巻号一覧
120 巻, 2 号
選択された号の論文の34件中1～34を表示しています
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Takashi Seki, Naoyuki Hayashi, Takeharu Nishimoto1996 年 120 巻 2 号 p. 207-214
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーRCC1 is a chromosomal protein that functions as a GEF of the nuclear G protein Ran, which GTPase activity is enhanced by RNA1 located in the cytoplasm. RCC1 has no preference for GTP or GDP-bound Ran, so that GTP-Ran formation in vivo is regulated by relative concentrations of GTP/GDP and regulatory proteins interacting with RCC1, Ran, and RNA1. Proteins possessing the special Ran-binding motif have been found to be conserved in species ranging from yeasts to mammalians. The finding of RanBP2/NUP358 clearly indicates the involvement of the Ran pathway in the nuclear pore transport function, in agreement with the finding that both rcc1- and rna1- show defects in this process. However, loss of RCC1 induces premature initiation of mitosis, resulting in G1 arrest with the micronuclei possessing mitotic condensed chromosomes. How both the cell cycle and nucleocytoplasmic transport are regulated by the RCC1-Ran pathway is a major question.抄録全体を表示PDF形式でダウンロード (2319K) -
Shuh Narumiya1996 年 120 巻 2 号 p. 215-228
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーRho, a Ras homologue of small GTPase, is present from yeast to mammals. It shuttles between the active GTP-bound form and the inactive GDP-bound form and works as a switch in stimulus-evoked cell adhesion and motility, enhancement of contractile responses, and cytokinesis. In these actions, Rho directs the reorganization of the actin cytoskeleton at a specific time and at a specific site in the cell. It also activates serum response factor possibly via a kinase cascade and mediates a growth signal to nuclei. Two signalling processes are known to lead to Rho activation: one is activation of certain types of G-protein-coupled receptors such as lysophosphatidic acid receptor, and the other is activation of other small GTPases including Ras, CDC 42, and Rac. Molecules catalyzing the GDP-GTP exchange of Rho, Rho guanine nucleotide exchange factors (Rho GEF), and those catalyzing the acceleration of GTP hydrolysis, Rho GTPase activating proteins (Rho GAP), were identified as Dbl- and Bcr-containing molecules, respectively. In addition, a molecule inhibiting guanine nucleotide exchange of Rho, Rho guanine nucleotide dissociation inhibitor (Rho-GDI), was isolated and characterized. More recently, putative Rho targets possibly mediating various Rho actions have been identified by their selective interaction with GTP-bound Rho. They include lipid kinases such as phosphatidyl-inositol-5-kinase and protein serine/threonine kinases such as PKN and p160ROCK. A model of the molecular mechanism of action of Rho constructed on the basis of these findings is presented. There are, however, still many unclarified links between cell stimulation, Rho activation and final Rho actions.抄録全体を表示PDF形式でダウンロード (2055K) -
Dong Luo, Nancy Mah, David Wishart, Yi Zhang, Fred Jacobs, Luis Martin1996 年 120 巻 2 号 p. 229-232
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe fused various polypeptide extensions to the C-termini of single chain Fv (scFv) and disulfide-stabilized Fv (dsFv) fragments to facilitate detection of bi-functional proteins or to add biological effector domains, which included the human metallothionein (HMT) motif and biotin mimetic sequence. These bi-functional proteins were expressed and secreted in a recombinant Pichia pastoris system and showed specific anti-idiotype binding activity, as determined by competitive radioimmunoassaying. However, the fusion protein constructed with dsFv- HMT, but not scFv-HMT, had lost this binding activity. The interruption of the structural conformation as a result in dsFv-HMT may be explained by the interactions between the cysteines engineered in dsFv domains and the cysteines in the HMT region.抄録全体を表示PDF形式でダウンロード (2752K) -
Kazuko Inoue, Toshihiko Arai, Masaaki Aoyagi1996 年 120 巻 2 号 p. 233-235
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーA real time biomolecular interaction assay system involving an optical sensor was applied to quantitative analysis of the binding of herpes simplex virus type 1 (HSV-1) to immobilized heparan sulfate, a cellular receptor component of HSV-1, and the neutralization antibody titer against this virus with a commercially available sulfonated human immuno-globulin preparation. The virus titer in a viral solution and the neutralizing antibody titer in the human immunoglobulin preparation could be successfully estimated in a short time with this system without any difficult cell culture.抄録全体を表示PDF形式でダウンロード (264K) -
Nobuhiko Miura, Nobuhiro Ishida, Masato Hoshino, Masatake Yamauchi, Ta ...1996 年 120 巻 2 号 p. 236-241
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe have cloned a cDNA that codes for a putative human UDP-galactose translocator (UGT) protein. The cDNA contained an open reading frame of 1, 179 base pairs encoding a novel protein of 393 amino acids. Introduction of the open reading frame sequence into a UGT-deficient mouse cell line, Had-1, complemented the genetic defect of the mutant, namely the inability to transport UDP-galactose from the cytosol to the Golgi lumen, as judged from the lectin-sensitivity spectrum of the transformant. To our knowledge, this is the first mammalian nucleotide-sugar translocator whose cDNA sequence has been described.抄録全体を表示PDF形式でダウンロード (646K) -
Nobuko Iritani, Hitomi Fukuda, Kayoko Tada1996 年 120 巻 2 号 p. 242-248
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe time courses of gene expression, and the nutritional regulation of gene expression of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, ATP citrate-lyase, malic enzyme, and glucose-6-phosphate dehydrogenase) in epididymal adipose tissue after refeeding food-deprived rats have been investigated and compared with those in liver (previously reported). The mRNA concentrations of lipogenic enzymes reached maximum levels at 24h after the refeeding in adipose tissue and at 8-16h in liver, while the enzyme induction reached maximum at 48-72h in both tissues. Moreover, the mRNAs were more strongly induced in adipose tissue than in liver, whereas the enzyme induction (except malic enzyme) was lower. In adipose tissue of rats fed a carbohydrate diet without protein, the mRNA concentrations of acetyl-CoA carboxylase, ATP-citrate lyase, malic enzyme, and fatty acid synthase reached comparable levels to those of the carbohydrate/protein diet group. The protein feeding increased the enzyme induction in adipose tissue. As regards reduction of gene expression, lipogenic enzyme mRNA concentrations were not so markedly reduced by starvation or polyunsaturated fatty acids in adipose tissue as in liver. The differences in regulation of lipogenic enzyme gene expression and induction between adipose tissue and liver can be ascribed to tissue specificity.抄録全体を表示PDF形式でダウンロード (2280K) -
Takayuki Nemoto, Tomonori Matsusaka, Minoru Ota, Takashi Takagi, David ...1996 年 120 巻 2 号 p. 249-256
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the α isoform of human HSP90 (HSP90α) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90α existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90α were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-His711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90α. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90α and the C-terminal 326 amino acids of GRP94 associated with HSP90α and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90α. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.抄録全体を表示PDF形式でダウンロード (4573K) -
Masayuki Nakanishi, Mikio Kakumoto, Kazuya Matsuura, Yoshihiro Deyashi ...1996 年 120 巻 2 号 p. 257-263
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーMouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, shows a strong coenzyme preference for NADP(H) over NAD(H), and is uniquely activated by fatty acids. Previous chemical modification and X-ray crystallography studies show that interactions responsible for the coenzyme specificity include salt linkages between the 2'-phosphate of NADPH and side-chains of Lys-17 and Arg-39 of the enzyme. Although Arg-39 is highly conserved in NADP(H)-dependent enzymes of the SDR family, Lys-17 is substituted with Arg in about half of the NADP(H)-dependent enzymes. The present study shows that mutations of Lys-17 to His (K17H) or Ser (K17S) and of Arg-39 to Ala (R39A) bring about decreases (from 5 to 90-fold) of the affinities for NADP(H), but minor changes in the affinity for NAD+. The binding energy arising from the mutations on the binding of the 2'-phosphate of NADP+ was decreased by 38-66% from the value of 4.8 kcal/mol calculated for the wild-type enzyme. In contrast, the mutation of Lys-17 to Arg (K17R) had little effect on the kinetic or thermodynamic properties. The activation by fatty acids was completely attenuated by the mutations of K17H and K17S, but not by K17R or R39A. These results indicate that the 2'-phosphate group of NADP(H) is recognized by both Lys-17 and Arg-39, of which Lys-17 is a component of the binding site for the activator, probably interacting with the negatively charged carboxylate group of fatty acids, and also suggest that the existence of a positively charged residue (either Lys or Arg) at position 17 is required for both NADP(H) specificity of the SDR family enzymes and fatty acid activation of the pulmonary carbonyl reductase.抄録全体を表示PDF形式でダウンロード (892K) -
Masato Sasaki, Jumpei Enami1996 年 120 巻 2 号 p. 264-270
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーTo identify new receptor tyrosine kinases (RTKs), we screened cDNAs from mouse mammary tumor cells and mouse brain. A homology search of the complete cDNA sequences obtained showed that one cDNA was a murine homologue of recently reported human sky [Ohashi, K. et al. (1994) Oncogene 9, 699-705]. Another cDNA obtained was also related to sky but had a 5' upstream sequence similar to brt [Fujimoto, J. and Yamamoto, T. (1994) Oncogene 9, 693-698]. Analysis of the 5' region of the sky genomic DNA revealed that brt-type and sky-type sequences are encoded by the sky gene in different exons. The upstream region of the sky-type coding exon is highly GC-rich and contains potential recognition sites for the Sp1 trans-acting factor, but lacks TATA and CAAT boxes, features commonly found in promoters of other RTKs. To examine whether this upstream region functions as a promoter, we fused it with chloramphenicol acetyltransferase (CAT) gene and transfected the construct into COS-7 cells. The results of the CAT assay showed that the sky upstream region retains a significant promoter activity. Furthermore, primer extension analysis revealed that the transcription starts at -240 nt upstream from the sky translation initiation codon. These observations suggest that the brt- and sky-types of mRNA are transcribed from a single sky gene by an alternative promoter usage.抄録全体を表示PDF形式でダウンロード (4281K) -
Hiroshi Itoh, Masaki Tomita, Takahiko Kobayashi, Hirofumi Uchino, Haru ...1996 年 120 巻 2 号 p. 271-275
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーExpression of inter-α-trypsin inhibitor light chain (ITI-LC, also known as bikunin or urinary trypsin inhibitor) was examined in various human tissues. By reverse-transcription polymerase chain reaction, the mRNA was detected not only in the liver, a known site of ITI-LC production, but also in the kidney, heart, lung, and pancreas. By RNA blot analysis, the mRNA was also detected in the pancreas and liver, but not in the kidney, heart, or lung. The ITI-LC protein was immunohistochemically detected along the surface of pancreatic acinar cells. These results indicate the apparent expression of the gene for ITI-LC in the pancreas. ITI-LC protein on the surface of pancreatic acinar cells may play an important role in preventing autodigestion by exocrine enzymes such as trypsinogen and chymotrypsinogen.抄録全体を表示PDF形式でダウンロード (2749K) -
Kyosuke Sato, Yasuzo Nishina, Kiyoshi Shiga1996 年 120 巻 2 号 p. 276-285
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーElectron-transferring flavoprotein (ETF) from pig kidney is composed of two subunits (α and β, molecular weights of 33, 000 and 29, 000) and two small molecules, FAD and AMP. In this study, in vitro refolding and unfolding of the subunits of ETF were carried out with urea as the denaturing reagent. The refolding reaction of α and β was revealed to proceed kinetically in two steps: D_??_I→N, where D, I, and N denote the denatured, intermediate, and native forms, respectively. The features of the I forms of α and β, described below, are consistent with the concept of the so-called “molten globule state, ” which is frequently observed in protein refolding. (i) The conversion between D and I was very rapid. (ii) The I form showed as much secondary structure as the N form as judged from the far-UV circular dichroism. (iii) The solvent accessibility of the I form, estimated by the analysis of equilibrium unfolding experiments, was intermediate between those of the D and N forms. (iv) The standard free energy of the I form is almost the same as that of the D form. The refolding reaction progressed more slowly and the environment of the tryptophan chromophore was changed more drastically in β refolding than in α refolding. We previously reported that the reconstitution of holoETF from denatured subunits is speeded up by increasing the AMP concentration. In this study, the effects of AMP, FAD, and the other subunit on the single subunit folding were examined, but no effect was detected. This result suggests that AMP plays a role in a later process, namely, assembly of the four components (refolded α and β, FAD, and AMP).抄録全体を表示PDF形式でダウンロード (1186K) -
F. Javier Alvarez, Angel Herráez, Juan C. Murciano, José ...1996 年 120 巻 2 号 p. 286-291
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーRat RBCs loaded with 125I-CA by hypotonic dialysis and isotonic resealing were evaluated as a carrier system. Loaded RBCs stored at 4°C remained unlysed (90% survival) allowing release of encapsulated 125I-CA for up to 4 days. Thereafter, cellular lysis increased significantly. IP-injected loaded RBCs reached the maximum level (50%) in circulation at 24h post-injection. Circulating loaded RBCs showed a half-life of 8-10 days, which was advantageous for carrier function. In contrast to IP-injected free CA, which remained in circulation for only a short time, encapsulated CA showed significant levels in circulation up to 10 days post-injection. The profile of organ uptake with time is essentially not altered for loaded with respect to native cells, being higher the removal of loaded cells and mainly localized in spleen. Nevertheless, liver is the organ with highest elimination capacity for both native and loaded cells, showing its maximum at 24h post-injection. Concomitantly, the concentration of 125I-CA in all organs studied was highest at this time. These data demonstrate that rat loaded RBCs can potentially be used as a carrier system for long-term dissemination of drug into the organism, with specially increased delivery to the spleen. They also support the use of the rat as an experimental model for biochemical and pharmacological studies in these therapeutic systems.抄録全体を表示PDF形式でダウンロード (774K) -
Sabato D'Auria, Mosè Rossi, Guido Barone, Francesca Catanzano, ...1996 年 120 巻 2 号 p. 292-300
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe β-glycosidase isolated from the extreme thermophilic archaeon Sulfolobus solfataricus, grown at 87°C, is a tetrameric protein with a molecular mass of 240 kDa. This enzyme is barely active at 30°C and has optimal activity, over 95°C, at pH 6.5. Its thermal stability was investigated at pH 10.1 and 10.6 by means of functional studies, circular dichroism and differential scanning calorimetry. There was no evidence of thermal activation of the enzyme and the temperature-induced denaturation was irreversible and not well represented by the two-state transition model. A more complex process occurred, involving the dissociation and unfolding of subunits, and subsequent nonspecific association and/or aggregation. Denaturation temperature was around 85°C, depending on protein concentration. The denaturation enthalpy change was between 7, 500 and 9, 800 kJ•mol-1, depending on the pH. The collapse of the native structure around 85°C was confirmed by circular dichroism measurements and time-dependent activity studies. Finally, preliminary inves-tigations were performed on the recombinant enzyme expressed in Escherichia coli.抄録全体を表示PDF形式でダウンロード (1103K) -
Chiaki Kato, Hideyuki Tamegai, Akihiko Ikegami, Ron Usami, Koki Horiko ...1996 年 120 巻 2 号 p. 301-305
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーEscherichia coli strain JD518, a cydD-deficient mutant, displayed temperature-sensitive and pressure-sensitive growth. The defective cydD gene in this strain was complemented by open reading frame 3 (ORF3), previously identified in DNA from a barotolerant bacterium, strain DSS12, allowing growth of the cydD mutant under high temperature and high pressure conditions. Spectrophotometrical analysis indicated that the cytochrome bd complex which is assembled by the CydD protein was expressed in E. coli strain JD518 carrying the ORF3 gene at the same level as occurred in the wild-type strain. Our results indicate that the cydD gene functions are required for cell stability under the condition of high pressure stress in bacteria.抄録全体を表示PDF形式でダウンロード (430K) -
Takako Sasaki, Hideki Yamada, Hideto Matsui, Yoshiko Dohi, Youki Hirak ...1996 年 120 巻 2 号 p. 306-312
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーIn order to elucidate possible effects of immunoglobulin on C1q metabolism at the anabolic steps, serum C1q levels and C1q mRNA of peritoneal exudate cells (PEC) and spleen cells were measured in female BALB/c mice implanted intraperitoneally with complement-(C)-fixing IgG2b- or non-C-fixing class IgG3-producing hybridomas and/or with immunoglobulin-non-productive myeloma cells (p3x63-Ag.8.653) (myeloma 653) (2×106/0.2 ml) or without any treatment as controls. In the IgG2b-hybridoma-treated mice, the serum C1q levels and C1q mRNA in PEC increased conspicuously as compared with those in the controls, but C1q mRNA in spleen cells was almost equal to that in the control mice. On the other hand, in the IgG3-hybridoma-treated mice, the serum C1q levels decreased signifi-cantly, but the extent of such decrease and the level of C1q mRNA in their PEC were almost equivalent to those in the myeloma 653-implanted mice. The serum C1q levels and C1q mRNA in PEC fluctuated similarly in mice injected intraperitoneally with highly purified IgG2b and/or IgG3 preparations. These results suggest some anabolic interaction, as well as catabolic interaction, between the C-fixing class of immunoglobulin and C1q.抄録全体を表示PDF形式でダウンロード (2672K) -
Eric Faure, Martin Best-Belpomme, Serge Champion1996 年 120 巻 2 号 p. 313-319
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーUsing constructs expressing the reporter gene under the control of the entire or deleted long terminal repeats (LTRs) of 1731, a Drosophila melanogaster retrotransposable element, we show that 1731-LTR is activated by X-irradiation in a dose- and time-dependent manner, and that a sequence located in the U3 region of these LTRs is required. The cis-acting element conferring X-responsiveness shows similarities to kappaB (κB)-like binding sequence. In response to X-irradiation, S2 Drosophila cells produced an extracellular factor which activates the 1731-LTR in nonirradiated cells. This factor was detected both when transfected cells were cocultured with inducing cells and when a conditioned medium taken from irradiated cultures was added.抄録全体を表示PDF形式でダウンロード (879K) -
Yoichi Nakabayashi, Koichi Nishigaki1996 年 120 巻 2 号 p. 320-325
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe cause of separation in gel electrophoresis between highly homologous ss (single-stranded) DNAs as observed in SSCP (single-strand conformation polymorphism) was pursued. Advancing our previous explanation [J. Biochem. 99, 663-671 (1986)], the mobility difference of ssDNAs was correlated to differences in their dynamic conformation (not to differences in their most stable structure), focusing on point-substituted sites. The contribution of semistable conformation dynamics was considered to be critical. Putative factors which may influence mobility (i.e., length of ssDNAs, location of substituted sites, and types of substitutions) were experimentally examined and critically discussed. Understanding of these phenomena should yield improvements in various techniques, such as SSCP, and in evaluation of the solution structures of DNA.抄録全体を表示PDF形式でダウンロード (2066K) -
Takeshi Ishimizu, Yoshihiko Sato, Toshihiro Saito, Yumi Yoshimura, Shi ...1996 年 120 巻 2 号 p. 326-334
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーS-allele-specific proteins (S-proteins) were separated and identified by two-dimensional (2D) gel electrophoresis from the style extract of 14 cultivars of Japanese pear, Pyrus pyrifolia Nakai, which exhibits gametophytic self-incompatibility. These S-proteins were 30-32 kDa basic proteins with putative pIs of 9.6-10.1 and were distinct from the other proteins, which were common for all cultivars examined. Each S-protein was assigned to a given S-genotype based on electrophoretic mobility and the partial amino acid sequence. For S1-to S7, -proteins, five different N-terminal amino acid sequences sharing the YFQFTQQY sequence were determined. Since the same N-terminal amino acid sequences were found for both S1- and S7-proteins, and for S3- and S5-proteins, the two S-proteins of each pair were distinguished based on their electrophoretic behavior. The internal amino acid sequences of S2- and S4-proteins, determined for Achromobacter protease I (API) digests, revealed that these proteins are S2- and S4-RNases, respectively. In the cultivar Nijisseiki, these two RNases were expressed from the white bud to mature flower stages when the cultivar acquires and enforces self-incompatibility. Osa-Nijisseiki, a self-compatible mutant of Nijisseiki, produced S2-RNase, but did not produce S4-RNase. The absence of S4-RNase was also observed in self-compatible offsprings derived from Osa-Nijisseiki. These results suggest that Japanese pear in the family Rosaceae possesses a gametophytic self-incompatibility system involving an S-RNase, and that a reduction or lack of expression of S4-RNase in the style is responsible for the self-compatibility of Osa-Nijisseiki.抄録全体を表示PDF形式でダウンロード (6449K) -
Naoko Norioka, Shigemi Norioka, Yoshimi Ohnishi, Takeshi Ishimizu, Chi ...1996 年 120 巻 2 号 p. 335-345
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe genes encoding three RNases were cloned from the style of a self-incompatible cultivar, Nijisseiki (S2S4), and its self-compatible mutant, Osa-Nijisseiki (S2S4sm, sm means stylar part mutant), of Japanese pear. For Nijisseiki, cDNAs coding for two S-RNases (S2-RNase and S4-RNase) and an RNase unrelated to self-incompatibility (non-S-RNase) were cloned from the stylar cDNA library. The cDNAs coding for S2-RNase, S4-RNase, and non-S-RNase include 678-, 684-, and 681-bp open reading frames, respectively. Their deduced amino acid sequences were composed of signal peptides and mature RNases (201-203 residues) which were verified by partial amino acid sequencing. The primary structures of mature proteins revealed that these RNases are of the RNase T2 type; only the two S-RNases have several potential N-glycosylation sites and 60% of their amino acid residues are identical, compared with 25% sequence identity with the non-S-RNase. Such a distinct difference in the primary structures between S-RNases and non-S-RNase has not previously been reported and may be a feature typical of S-RNases in the family Rosaceae. Similar experiments were performed for Osa-Nijisseiki. The cDNAs coding for S2-RNase and non-S-RNase were similarly cloned from the stylar cDNA library. However, the cDNA coding for S4-RNase was neither amplified by PCR nor cloned from the library, suggesting that the mutation of self-incompatible Nijisseiki to self-compatible Osa-Nijisseiki is due to a failure of expression of S4-RNase. These results lead to the idea that Osa-Nijisseiki is a variant of Nijisseiki in which the S4-allelic gene in the S-locus is exclusively mutated or deleted, causing severely impaired or suppressed expression of its gene product, S4-RNase, at the style.抄録全体を表示PDF形式でダウンロード (2328K) -
Tetsuo Ishida, Mitsuhiro Narita, Mitsuhiro Nozaki, Kihachiro Horiike1996 年 120 巻 2 号 p. 346-352
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーBovine dopamine β-monooxygenase is a tetramer consisting of two disulfide-linked dimers. To examine the role(s) of the intersubunit disulfide bonds in the protein structure and activity, the enzyme was treated with DTT at pH 7.5 and 25°C under nondenaturing conditions. A 15-min incubation with 0.5mM DTT selectively cleaved half of the intersubunit disulfide bonds. The cleavage did not affect the activity or tetrameric structure of the enzyme. Upon chemical modification of the reduced cysteine residues with 0.1M iodoacetamide (IAA) for 60min, half of the tetramer was converted to a dimeric species. The resulting dimeric and tetrameric species exhibited similar kinetic properties, and the Vmax values were decreased by 30% compared to that of the native enzyme. Upon treatment with IAA alone, no dimer species was detected but the enzyme lost 30% of the original activity. Cys514 and Cys516 were selectively modified by the treatment with DTT and IAA. From these results, we concluded that: (i) chemical modification of the intermolecular disulfide bonds strongly destabilizes the intersubunit interaction; (ii) breakage of the intersubunit interaction does not affect the activity. The reduction mechanism of the intersubunit disulfide bonds and the roles of the intersubunit interactions are discussed.抄録全体を表示PDF形式でダウンロード (957K) -
Atsushi Takeda, Yutaka Masuda, Takako Yamamoto, Takahiro Hirabayashi, ...1996 年 120 巻 2 号 p. 353-359
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe isolated and characterized almost the entire cDNA encoding BLM hydrolase from rat spleen cDNA libraries. The cDNA encoded a polypeptide composed of 454 amino acids, that had a slightly larger molecular mass than that was previously estimated by SDS-PAGE for purified BLM hydrolase subunit. Amino acid sequence alignments showed that rat BLM hydrolase is homologous to that of rabbit (94% identity of partial amino acid sequence), yeast cysteine protease (39% identity), and the pepC gene products of three bacteria (34-40% identity). In addition, it contained the three regions that are conserved in other cysteine proteases and thought to constitute the catalytic center. These results indicated that rat BLM hydrolase is a member of the papain superfamily of cysteine proteases. Sequencing revealed several putative sites phosphorylated by different types of protein kinases, but no signal sequence, transmembrane domain, N-linked glycosylation site or DNA-binding motif. The yeast homolog is a DNA-binding cysteine protease [Xu et al. (1994) J. Biol. Chem. 269, 21177-21183]. We demonstrated that rat BLM hydrolase also binds the singlestranded form of the Gal4 DNA-binding site oligonucleotide with high affinity compared with that of the double-stranded form. Northern blots revealed that the level of BLM hydrolase mRNA expression was very low in the rat skin, lung, and skeletal muscle. Furthermore, BLM hydrolase mRNA was ubiquitously expressed in the human cell lines, HeLa, SKG-IIIa, FL, KB, HEp-2, U373 GM, P3HR-1, Raji, THP-1, Jurkat, and Molt-4. These results suggested that BLM hydrolase plays important physiological roles, including the metabolism of antibiotics.抄録全体を表示PDF形式でダウンロード (3496K) -
Fuminori Tokunaga, Toshiro Tsukamoto, Takehiko Koide1996 年 120 巻 2 号 p. 360-368
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーProtein C is a zymogen of an anticoagulant vitamin K-dependent serine protease. Inherited protein C deficiency is often associated with a high risk for venous thromboembolism. It is characteristic of protein C deficiency that most single amino acid replacements result in type I (secretion defect) deficiency. To determine the molecular and cellular bases of protein C deficiency, we expressed recombinant human protein C mutants in which Arg15 was mutated to either Gly, Trp, Gln, Leu, or Pro by a single base exchange. Arg15 is one of the conservative residues in the γ-carboxyglutamic acid (Gla) domains of the vitamin K-dependent coagulation factors, and is also one of the high frequency multiple mutation sites in protein C deficiency. In transient expression studies using human kidney 293 cells, the relative amounts of Arg15 mutants secreted into the medium and determined by enzymelinked immunosorbent assay (ELISA) were as follows: Gly, 42%; Trp, 14%; Gln, 54%; Leu, 22%; and Pro, 13%, the amount of wild-type (Wt) protein C being taken as 100%. Thus, the order of the secreted amounts of the recombinant mutants was determined to be Wt>Gln>Gly>Leu>Trp, Pro. Pulse-chase experiments using both transiently-transfected and a pool of stably-transfected 293 cells, and stably-transfected BHK cells showed the same order of secretion efficiency. Since this order correlated well with that of the hydrophobicity scale of amino acid side chains, a conformational alteration of the Gla domain resulting in impaired secretion may be dependent on the hydrophobicity of the replaced amino acid. In transient cells, the relative radioactivities of pulse-labeled bands of all recombinant protein C were almost equal, suggesting that the same translational efficiency for Wt and all Arg15 mutants. All of the Arg15-mutated protein C precursors were shown to be located in the same organelle as protein disulfide isomerase (PDI), an endoplasmic reticulum-resident protein, and were sensitive to endoglycosidase H digestion. These results suggest that mutations of the highly conserved Arg15 in the Gla domain of protein C caused a secretion defect to variable degrees depending on replaced amino acid residue.抄録全体を表示PDF形式でダウンロード (4128K) -
Seiji Ishii, Hiroyuki Mizuguchi, Junji Nishino, Hideyuki Hayashi, Hiro ...1996 年 120 巻 2 号 p. 369-376
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーTo identify functional residues of rat liver L-aromatic amino acid decarboxylase (AADC), we aligned the sequences of 13 group II amino acid decarboxylases and performed mutational analysis on the residues that were invariant or conservatively substituted. Replacements of His192, Asp252, Asp271, Ser296, Lys303, Tyr332, and Arg355 with alanine residues decreased the AADC activity (kcat/Km) by more than 104-fold. Conservative replacements of [Asp252→aGlu], [Lys303→Arg], and [Tyr332→Phe] also resulted in decreases in activity by more than 104-fold, indicating that both the chemical properties and the shape of these residues are essential for catalysis. The presence of a Schiff base between the amino group of Lys303 and the coenzyme pyridoxal 5'-phosphate is important for catalysis, probably at the transaldimination step. The enzyme activity was essentially unaffected by conservative mutation of [Arg355→Lys], showing that the presence of a basic group at position 355 is necessary and sufficient for the catalysis. Replacement of [Thr246→Ala], [His269→Ala], and [Trp363→Leu] yielded mutant enzymes that were 1-8% as active as the wild-type enzyme; these residues are not essential for the catalysis but are considered to contribute to the activity through conformational or other effects. The roles of the catalytically important residues of the group II amino acid decarboxylases probed in this study were discussed in the light of their relationship with the residues of other pyridoxal enzymes.抄録全体を表示PDF形式でダウンロード (1138K) -
Atsuko Yamashita, Hideto Miyoshi, Taku Hatano, Hajime Iwamura1996 年 120 巻 2 号 p. 377-384
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe electron-transfer activities of bovine heart rnitochondrial complexes I, II, and III, but not complex IV, were simultaneously inhibited by 2-alkyl-4, 6-dinitrophenols to a different extent. The extent of inhibition of NADH and succinate oxidase activities by dinitrophenols was compared with that of individual complex activities using submitochondrial particles. The extent of inhibition of succinate oxidase activity by 1-methyipropyl and 1-methylbutyl derivatives was much larger than that of NADH oxidase activity. This large inhibition of succinate oxidase activity seemed not to be explainable by the extent of inhibition of individual complex activities (i.e., complexes II and III activities), based upon the homogeneous ubiquinone pool model. On the other hand, other dinitrophenols (n-propyl, 1-methylpentyl, 1-methylhexyl, and tert-butyl derivatives) very similar to the above compounds did not elicit such anomalous inhibitory action, indicating that the action of 1-methylpropyl and 1-methylbutyl derivatives is highly specific to their structure. The anomalous inhibition by these two compounds was also observed with the isolated succinate-cytochrome c oxidoreductase, in which there is no ubiquinone pool behavior [Rich, P. R. (1984) Biochim. Biophys. Acta 768, 53-79]. However, when the succinate-cytochrome c reductase of which the activity had been partially restored by adding phospholipid and exogenous quinone to the phospholipid- and ubiquinone-depleted succinate-cytochrome c reductase was assayed, the anomalous inhibitory action of interest was undetectable. These results indicated that electron-transfer between complexes II and III, which is mediated not only by free-form, but also by protein-bound ubiquinone, occurs in the mitochondrial membrane. The fact that the anomalous inhibition of succinate oxidase activity of submitochondrial particles was sensitive to changes in the external osmotic pressure which affected the total area of the particle supports this notion.抄録全体を表示PDF形式でダウンロード (1102K) -
Naofumi Uozumi, Tadashi Teshima, Toshihiro Yamamoto, Atsushi Nishikawa ...1996 年 120 巻 2 号 p. 385-392
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーAn assay method for GDP-L-Fuc: N-acetyl-β-D-glucosaminide α1-6fucosyltransferase (α1-6FucT; EC 2. 4. 1. 68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine γ-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. _??_ The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have α1-6fucose at the reducing end GlcNAc. This method is highly specific for α1-6FucT and is applicable for various experiments, including purification and cell culture ones.抄録全体を表示PDF形式でダウンロード (808K) -
Hitoo Iwase, Atsushi Tanaka, Yoshiyuki Hiki, Tohru Kokubo, Ikuko Ishii ...1996 年 120 巻 2 号 p. 393-397
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーIn our previous study, gas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA1. All O-linked oligosaccharide chains are known to be present in the hinge portion. However, the number of O-linked oligosaccharide chains on IgA1 remained unclear. In order to determine the number of linked sugar chains, we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the hinge glycopeptide prepared from human serum IgA1. MALDI-TOFMS did not show clear peaks, probably due to the microheterogeneity of the structure of each sugar chain. However, elimination of peripheral sialic acid and galactose residues by sequential treatment with neuraminidase and β-galactosidase gave clear mass spectra with several sharp peaks. On the basis of these spectra, we conclude that IgA1 prepared from normal human serum carries different numbers of sugar chains. There are two major populations, one contains five GalNAc residues and the other four GalNAc residues. On the other hand, the hinge glycopeptide prepared from myeloma IgA1 was composed mainly of one population containing four GalNAc residues. Earlier, we reported incomplete glycosylation of IgA1 isolated from the serum of an IgA1 myeloma patient. In this experiment, the presence of four O-linked oligosaccharides per heavy chain of IgA1 from a myeloma patient was found. The reason why only four out of five sites on the hinge glycopeptide were fully glycosylated in the IgA1 from the IgA1 myeloma patient is not clear.抄録全体を表示PDF形式でダウンロード (534K) -
Kuniaki Okamoto, Tomoko Kadowaki, Koji Nakayama, Kenji Yamamoto1996 年 120 巻 2 号 p. 398-406
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーLys-gingipain (KGP), so termed due to its peptide cleavage specificity for lysine residues, is a cysteine proteinase produced by the Gram-negative anaerobic bacterium Porphyromonas gingivalis. Mixed oligonucleotide primers designed from the NH2-terminal sequence of the purified enzyme were used to clone the KGP-encoding gene (kgp) from the organism. The nucleotide sequence of kgp had a 5, 169-bp open reading frame encoding 1, 723 amino acids with a calculated molecular mass of 218 kDa. As the extracellular mature enzyme had an apparent molecular mass of 51 kDa in gels, the precursor of KGP was found to comprise at least four domains, the signal peptide, the NH2-terminal prodomain, the mature proteinase domain, and the COOH-terminal hemagglutinin domain, and to be proteolytically processed during its transport. Importantly, the COOH-terminal region contained three direct repeats of two different amino acid sequences, LKWD (or E) AP and YTYTVYRDGTKI, and the subdomains located between the two repeats exhibited strong similarity to those of Arg-gingipain (RGP), another major cysteine proteinase produced by the organism and having cleavage specificity for arginine residues, although the arrangement of the subdomains was not necessarily identical in the two enzymes. Since the KGP activity was greatly decreased in RGP-deficient mutants and since the most probable site of the propeptide cleavage was present in the homologous sequence highly susceptible to proteolysis by RGP, the precursor of KGP is likely to be processed by RGP to form the mature enzyme.抄録全体を表示PDF形式でダウンロード (2928K) -
Akira Hirasawa, Senarath B.P. Athauda, Kenji Takahashi1996 年 120 巻 2 号 p. 407-414
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーPepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trionyx sinensis) by a series of chromatographies on DEAF-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43, 000 Da on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl-L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymosin.抄録全体を表示PDF形式でダウンロード (2628K) -
Hiroaki Masuda, Kazuhiko Tanaka, Michiko Takagi, Kinya Ohgami, Tetsuo ...1996 年 120 巻 2 号 p. 415-424
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーcDNA clones encoding a calponin isoform with 309 amino acids have been isolated from human heart. The deduced amino acid polypeptide (Mr 33, 697) showed a neutral isoelectric point of 7.1. The mRNA, expressed in cultured smooth muscle cells as well as in fibroblasts, vascular endothelial cells, and keratinocytes, contains a 3' untranslated region of 1.2 kilobases that includes an Alu repetitive sequence in the antisense direction. On the basis of the nucleotide sequence identity to an expressed sequence tag, HUM21ES93 [Cheng, J. -F., Boyartchuk, V., and Zhu, Y. (1994) Genomics 23, 75-84], the human neutral calponin gene is assigned to chromosome 21q11.1. The amino acid sequence indicates that this protein is the human equivalent of mouse calponin-h2 (94.8% identity) [Strasser, P., Gimona, M., Moessler, H., Herzog, M., and Small, J. V. (1993) FEES Lett. 330, 13-18]. Three tandem repeats of 29 amino acids, a Vav-homologous region and an actin-binding sequence, originally identified in the basic calponin isoform, are conserved. There are two consensus phosphorylation sites for tyrosine kinase. An immunoreactive form of the neutral calponin appears to be localized with vinculin in the cell-to-cell junctions of cardiomyocytes. Mouse calponin-h2 is also expressed in both embryonic and adult heart. These results indicate that the human neutral calponin is a non-smooth muscle isoform, and may play a physiological role in cytoskeletal organization.抄録全体を表示PDF形式でダウンロード (4501K) -
Natalia Battchikova, Marianne Koivulehto, Alexander Denesyuk, Leonid P ...1996 年 120 巻 2 号 p. 425-432
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーAspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments. DNA sequencing showed an open reading frame of 432 amino acids (Mr 47, 439) exhibiting moderately low homology with AspATs from other sources. Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved. The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain. The functional N-terminal arm was about two times longer than those of AspATs from other sources. According to the molecular model, the unique regions of B. circulans AspAT are all located together, forming a continuous network of contacts. Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs.抄録全体を表示PDF形式でダウンロード (1019K) -
Gerry M. Castillo, Joel A. Cummings, Catherine Ngo, Wenhua Yang, Alan ...1996 年 120 巻 2 号 p. 433-444
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーCo-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (Aβ) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar Aβ amyloid in brain [Snow, A. D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a _??_220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100g of EHS tumor were routinely extracted using 4M guanidine-HCI, followed by anion-ex-change and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with Aβ (1-40) into rodent hippocampus, led to deposition of fibrillar Aβ amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of Aβ amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.抄録全体を表示PDF形式でダウンロード (5206K) -
Ohoshi Murayama, Hitoshi Nishida, Kiyotoshi Sekiguchi1996 年 120 巻 2 号 p. 445-451
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーIntegrin α6β1 is a major adhesion receptor for the basement membrane, specifically binding to laminin-1. To identify the peptide sequences recognized by α6β1, we screened a 15-mer phage display library by panning with α6β1 purified from human placenta. DNA sequencing of 73 randomly picked phage revealed that three clones were dominantly enriched after repeated panning with α6β1. None of the peptide sequences displayed on these phage showed significant homology to laminin-1. A synthetic peptide modeled after the sequence displayed by one of these phage, designated P3, was found to strongly inhibit the binding of laminin-1 to α6β1. This inhibitory effect of the P3 peptide seems to be specific for α6β1, since it did not affect the binding of fibronectin to integrin α5β1. A synthetic peptide with a scrambled P3 amino acid sequence barely inhibited the binding of laminin-1 to α6β1. When coated on a substratum after conjugation with bovine serum albumin, the P3 peptide was capable of promoting cell spreading in an α6β1 -dependent manner, although the peptide with the scrambled sequence showed activity similar to that of a control peptide. These results taken together indicate that the P3 peptide is a novel ligand for integrin α6β1 with potent cell spreading activity.抄録全体を表示PDF形式でダウンロード (2992K) -
Tomoko Yamanushi, Aiko Hirata, Toshihiko Oka, Akihiko Nakano1996 年 120 巻 2 号 p. 452-458
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーSAR1 encodes a low molecular weight GTPase that is essential in the early process of vesicular transport in the secretory pathway. By random and site-directed mutagenesis of the SAR1 gene, we have obtained three temperature-sensitive mutants, N132I, E112K, and D32G. They all show a defect in transport from the endoplasmic reticulum to the Golgi apparatus, and accumulate endoplasmic reticulum membranes at the restrictive temperature. This is consistent with our previous observations in vivo on a galactose-shutoff mutant as well as the in vitro results, and provides powerful tools for further genetic analyses.抄録全体を表示PDF形式でダウンロード (3965K) -
Teruo Yasunaga, Shihoko Motoyama, Takeru Nose, Hiroaki Kodama, Michio ...1996 年 120 巻 2 号 p. 459-465
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe 3-nitro-2-pyridinesulfenyl (Npys) group bound to a mercapto group is a highly activated electrophilic reagent, which only reacts with a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. We incorporated the Npys group into enkephalin analogs to affinity label μ and δ opioid receptors. When rat brain membranes were incubated with [D-Ala2, Leu(CH2SNpys)5]enkephalin, and assayed for the inhibition of binding of DAGO and DSLET enkephalin analogs to opioid receptors, the number of receptors decreased sharply, depending upon the concentration of this SNpyscontaining enkephalin. It was found that this enkephalin analog occupies μ receptors highly specifically (EC50=51 nM) and almost 100 times more selectively than δ receptors. In contrast, [D-Ala2, Leu5] enkephalyl-Cys(Npys)6 attached covalently to δ receptors (EC50=34 nM) about 150 times more selectively than to μ receptors. Although N-ethylmaleimide also inhibited the binding of DAGO and DSLET, four to six orders of magnitude higher concentrations were required as compared to SNpys-containing enkephalins. When enkephalin-bound rat membranes were treated with dithiothreitol, the loss of receptors was reversed, depending upon the concentration of and incubation time with dithiothreitol. The recovery was much faster (about 1, 000 times) for δ receptors than for μ receptors. The present results indicated that both μ and δ receptors in rat brain consist of a free mercapto group near the enkephalin binding site and that SNpys-containing enkephalins can label these mercapto groups discriminatively. The disulfide bond between [D-Ala2, Leu5]enkephalyl-Cys6 and δ receptors appears to be exposed, while that between [D-Ala2, Leu(CH2-SNpys)5]enkephalin and μ receptors is shielded.抄録全体を表示PDF形式でダウンロード (2027K)
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