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E Matsui

Publications and source records attributed to E Matsui.

At least 19 recordsLinked to original sources
PubMed2000

Human Cdc7-related kinase complex. In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks.

huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D. , Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

Antigens, Viral, Tumor↗
PubMed2000

The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii.

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 A. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting two domains, and a long loop transformed from an alpha-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(233) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe(121) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.

Amino Acid Sequence↗
PubMed2000

Author reply

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Journal Article↗
PubMed2000

Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii.

A beta-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85 degrees C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100 degrees C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90 degrees C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k(cat)/K(m) values high enough for hydrolysis of beta-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of beta-linked glucose dimer more hydrophilic than aryl- or alkyl-beta-D-Glcp.

Amino Acid Sequence↗
PubMed2000

Reduced IFNgamma production in response to IL-12 stimulation and/or reduced IL-12 production in atopic patients.

BACKGROUND: Several studies have shown that interleukin (IL)-4 and interferon-gamma (IFNgamma) are important for the regulation of IgE production and that IL-12 induces IFNgamma. OBJECTIVE: IFNgamma production in response to IL-12 stimulation and IL-12 production were investigated in peripheral blood mononuclear cells (PBMCs) of atopic patients with various levels of serum IgE. METHODS: Cytokine production from PBMCs was measured following stimulation with a nonspecific stimulator (phytohemagglutinin: PHA), a specific stimulator (Der f 1) and IL-12 in 17 healthy controls and 23 atopic patients with various serum IgE levels. RESULTS: The IFNgamma production by IL-12-stimulated PBMCs in the atopic group was lower than that in the control group. Furthermore, the serum IgE level was negatively correlated with IFNgamma production by PBMCs stimulated with IL-12 (P < 0.001), and with IL-12 production by PBMCs stimulated with Der f 1 (P < 0.001). Although the IFNgamma concentrations by PHA-stimulated PBMCs were correlated with those by IL-12-stimulated PBMCs, there were differences in several patients. CONCLUSIONS: Our results indicated that atopic patients may have some abnormality in the IL-12-IFNgamma loop. It was shown that the elevation in IgE levels in atopic patients may be due to reduced IFNgamma production in response to IL-12 stimulation and/or due to reduced IL-12 production.

Adolescent↗
PubMed1999

Mutations of the IL-12 receptor beta2 chain gene in atopic subjects.

Interleukin-12(IL-12) promotes cell-mediated Th1 responses and production of IFN-gamma that downregulates IgE production. The signal of IL-12 is transduced through the IL-12 receptor (IL-12R) and Stat4. Twenty-four of 75 atopic individuals with high levels of IgE showed insufficient IFN-gamma production by peripheral blood mononuclear cells (PBMCs) following stimulation with IL-12 but not that with phytohemagglutinin (PHA). Interestingly, 10 of the above 24 subjects were found to be heterozygous for truncated (2496 del 91) or missense (1577 A to G and 2799 A to G) mutations of IL-12R beta2 chain gene (IL-12R beta2). Insufficient phosphorylation of Stat4 was also demonstrated in these 10 individuals. This is the first report showing that reduced IFN-gamma production following IL-12 stimulation is associated with the heterozygous IL-12R beta2 mutations in atopic subjects.

Adolescent↗
PubMed1999

Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically.

The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.

Amino Acid Sequence↗
PubMed1999

Multiple chondromatous hamartomas of the lung: a case report and review of the literature with special reference to Carney syndrome.

BACKGROUND: Multiple chondromatous hamartomas of the lung, which are very rare, are a feature of Carney syndrome. The relation between the two entities is not clear. METHODS: A patient with multiple chondromatous hamartomas of the lung is described in this article. The literature was reviewed with special reference to the relation between multiple chondromatous hamartomas of the lung and Carney syndrome as well as the triad of gastric epithelioid leiomyosarcoma, functioning extra-adrenal paraganglioma, and pulmonary chondroma. RESULTS: A total of 15 cases of multiple chondromatous hamartomas of the lung have been published worldwide. Two cases exhibited two other features of Carney syndrome, namely, gastric leiomyogenic neoplasms and extra-adrenal paragangliomas, and three other cases demonstrated only gastric leiomyomatous neoplasms. These five patients were all young females. CONCLUSIONS: Some patients with multiple chondromatous hamartomas of the lung have a history of Carney syndrome. Patients with multiple chondromatous hamartomas require further examination of other sites, particularly the stomach and nervous system.

Chondromatosis↗
PubMed1999

Comparison of endoscopic features of early-stage squamous cell lung cancer and histological findings.

Seventy cases with early-stage central-type squamous cell carcinoma were treated surgically between 1984 and 1993 in seven participating institutes. We classified endoscopic features of early-stage central-type squamous cell carcinoma into three types (hypertrophic type, nodular type and polypoid type). After surgery we investigated the relationship between endoscopic features and both the area of superficial extent and depth of carcinoma invasion based on histopathological investigations of the surgical specimens. In 66.7% of the hypertrophic type lesions cancer cells did not invade into the cartilaginous layer, and only 4.8% of this type showed tumour invasion beyond the bronchial cartilage. On the other hand, a few nodular and polypoid type cases showed in-situ carcinoma or carcinoma with invasion from the subepithelial layer to the muscle layer, and in approximately 20% the these types we observed carcinoma invasion beyond the cartilaginous layer, which was not suitable for photodynamic therapy. Also, concerning the greatest dimension 24 out of 35 lesions (68.6%) less than 10 mm in the greatest dimension were evaluated as either in-situ carcinoma or micro-invasive tumour within the muscle layer. The endoscopic features can provide a basis for the determination of therapeutic strategy in early-stage central-type lung cancer.

Aged↗
PubMed1999

Expression of the BLM gene in human haematopoietic cells.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by stunted growth, sun-sensitive erythema and immunodeficiency. Chromosomal abnormalities are often observed. Patients with BS are highly predisposed to cancers. The causative gene for BS has been identified as BLM. The former encodes a protein, which is a homologue of the RecQ DNA helicase family, a family which includes helicases such as Esherichia coli RecQ, yeast Sgs1, and human WRN. WRN is encoded by the gene that when mutated causes Werner's syndrome. The function of BLM in DNA replication and repair has not yet been determined, however. To understand the function of BLM in haematopoietic cells and the cause of immunodeficiency in BS, expression of the BLM gene in various human tissues and haematopoietic cell lines was analysed and the involvement of BLM in immunoglobulin rearrangement examined. In contrast to WRN, BLM was expressed strongly in the testis and thymus. B, T, myelomonocytic and megakaryocytic cell lines also expressed BLM. All of the examined sequences at the junction of the variable (V), diversity (D) and joining (J) regions of the immunoglobulin heavy-chain genes were in-frame, and N-region insertions were also present. The frequency of abnormal rearrangements of the T cell receptor was slightly elevated in the peripheral T cells of patients with BS compared with healthy individuals, whereas a higher frequency of abnormal rearrangements was observed in the cells of patients with ataxia-telangiectasia (A-T). In DND39 cell lines, the induction of sterile transcription, which is required for class switching of immunoglobulin heavy-chain constant genes, was correlated with the induction of the BLM gene. Taking into consideration all these results, BLM may not be directly involved in VDJ recombination, but is apparently involved in the maintenance of the stability of DNA.

Adenosine Triphosphatases↗
PubMed1999

A fission yeast gene, him1(+)/dfp1(+), encoding a regulatory subunit for Hsk1 kinase, plays essential roles in S-phase initiation as well as in S-phase checkpoint control and recovery from DNA damage.

Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G(1)/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1(+) from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1(+) is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G(1). The protein level is low at START in G(1), increases at the G(1)/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G(1)/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1(+) is identical to rad35(+). Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

Amino Acid Sequence↗
PubMed1999

Plasma adrenomedullin and natriuretic peptides in patients with essential or malignant hypertension.

Adrenomedullin (AM), a potent vasodilator and natriuretic peptide, is found in human blood. To investigate the pathophysiological role of AM in essential and malignant hypertension (EHT and MHT), we measured the plasma concentrations of AM in patients with EHT of WHO stage I or II (n = 42) and in those with MHT (n = 9) by a specific radioimmunoassay, and compared these concentrations with those in normotensive controls (n = 46). The plasma concentrations of atrial and brain natriuretic peptides (ANP and BNP) in these subjects were also measured by immunoradiometric assays, and their relations to plasma AM were examined. The plasma AM level in the EHT patients (7.15+/-0.21 pmol/l, mean+/-SEM) was significantly (p < 0.01) higher than that in the normotensive controls (6.14+/-0.25 pmol/l), and a further elevation was observed in the MHT patients (14.1+/-3.8 pmol/l). Similar elevations of plasma ANP and BNP were seen in the two patient groups. The plasma AM level significantly (p < 0.01) correlated with not only the systolic (r = 0.44) and diastolic (r = 0.46) blood pressures, but also with the plasma levels of ANP (r = 0.43) and BNP (r = 0.43). The elevated plasma concentration of AM in the MHT patients decreased significantly (p < 0.05) after antihypertensive treatment, and the plasma ANP and BNP levels similarly declined. These results suggest that AM may participate, along with ANP and BNP, in mechanisms counteracting a further elevation of blood pressure in patients with EHT and MHT.

Adrenomedullin↗
PubMed1998

Acylamino acid-releasing enzyme from the thermophilic archaeon Pyrococcus horikoshii.

When the genome of the thermophilic archaeon Pyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coli with the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 degreesC, with its optimum temperature over 90 degreesC. The specific activity of the enzyme increased 7-14-fold with heat treatment, suggesting the modification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of Nalpha-acylated residues in short peptide sequence analysis at high temperatures.

Amino Acid Sequence↗
PubMed1998

Reduced interferon gamma production by antigen-stimulated cord blood mononuclear cells is a risk factor of allergic disorders--6-year follow-up study.

BACKGROUND: It is not sufficient to predict 'high allergic risk newborns' on the basis of increased IgE concentrations of cord blood alone, because a raised cord blood IgE concentration is specific but not sensitive in the prediction of the development of allergic disorders. Warner et al. have reported that interferon gamma (IFN-gamma) production by allergen-triggered cord blood cells is a predictor of atopic eczema, based on the 1-year follow-up study. OBJECTIVE: We examined whether IFN-gamma production by antigen-stimulated cord blood mononuclear cells (CBMCs) is a risk factor of allergic disorders, based on the 6-year follow-up study. METHODS: The relationships among cord blood IgE concentrations, IFN-gamma and interleukin-2 (IL-2) productions by antigen-stimulated CBMCs, and the development of allergic disorders in 21 infants for 6 years were investigated. RESULTS: Atopic dermatitis, atopic dermatitis and allergic rhinitis, or atopic dermatitis and bronchial asthma developed in seven of 21 subjects. The maximal IFN-gamma concentration in culture supernatants of ovalbumin (OA) or bovine serum albumin (BSA) stimulated CBMCs in infants who developed allergic disorders was significantly lower (P < 0.01) than that in infants who did not develop any allergic disorders. In contrast, the maximal IL-2 concentration in culture supernatants of OA- or BSA-stimulated CBMCs in infants who developed allergic disorders was not lower than in infants who did not develop any allergic disorders. Moreover, the IFN-gamma concentrations were negatively correlated with cord blood IgE concentrations. CONCLUSION: Reduced IFN-gamma production by antigen-stimulated CBMCs is a risk factor of allergic disorders.

Antigens↗
PubMed1998

Expression of Pax5 gene in human haematopoietic cells and tissues: comparison with immunodeficient donors.

In mice, Pax5 gene is indispensable for B cell development. Pax5-deficient mice fail to produce mature B cells owing to complete arrest of B cell development at a precursor stage. However, the lineage and stage of human Pax5 gene expression have remained elusive. In this investigation expression of the human Pax5 gene was studied. Pax5 gene expression was detected in B cell lines but not in myeloma cell lines. CD19 expression was correlated with Pax5 gene expression. Adult spleen and bone marrow and fetal spleen and liver showed strong Pax5 gene expression, as did the corresponding mouse tissues, as reported previously. In common variable immunodeficiency (CVID) peripheral blood lymphocytes (PBL) with a decreased number of B cells, no Pax5 gene expression was detected. Some CVID PBL stimulated with IL-2, IL-10 and anti-CD40 monoclonal antibody, expressed the Pax5 gene. Defect of Pax5 gene expression in CVID may be caused by regulatory T cell disorder.

Adolescent↗
PubMed1998

Overexpression of CD44 variant transcripts in rat transplantable thyroid carcinoma lines demonstrating lung metastasis.

Expression of CD44 isoforms has been reported to be involved in tumor invasion and metastasis in both rodents and man. We earlier documented establishment of rat transplantable thyroid carcinoma lines in vivo from primary lesions induced by a chemical carcinogen. Recently, two lines (L1a-M4 and L2a-M6) were found to spontaneously metastasize to the lung after subcutaneous transplantation. To determine whether CD44 splice variants contribute to their metastatic spread, carcinoma lines with and without lung metastasis were evaluated quantitatively and qualitatively using RT-PCR followed by hybridization and immunohistochemical analyses. The L1a-M4 and L2a-M6 metastatic lines showed significant overexpression of CD44 variant transcripts containing variant exons v4-v6 or v9-v10/v8-v10, respectively, with concomitant reduced levels of standard transcripts. Investigation of the precise composition of alternatively spliced mRNA in normal tissues and carcinoma lines using an exon-specific RT-PCR method, revealed major chain variant transcripts containing v2/v3, v4-v6, v7-v10 and v8-v10 in all specimens. Applying the same RT-PCR analysis to mRNAs derived from cultured cell lines, demonstrated essentially the same pattern. The results suggest that quantitative increase rather than qualitative change in CD44 variant isoforms is associated with the pathogenesis of lung metastasis of rat thyroid carcinomas.

Alternative Splicing↗
PubMed1997

BLM (the causative gene of Bloom syndrome) protein translocation into the nucleus by a nuclear localization signal.

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, sun sensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS, and BLM protein is homologous to DNA helicase. There are two putative nuclear localization signals (NLSs) within amino acid residues 1334-1349 in the C-terminus of the BLM protein, which has the distinctive structure of two basic residue arms separated by a spacer. The entire coding or deleted BLM sequences of various sizes were ligated into an enhanced green fluorescent protein (EGFP) vector and transfected into HeLa cells. The EGFP vector harboring the entire BLM coding sequence was transported to the nucleus. The BLM protein truncated at 1341 amino acid, containing an intact helicase domain and only one proximal arm, was not transported to the nucleus. The BLM protein truncated at 1357 amino acid, containing an intact helicase domain and two arms, was transported to the nucleus. The EGFP vector harboring DNA fragments encoding a protein having only the distal arms of basic amino acids in the C-terminus was also transported to the nucleus. The truncated BLM proteins corresponding to previously reported mutated BLM proteins were retained in the cytoplasm or both the cytoplasm and the nucleus as was the EGFP vector with no insert. These results show that the BLM protein translocates into the nucleus and that the distal arm of the bipartite basic residues in the C-terminus of the BLM protein is essential for targeting the nucleus.

Adenosine Triphosphatases↗