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O Koiwai

Publications and source records attributed to O Koiwai.

At least 37 records · Page 2Linked to original sources
PubMed1994

Gelatinases and metalloproteinase inhibitor secreted by murine colonic carcinoma cells with differing metastatic potential.

Gelatinase activity and inhibitory activity against collagenase were measured in serum-free medium conditioned by murine colonic carcinoma cells with different spontaneous metastatic potentials to the lung. The medium conditioned with poorly metastatic NM11 cells gave higher inhibitory activity than that conditioned with highly metastatic LuM1 cells, while the level of secreted gelatinases in the same medium was lower in NM11 medium than in LuM1 case. Northern analysis showed the higher gene expression of both tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in NM11 cells than in LuM1 cells, suggesting that both TIMPs are responsible for the increase of inhibitory activity in NM11 conditioned medium. Examination of the balance of gelatinases and inhibitor revealed that the amount of inhibitor exceeded that of gelatinases in the medium conditioned with NM11 cells. In contrast, the medium conditioned with LuM1 cells contained excess amounts of gelatinases. The results indicated a close correlation between the balance of gelatinases and inhibitors and the metastatic behavior of murine tumor cells.

Animals↗
PubMed1994

A new type of defect in the gene for bilirubin uridine 5'-diphosphate-glucuronosyltransferase in a patient with Crigler-Najjar syndrome type I.

Crigler-Najjar syndrome (CN) type I, which is characterized by the complete absence of bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT) activity, is inherited as an autosomal recessive trait associated with unconjugated hyperbilirubinemia. Phenobarbital has no effect on the bilirubin concentration in the serum of patients with CN type I. Recently, cDNA for two human liver bilirubin UGT (UGT1A and UGT1D) were isolated, and their genetic organization was determined. The UGT1A (UGT1*1) and UGT1D (UGT1*4) genes each have a unique exon 1, whereas exons 2-5 are common to both genes. It has been predicted that some defect in the exons common to both genes is responsible for the absence of UGT1A and UGT1D activities in CN type I, and five cases with such a mutation have been reported. We describe here a new type of defect in the gene for bilirubin UGT in a patient with CN type I, namely, an abnormality in the exon 1 that is characteristic of the UGT1A gene. This mutation is a single nucleotide substitution, that is, C is changed to A at base position 840 in UGT1A cDNA, and this change results in a stop codon. Our patient is homozygous for the defect, and his nonconsanguineous parents and elder brother, who are clinically normal, are heterozygous for the defective allele. No mutation was detected in any exons of the UGT1D gene. Our results suggest that a homozygous nonsense or deletion mutation is detected not only in the exons common to UGT1A and UGT1D genes but also in unique exon 1 of UGT1A in CN type I.

Amino Acid Sequence↗
PubMed1993

Identification of defect in the genes for bilirubin UDP-glucuronosyl-transferase in a patient with Crigler-Najjar syndrome type II.

Crigler-Najjar syndrome (CN) type II is characterized by severe chronic nonhemolytic unconjugated hyperbilirubinemia due to reduced hepatic bilirubin UDP-glucuronosyl-transferase (UGT) activity. Two bilirubin UGT isozymes, UGT1A and UGT1D, have been identified. We analyzed the DNA sequence of the bilirubin UGT genes in a 5-year-old Japanese male patient with CN type II, who had consanguineous parents. Point mutations were found on exons 1 of the UGT1A and UGT1D genes. The abnormalities were single nucleotide substitutions of G by A and of T by C at base position 211 of UGT1A cDNA and at base position 395 of the UGT1D, respectively. We found another single nucleotide substitution of T by G on exon 5 common to both genes at base position 1456 of the UGT1A cDNA or 1459 of the UGT1D cDNA. These three mutations result in changes of glycine to arginine and of tyrosine to aspartic acid at amino acid positions 71 and 486 of the UGT1A protein, and of leucine to proline and of tyrosine to aspartic acid at amino acid positions 132 and 487 of the UGT1D protein, respectively. Our patient was homozygous for all defects and his parents and elder brother were heterozygous for all defective alleles. The findings suggest that the CN Type II is inherited as an autosomal recessive trait.

Adult↗
PubMed1993

Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct.

A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis. The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon. This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21. The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E. coli BL21 cells that express T7 RNA polymerase. The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside. The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I. Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E. coli cells.

Amino Acid Sequence↗
PubMed1993

Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene.

The mouse cDNA encoding DNA topoisomerase I (TopoI) was cloned and the nucleotide sequence of 3512 bp was determined. The cDNA clone contained an open reading frame encoding a protein of 767 amino acids (aa), which is 2 aa longer than its human counterpart. Overall aa sequence homology between the mouse and human, and between the mouse and yeast (Saccharomyces cerevisiae) sequences was 96% and 42%, respectively. The mouse TopI gene was mapped at position 54.5 on chromosome 2 from linkage analyses of a three-point cross test with Geg, Ada, and a as marker genes.

3T3 Cells↗
PubMed1993

[Genetic defect of the hyperbilirubinemic Gunn rat, a model for Crigler-Najjar syndrome type I].

The hyperbilirubinemic Gunn rat lacks hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin and has been used as an animal model for human Crigler-Najjar syndrome type I. Rat liver bilirubin UDPGT cDNA was isolated. The cDNA shared an identical 913-bp sequence (corresponding to the C-terminal 247 amino acid residues) with that for phenol UDPGT whose activity was also deficient in the Gunn rat. The bilirubin UDPGT gene was mapped at the position of 37 on mouse chromosome 1 by analyzing restriction endonuclease fragment length variations using the rat bilirubin UDPGT cDNA as a probe. The genetic defect of bilirubin UDPGT in the mutant rat was proved to be a -1 frameshift mutation. The mutation was found not only to be located in the region where the cDNA for bilirubin UDPGT shared the identical sequence with that for phenol UDPGT but also to occur in the same position in the two cDNAs from the mutant.

Amino Acid Sequence↗
PubMed1992

Gastric procathepsin E and progastricsin from guinea pig. Purification, molecular cloning of cDNAs, and characterization of enzymatic properties, with special reference to procathepsin E.

Procathepsin E and progastricsin were purified from the gastric mucosa of the guinea pig. They were converted to the active form autocatalytically under acidic conditions. Each active form hydrolyzed protein substrates maximally at around pH 2.5. Pepstatin inhibited cathepsin E very strongly at an equimolar concentration, whereas the inhibition was much weaker for gastricsin. Molecular cloning of the respective cDNAs permitted us to deduce the complete amino acid sequences of their pre-proforms; preprocathepsin E and preprogastricsin consisted of 391 and 394 residues, respectively. Procathepsin E has unique structural and enzymatic features among the aspartic proteinases. Lys at position 37, which is common to various aspartic proteinases and is thought to be important for stabilizing the activation segment, was absent at the corresponding position, as in human procathepsin E. The rate of activation of procathepsin E to cathepsin E is maximal at around pH 4.0. It is very different from the pepsinogens and may be correlated with the absence of Lys37. Native procathepsin E is a dimer, consisting of two monomers covalently bound by a disulfide bridge between 2 Cys37. Interconversion between the dimer and the monomer was reversible and regulated by low concentrations of a reducing reagent. Although the properties of the dimeric and monomeric cathepsins E are quite similar, a marked difference was found between them in terms of their stability in weakly alkaline solution: monomeric cathepsin E was unstable at weakly alkaline pH whereas the dimeric form was stable. The generation of the monomer was thought to be the process leading to inactivation, hence degradation of cathepsin E in vivo.

Amino Acid Sequence↗
PubMed1992

Mapping of the mouse bilirubin UDP-glucuronosyltransferase gene (Gnt-1) to chromosome 1 by restriction fragment length variations.

Restriction endonuclease fragment length variations (RFLVs) were detected by using a rat cDNA probe for the bilirubin UDP-glucuronosyltransferase (UDPGT) gene between two mouse strains, 129/Sv and MOL-MIT. RFLVs of the gene were found by EcoRI and PvuII digestions. From linkage analyses of the three-point cross test using Elo and En-1 as marker genes, the bilirubin UDPGT gene was mapped at position 37 on chromosome 1. Bilirubin and phenol UDPGTs have been suggested to be expressed by a single gene by alternative splicing in human and rat. The mouse bilirubin UDPGT gene was named Gnt-1.

Animals↗
PubMed1991

Development-dependent expression of isozymogens of monkey pepsinogens and structural differences between them.

The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A-(1-4) and progastricsin. Their respective relative levels and specific activities changed significantly during development. The sequential expression of genes for type-A pepsinogens was particularly noteworthy. Pepsinogen A-3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A-2 became predominant at the 4-month stage, and pepsinogen A-1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A-1, A-2 and A-3 were similar but not identical to those of pepsinogen A-4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type-A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein-coding regions being identical. Northern analysis revealed that the level of expression of genes for type-A pepsinogens and for progastricsin was significant at the fetal stage and increased with development.

Aging↗
PubMed1991

Genetic defect of bilirubin UDP-glucuronosyltransferase in the hyperbilirubinemic Gunn rat.

The genetic defect of bilirubin UDP-glucuronosyltransferase (UDPGT) in the hyperbilirubinemic Gunn rat was proved to be a -1 frameshift mutation. The mutation was found not only to be located in the region where bilirubin UDPGT cDNA shared an identical sequence with 3-methylcholanthrene (3M C)-inducible UDPGT cDNA but also to occur in the same position on the two cDNAs from the mutant rat. At the 5' end of the identical region there was a consensus sequence for splicing, of which position coincided with the boundary between the 2nd and 3rd exon of the testosterone UDPGT gene. These results strongly suggest that mRNAs for bilirubin and 3M C-inducible UDPGTs are produced from a single primary-transcript after an alternative splicing and the defects of bilirubin and 3M C-inducible UDPGTs in the mutant rat are caused by a point mutation on a common exon.

Animals↗
PubMed1991

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.

Amino Acid Sequence↗
PubMed1991

A molecular genetic linkage map of mouse chromosome 19, including the lpr, Ly-44, and Tdt genes.

The mouse lpr gene, which is an autosomal recessive gene causing autoimmune disease with features of human systemic lupus erythematosus and eventually death from severe immune-complex glomerulonephritis, has been mapped on chromosome 19. To determine its exact chromosomal location, a three-point backcross was carried out by mating (MRL/MpJ-lpr/lpr x MOL-MIT)F1 x MRL/MpJ-lpr/lpr using the genes Ly-44 (lymphocyte differentiation antigen-44) and Tdt (terminal deoxynucleotidyl transferase) as markers. The following order of genes is proposed, with the distances between genes given in parentheses: centromere-Ly-44 (19.3 cM)-lpr (6.1 cM)-Tdt-telomere. The Ly-44a and Tdta alleles are found in all laboratory strains and in the wild Western European subspecies, domesticus and brevirostris. In contrast, the Ly-44b and Tdtb alleles are found in some Asian subspecies, Chinese mice of wild origin, yamashinai and molossinus. Furthermore the third Tdt allele, Tdtc, is detected in castaneus.

Animals↗
PubMed1991

Expression of terminal deoxynucleotidyltransferase gene in a case of adult T-cell leukemia.

A 37-year-old male from Kagoshima Prefecture was admitted with adult T-cell leukemia (ATL). Monoclonal integration of HTLV-1 proviral DNA was found, but the integration site was different from that of terminal deoxynucleotidyltransferase (TdT)-positive MT-1 cells (an ATL cell line). The ATL cells expressed enzymatically active TdT and exhibited 2100 b TdT mRNA, which corresponds to the thymus type of TdT mRNA. The same size of TdT mRNA was also detected in MT-1. Southern blot analyses revealed no differences in the gene structure of the promoter region of TdT genes between this ATL case and TdT-positive lymphoblastic leukemia cells. There is little possibility that cis-acting viral elements promote TdT gene expression by proviral integration. The activation of TdT gene in ATL may be mediated by other trans-acting factors.

Adult↗
PubMed1990

Structure and development of rabbit pepsinogens. Stage-specific zymogens, nucleotide sequences of cDNAs, molecular evolution, and gene expression during development.

In order to clarify the structure and development of rabbit pepsinogens, purification and molecular cloning of these proteins were performed at various developmental stages. Several pepsinogens were isolated, and they were classified as pepsinogens F and M, and into pepsinogen groups I, II, and III. The relative levels and specific activities of the various pepsinogens changed significantly during development. Pepsinogens F and M were present only at the early postnatal stage, and their level was higher than those of other pepsinogens at this stage. Pepsinogens in groups I, II, and III were the predominant zymogens at the late postnatal stage. cDNA clones encoding all of these pepsinogens were obtained, with the exception of pepsinogens I and M, and the nucleotide sequences were determined. Each cDNA contained a leader region (signal peptide), a pro-region (activation segment), and a pepsin region, of 15, 44, and 328 residues, respectively, with the exception of the cDNA for pepsinogen F in which the pro- and pepsin regions were composed of 43 and 330 residues, respectively. Pepsinogens in groups II and III exhibited a high degree of similarity with one another, whereas many substitutions were found in pepsinogen F. A unique substitution in the activation segment of pepsinogen F, namely, Gly----Asp at position 21, was found, which made the structural features of this segment more specific. A phylogenic tree was constructed from the differences in nucleotide sequences and showed clearly that each pepsinogen in groups II and III could be classified as pepsinogen A, a major pepsinogen in mammals. Pepsinogen F diverged significantly from these groups and may be a new type of pepsinogen. Northern analysis revealed that the expression of the gene for pepsinogen F was restricted to the early postnatal stage, and the expression of genes for pepsinogens in groups II and III was detected predominantly at later stages, a result that shows the switching of gene expression from fetal pepsinogen to adult pepsinogens during development.

Aging↗
PubMed1990

Isolation and sequencing of rat liver bilirubin UDP-glucuronosyltransferase cDNA: possible alternate splicing of a common primary transcript.

A 1763-bp cDNA for rat liver bilirubin UDP-glucuronosyltransferase (UDPGT) was isolated. Bilirubin UDPGT activity was demonstrated by transfection of the pcDL1 vector carrying the cDNA into COS7 monkey kidney cells. The cDNA shares an identical 913-bp sequence (corresponding to the C-terminal 247 amino acid residues) with that for rat liver 3-methylcholanthrene-inducible 4-nitrophenol UDPGT including the locus where a -1 frameshift mutation was found in the 4-nitrophenol UDPGT cDNA from the jaundiced homozygous Gunn rat. The result suggests that both the UDPGTs are derived from a common primary-transcript and that the multiple defects of UDPGT isoenzymes observed in the homozygous Gunn rat may be produced by a single-mutated-locus after an alternative splicing of the 5' end region.

Animals↗
PubMed1989

Demonstration of promoter function for the 5'-flanking region of the human gene for terminal deoxynucleotidyl transferase.

Promoter activity was detected by transfection of pBS vectors that carried the 5'-flanking region of the gene for human terminal deoxy-nucleotidyltransferase (TdT) and the entire human TdT cDNA into COS7 monkey kidney cells, with direct assay of the expressed enzymatically active TdT. The synthesis of TdT was also detected immunologically using polyclonal rabbit antibodies against bovine TdT. One fifth of the TdT activity detected in normal thymocytes was detected in transfected COS7 cells. This results suggests that a lymphoid-specific regulatory system is present in B or T-cells to promote the effective expression of TdT.

Animals↗
PubMed1989

Gene expression of terminal deoxynucleotidyl transferase in neoplastic cells of leukemia and lymphoma.

Expression levels of terminal deoxynucleotidyl transferase (TdT) mRNA in fresh leukemia and lymphoma cells were measured by northern blotting analysis. Bands of 2.1 kb mRNA were detected in all of eight cases of TdT activity-positive leukemias: two cases of null-cell acute lymphoblastic leukemia (null-ALL), two of common ALL, one of pre-B ALL, one of T-ALL, and two of chronic myelogenous leukemia in blastic crisis. One of the null-ALL and one of the common ALL cases also showed large TdT mRNA (3.3 kb). Since all TdT activity-positive samples exhibited TdT mRNA, the TdT gene might be mainly regulated at the transcription level in leukemic cells. An elevated level of 2.1 kb TdT mRNA was also detected in one lymphoma case, where neither TdT activity nor immunoreactive TdT was detected. The extensive chromosomal abnormality demonstrated in this case might be associated with the translational anomaly of TdT.

Blotting, Northern↗