalcochinin-8'-O-β-glucoside β-glucosidase (dalcochinase) from the Thai rosewood (Dalbergia cochinchinensis Pierre) has aglycone specificity for isoflavonoids and can hydrolyze both β-glucosides and β-fucosides. To determine its structure and evolutionary lineage, the sequence of the enzyme was determined by peptide sequencing followed by PCR cloning. The cDNA included a reading frame coding for 547 amino acids including a 23 amino acid propeptide and a 524 amino acid mature protein. The sequences determined at peptide level were found in the cDNA sequence, indicating the sequence obtained was indeed the dalcochinase enzyme. The mature enzyme is 60% identical to the cyanogenic β-glucosidase from white clover glycosyl hydrolase family 1, for which an Xray crystal structure has been solved. Based on this homology, residues which may contribute to the different substrate specificities of the two enzymes were identified. Eight putative glycosylation sites were identified, and one was confirmed to be glycosylated by Edman degradation and mass spectrometry. The protein was expressed as a prepro-α-mating factor fusion in Pichia pastoris, and the activity of the secreted enzyme was characterized. The recombinant enzyme and the enzyme purified from seeds showed the same Km for pNP-glucoside and pNP-fucoside, had the same ratio of Vmax for these substrates, and similarly hydrolyzed the natural substrate, dalcochinin-8'-β-glucoside.抄録全体を表示PDF形式でダウンロード (1814K) -
Ayano Matsumoto, Naoya Kojima, Fujio Takeuchi, Tsugao Mizuochi2000 年 128 巻 6 号 p. 1009-1016
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーRheumatoid factors (RFs) are autoantibodies, which recognize antigens on a constant region of immunoglobulin G (IgG). Among various RF classes, RF of the IgG class (IgGRF) forms immune complexes in rheumatoid joints and is implicated in the pathogenesis of rheumatoid arthritis (RA). To characterize the formation of IgGRF immune complexes, in the present study, IgGRF was isolated from sera of RA patients, and its interaction with immobilized IgG was analyzed and compared to that of IgMRF or IgARF by means of surface plasmon resonance. On gel filtration, the IgGRF was eluted as a single peak corresponding to IgG, excluding the possible formation of self-associating IgGRF complexes in solution. Sensorgrams of the interaction of IgGRF with immobilized IgG revealed that it clearly bound to the IgG at 6°C, but not at 30°C. The degree of interaction decreased inversely with an increase in temperature, suggesting that IgGRF is much more reactive at lower temperatures. In contrast, the interaction of IgARF and IgMRF with IgG at 6°C was similar to that at 30°C. The association rate constant (ka) of IgGRF decreased with an increase in temperature, while those of IgARF and IgMRF were similar under various thermal conditions. The dissociation rate constant (kd) of IgGRF was greatly reduced at 25°C, but those of IgARF and IgMRF slightly increased with an increase in temperature. These results suggested that the mode of interaction of IgGRF with IgG differed from in the cases of IgMRF and IgARF. The kinetic properties of the IgGRF-IgG interaction may facilitate elucidation of the IgGRF immune complex formation in rheumatoid joints.抄録全体を表示PDF形式でダウンロード (1831K) -
Yumiko Itoh, Kimiko Inuzuka, Isao Kohno, Hideo Wada, Hiroshi Shiku, Na ...2000 年 128 巻 6 号 p. 1017-1024
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーβ2-Glycoprotein I (β2G-PI) consists of five tandem repeated domains (I, II, III, IV, and V). The nicked form of (N-β2CPI ) which was cleaved by plasmin in vitro at Lys 317-Thr 318 in domain V, showed reduced affinity for the negatively charged phospholipids, especially cardiolipin (CL). Recently, the N-β2 was detected in the plasma of patients with disseminated intravascular coagulation syndrome (DIC) by an immunological method. In the present study, we prepared monoclonal antibodies for the nicked form, and demonstrated that the concentrations of this form of β2GPI, which were analyzed by a sandwich ELISA using two specially prepared monoclonal antibodies, were significantly increased in the plasma of patients with leukemia (n=51, mean±SD: 162.0±118.3ng/ml) and with lupus anticoagulant (LA) (n=40, mean±SD: 3, 041.5±16, 579.7ng/ml), compared to the normals (n=33, mean±SD: 1.04±1.54ng/ml). We found a significant correlation between the concentrations of N-β2GPI and those of typical molecular markers for a fibrinolytic state such as plasmin-α2 plasmin inhibitor complex (PIC) and D-dimer in patients with leukemia, but not in patients with LA. These results suggested that the generation of N-β2GPI was caused by plasmin in the patients with leukemia, and by unknown proteases in the patients with LA. In the patients with LA, the levels of N-β2GPI tended to be higher in those without thrombosis than in those with thrombosis.抄録全体を表示PDF形式でダウンロード (1862K) -
Toshiyuki Takeuchi, Seilchi Nakamura, Atsushi Kayasuga, Shun-ichi Isa, ...2000 年 128 巻 6 号 p. 1025-1031
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーLike such hepatic genes as those for albumin and aldolase B, the rat catalase gene shows markedly reduced expression in carcinogenesis of hepatocytes. Strong silencer activity has been widely observed in the 5'-flanking region of the gene, downstream from the G-rich sequence identified in a previous study. In this study, we identified and characterized multiple elements involved in negative regulation of catalase gene expression by reporter assay and gel shift assay. One of the silencer elements is located 3 kb upstream of the gene and has GATATCCCGATATC as core sequence. The observation that protein binding to the element is abundantly expressed in dedifferentiated hepatoma cell lines, but scarcely in well-differentiated cell lines suggests that this element is involved in negative regulation of the catalase gene expression in hepatocarcinogenesis. This element was targeted by a novel 20-kDa nuclear protein, which is designated HNRF (hepatocarcinogenesis-related negative regulatory factor).抄録全体を表示PDF形式でダウンロード (2421K) -
Shou Taltashima, Mari Kono, Nobuyuki Kurosawa, Yukiko Yoshida, Yuriko ...2000 年 128 巻 6 号 p. 1033-1043
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe genomic organization of the gene encoding the mouse GD3 synthase (ST8Sia I) has been determined. The mouse ST8Sia I gene spans over 100 kilobases of genomic DNA with a unique genomic structure of 5 exons. Analysis of the sequence immediately upstream of the transcription initiation site revealed that the ST8Sia I promoter contained no canonical TATA- or CCAAT-box, but contained a putative Sp1 binding site. Transient transfection experiments demonstrated functional promoter activity of the ST8Sia I promoter in an ST8Sia I-expressing cell line, P19, but not in an ST8Sia I-nonexpressing cell line, NIH3T3. Mobility shift assay and mutation analysis of the promoter region indicated that the Sp1 binding site is involved in the transcriptional regulation of the ST8Sia I gene in P19 cells. Here, the genomic structural analyses of mouse sialyltransferase genes are summarized and the genomic structures of these genes are compared.抄録全体を表示PDF形式でダウンロード (1443K) -
Ryo Yoshimoto, Masatoshi Hori, Hiroshi Ozaki, Hideaki Karaki2000 年 128 巻 6 号 p. 1045-1049
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーAcidic calponin is an actin binding protein expressed in smooth muscle and brain. Although the role of smooth muscle calponin (basic calponin) has been well studied, few studies have been performed on acidic calponin. In the present study, we demonstrated that acidic calponin binds to filamentous actin, but not monomeric actin. A co-sedimentation assay indicated that acidic calponin binds to actin with an apparent binding constant of 4×105M-1. In the presence of an excess amount of calmodulin, the binding of acidic calponin to actin was inhibited. The binding of acidic calponin to calmodulin was Ca2+-dependent with Kd of 31 μM. We next investigated whether or not acidic calponin could be a substrate for μ-calpain in vitro, since it has been shown that basic calponin is cleaved by μ-calpain. The results showed that acidic calponin was also cleaved by μ-calpain. Neither the proteolytic pattern nor velocity of acidic calponin was different in the absence or presence of calmodulin. When acidic calponin had bound to actin, however, the susceptibility of the acidic calponin to μ-calpain was significantly reduced, which was reversed by the addition of calmodulin. Our results suggest that acidic calponin might be involved in the μ-calpain-regulated actin cytoskeleton.抄録全体を表示PDF形式でダウンロード (1884K) -
Kai Hilpert, Gerd Hansen, Helga Wessner, Jens Schneider-Mergenenr, Wol ...2000 年 128 巻 6 号 p. 1051-1057
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーA new method is presented that uses parallel peptide array synthesis on cellulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibitor was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while inhibition was measured by an assay in microtiter plates with punched out peptide spots. The importance of each residue for binding or inhibition was determined by substitutional analyses, exchanging every original amino acid with all other 19 coded amino acids. Seven hundred eighty individual peptides were investigated for binding behavior to porcine pancreatic elastase, and 320 individual peptides were measured in inhibition experiments. The results provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase. Combining these data with length analysis we designed new peptides in a stepwise fashion which in the end not only inhibited elastase 400 times more strongly than the original peptide, but are highly specific for the enzyme. In addition, the optimized inhibitor peptide was protected against exopeptidase attack by substituting D-amino acids at both termini.抄録全体を表示PDF形式でダウンロード (2221K) -
Jeong Goo Lee, Seong Pil Cho, Heon Sik Lee, Cheong Ho Lee, Kyung Sook ...2000 年 128 巻 6 号 p. 1059-1072
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーSaccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Citlp and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Citlp, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H. S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Citlp and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Citl, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).抄録全体を表示PDF形式でダウンロード (4357K) -
Toshimichi Sakaihara, Atsuko Honda, Seiji Tateyama, Hiroshi Sagami2000 年 128 巻 6 号 p. 1073-1078
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーPolyisoprenoid alcohols occurring in spinach leaves were analyzed by a two-plate TLC method. Z, E-mixed polyprenols (C55-60), glycinoprenols (C50-55), and solanesol (C45) were mainly found in chloroplasts, whereas dolichols (C70-80) were mainly found in microsomes. Analysis of enzymatic products derived from [1-14C] isopentenyl diphosphate and farnesyl diphosphate (FPP) with subcellular fractions revealed that chloroplasts and microsomes had the ability to synthesize Z, E-mixed polyprenyl (C50-65) and all E-polyprenyl (C45-50) diphosphates, and Z, E-mixed polyprenyl (C70-65) diphosphates, respectively. FPP and geranylgeranyl diphosphate (GGPP) were both accepted for these enzymatic reactions, the former being a better substrate than the latter. NMR analysis of naturally occurring spinach Z, E-mixed polyprenol (C55) and dolichol (C75) revealed that the number of internal trans isoprene residues in the former was three in comparison with two internal trans residues found for the latter. These results indicate that two kinds of polyprenyl diphosphate synthases occur in spinach: One is the chloroplast enzyme involved in the synthesis of the shorter-chain (C50-65) Z, E-mixed polyprenols and the other is the microsomal enzyme involved in the synthesis of longer-chain (C70-85) Z, E-mixed polyprenols, which is converted to dolichols.抄録全体を表示PDF形式でダウンロード (3136K) -
Shinji Koga, Issei Yoshioka, Haruhiko Sakuraba, Mamoru Takahashi, Shin ...2000 年 128 巻 6 号 p. 1079-1085
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe ADP-dependent (AMP-forming) glucokinases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis catalyze the phosphorylation of glucose using ADP as the essential phosphoryl group donor. Both enzymes were purified to homogeneity and characterized with regard to each other. The enzymes had similar enzymological properties as to substrate specificity, coenzyme specificity, optimum pH, and thermostability. However, a difference was observed in the subunit composition; while the T. litoralis enzyme is a monomer with a molecular mass of 52 kDa, the P. furiosus enzyme has a molecular mass of about 100 kDa and consists of two subunits with identical molecular masses of 47 kDa. The genes encoding these enzymes were cloned and sequenced. The gene for the P. furiosus enzyme contains an open reading frame for 455 amino acids with a molecular weight of 51, 265, and that for the T. litoralis enzyme contains an open reading frame for 467 amino acids with a molecular weight of 53, 621. About 59% similarity in amino acid sequence was observed between these two enzymes, whereas they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, two phosphate binding domains, and adenosine and glucose binding motifs commonly conserved in the eukaryotic hexokinase family were not observed.抄録全体を表示PDF形式でダウンロード (1185K) -
Nobunao Wakabayashi, Ryoichiro Kageyama, Toshiyuki Habu, Toshihide Doi ...2000 年 128 巻 6 号 p. 1087-1095
発行日: 2000年
公開日: 2008/11/18
ジャーナル フリーThe regulatory mechanisms of mammalian hairy and Enhancer of split homologue-1 (HES-1) genes were examined in mouse P19 embryonic carcinoma cells (P19 cells). Undifferentiated P19 stem cells expressed a basal level of the HES-1 gene, whereas the expression of this gene was increased upon induction of the cells to a neural cell lineage using retinoic acid (RA). Reporter co-transfection analysis identified an activating region within the upstream promoter region of HES-1 from nucleotides -201 to -172. This activating region, called activating region X (ARX), shows a high GC content and contains both an AP-2 binding motif and a CCAAT box. An electrophoretic mobility shift assay using nuclear proteins extracted from P19 cells showed that ARX forms a specific DNA-protein complex. Importantly, ARX-dependent transcription, as well as ARX-binding activity, was significantly increased in P19 cells treated with RA. These results indicate that ARX transduces signals that up-regulate HES-1 gene expression in response to RA-treatment. Thus, a novel cis-acting element involved in HES-1 gene regulation that plays a role in RA-induced neural differentiation of P19 cells has been identified.抄録全体を表示PDF形式でダウンロード (3438K)
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