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H Takeshita

Publications and source records attributed to H Takeshita.

At least 19 recordsLinked to original sources
PubMed1999

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis.

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.

Amino Acid Substitution↗
PubMed1998

Identification of the three non-identical subunits constituting human deoxyribonuclease II.

We purified DNase II from human liver to apparent homogeneity. The N-terminal amino acid sequences of each of three components constituting the purified mature enzyme were then separately determined by automatic Edman degradation. A combination of this chemical information and the previously reported nucleotide sequence of the cDNA encoding human DNase II [Yasuda et al. (1998) J. Biol. Chem. 273, 2610-2626] allowed detailed elucidation of the enzyme's subunit structure: human DNase II was composed of three non-identical subunits, a propeptide, proprotein and mature protein, following a signal peptide. Expression analysis of a series of deletion mutants derived from the cDNA of DNase II in COS-7 cells suggested that although a single large precursor protein may not be necessary for proteolytic maturation, the propeptide region L17-Q46 may play an essential role in generating the active form of the enzyme.

Amino Acid Sequence↗
PubMed1998

Relation between cellular doxorubicin binding ability to nuclear DNA and histologic response to preoperative chemotherapy in patients with osteosarcoma.

BACKGROUND: Although chemosensitivity to antiosteosarcoma agents is the most important prognostic factor in human osteosarcoma, none of the many chemosensitivity tests reported previously are reliable and clinically useful. In this study, the authors investigated the reliability and clinical availability of doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) binding assay (ABA) as a new chemosensitivity test for osteosarcoma. METHODS: Doxorubicin (adriamycin [ADM]) binding ability (%AB) to nuclear DNA in isolated osteosarcoma cells was assessed by ABA in 14 patients with primary osteosarcoma who were treated with preoperative chemotherapy containing ADM and 6 patients with relapsed osteosarcoma after intensive chemotherapy. Histologic responses to preoperative chemotherapy were evaluated by percentage of tumor necrosis (%necrosis). RESULTS: Four of the 14 patients with primary osteosarcoma had %AB > 80% (97.3+/-3.7%) and demonstrated good histologic responses (>90% of %necrosis) to preoperative chemotherapy, whereas the remaining 10 patients had %AB < 80% (38.9+/-21.0%) and demonstrated poor responses. Patients with recurrent osteosarcoma that was clinically evaluated to be resistant to previous chemotherapy also had low %AB (34.2+/-28.3%). CONCLUSIONS: Because the results of the current study revealed that ABA is useful for predicting chemosensitivity to chemotherapy with ADM as well as chemotherapy without ADM for patients with osteosarcoma, and because ABA technically is simple and results can be assessed rapidly, the authors conclude that ABA is a clinically useful chemosensitivity test for patients with osteosarcoma.

Adolescent↗
PubMed1998

Affinity maturation in Lyn kinase-deficient mice with defective germinal center formation.

Lyn kinase-deficient (lyn-/-) mice show several abnormalities such as reduced numbers of circulating B cells, hyper-IgM, and low proliferative responses induced by CD40 ligand. Lyn-/- mice also develop splenomegaly, produce autoreactive Abs with age, and finally develop glomerulonephritis. Another abnormality observed in lyn-/- mice is that their disability to form germinal centers (GCs). It has been considered that GCs play an important role in affinity maturation and differentiation to B cell memory upon immunization with thymus-dependent Ag. Since Lyn kinase has been thought to be downstream of the signals from the B cell Ag receptor as well as CD40, we studied whether or not lyn-/- mice could exhibit normal Ag-specific class switching and affinity maturation following somatic hypermutation. The mice were immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (NP-CG). Production of NP-specific IgG1 Abs was slightly reduced but clearly detectable. The affinity of Abs produced was comparable to that in wild-type mice. Furthermore, somatic hypermutation occurred in the heavy-chain variable region at the same level as that in wild-type mice. Therefore, we conclude that isotype switching and affinity maturation occur normally in lyn-/- mice without the formation of GCs. The results lead to a speculation that Lyn may not play a role in induction of isotype switching or affinity maturation, despite being downstream of the signals from the B cell Ag receptor complex and CD40, and that GC architecture may not be absolutely essential for affinity maturation.

Amino Acid Sequence↗
PubMed1998

Actin organization associated with the expression of multidrug resistant phenotype in osteosarcoma cells and the effect of actin depolymerization on drug resistance.

We have previously reported that P-glycoprotein (Pgp)-overexpressing multidrug resistant (MDR) osteosarcoma cells were functionally more differentiated than their parent cells. The present study showed that in the parent cells, the actin filaments were sparsely distributed or were diffusely spread throughout the cytoplasm, whereas the MDR osteosarcoma cells exhibited a remarkable increase in well-organized actin stress fibers. Furthermore, dihydrocytochalasin B, a specific inhibitor of actin polymerization, dramatically disrupted this network of stress fibers, increased the intracellular accumulation of doxorubicin (DOX) and modified the resistance against DOX. These results indicate that the organization of actin filaments associated with cellular differentiation may be involved in the expression of Pgp function in the MDR osteosarcoma cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Chromosomal localization of a human deoxyribonuclease II gene (DNASE2) to 19p13.2-p13.1 using both the polymerase chain reaction and fluorescence in situ hybridization analysis.

Recently obtained information on the cDNA encoding human deoxyribonuclease II (DNase II) (T. Yasuda et al., 1998, J. Biol. Chem. 273, 2610-2616) has made it possible to demonstrate the precise position of the the human DNase II gene (DNASE2) on human chromosomes. Two different sets of oligonucleotide primers specific for human DNase II cDNA sequences were used to amplify unique DNA fragments in the human DNase II gene from a panel of human x rodent hybrid cell lines carrying different human chromosomes. Based on this analysis, DNASE2 was assigned to human chromosome 19. Furthermore, regional localization of the gene to 19p13.2-p13.1 was achieved by fluorescence in situ hybridization analysis using a full-length cDNA probe corresponding to the entire open reading frame.

Chromosome Mapping↗
PubMed1998

Layer-by-layer construction of enzyme multilayers on an electrode for the preparation of glucose and lactate sensors: elimination of ascorbate interference by means of an ascorbate oxidase multilayer.

A layer-by-layer structure of enzyme multilayers composed of glucose oxidase (GOx) or lactate oxidase (LOx) and ascorbate oxidase (AOx) was prepared on the surface of a platinum electrode. The amperometric response to glucose or lactate was studied in the presence of ascorbic acid as a possible interference. An alternating and repeated deposition of avidin and the biotin-labeled enzymes resulted in the layer-by-layer structure of GOx/AOx and LOx/AOx multilayers. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of monomolecular layers of the proteins. The GOx/AOx and LOx/AOx enzyme multilayers were useful to eliminate ascorbic acid interference in the glucose and lactate biosensors, because ascorbic acid can be converted to an electrochemically inert form, dehydroascorbic acid, before being oxidized directly on the Pt electrode. Thus, the GOx/AOx or LOx/AOx multilayer-modified biosensors can be used to determine the normal blood level of glucose (5 mM) and lactate (1 mM) in the presence of a physiological level of ascorbic acid (0.1 mM). The effects of the number of the AOx layers and geometry of the enzyme layers in the multilayer on the performance characteristics of the biosensors are discussed.

Ascorbate Oxidase↗
PubMed1998

Molecular cloning of the cDNA encoding human deoxyribonuclease II.

A rapid amplification of cDNA ends method, using degenerate oligonucleotides based upon the N-terminal amino acid sequence of human hepatic deoxyribonuclease II (DNase II), allowed a novel cDNA encoding DNase II to be constructed from thyroid gland RNA. The composite nucleotide sequence (1593 bases) included an open reading frame of 1080 bases, which encoded a single polypeptide of 360 amino acids (signal peptide, 16; propeptide, 91; mature protein, 253). Although the sequence of DNase II showed no significant homology to other mammalian proteins, its cDNA structural organization resembled those of the lysosomal cathepsin families. The two parts of the cDNA corresponding to the propeptide and the mature protein were expressed in Escherichia coli, and the recombinant polypeptides thus obtained were strongly stained with an anti-DNase II antibody on Western blotting. DNase II is ubiquitously expressed in human tissues, and the DNase II gene (DNASE2) was assigned to chromosome 19.

Amino Acid Sequence↗
PubMed1998

Aberrations of chromosomes 1 and 17 in six human osteosarcoma cell lines using double-target fluorescence in situ hybridization.

Analysis of six human osteosarcoma cell lines was performed by using double-target fluorescence in situ hybridization (FISH). FISH was applied to interphase nuclei, not to metaphase chromosomes. In this study, numerical aberrations of chromosomes 1 and 17 or structural chromosomal aberrations of chromosome arm 1p or 17p, in which it has been suggested that there are one or more tumor suppressor genes in various malignant tumors, were examined with this technique. All six of the human osteosarcoma cell lines studied had extra copies of chromosomes 1 and 17. A high frequency of deletions (> 60%) in chromosome 1 was found in two cell lines and deletions of chromosome 17 were found in one cell line.

Bone Neoplasms↗
PubMed1998

Structure and organization of the human deoxyribonuclease II (DNase II) gene.

The structure of the human gene for deoxyribonuclease II (DNase II; EC 3.1.22.1) was determined using several specific primers based on the human DNase II cDNA sequence [Yasuda et al. (1998). J. Biol. Chem. 273, 2610-2616] in a polymerase chain reaction-based strategy. The gene spanned about 6 kb and consisted of 6 exons. No canonical TATA or CAAT boxes could be identified within the 1341 nucleotides upstream of the putative transcription start site, although the 5'-flanking region contained a CpG island and several putative binding motifs for transcription factors Sp1 and ETF. These properties indicate that the DNase II gene is a housekeeping gene and this is compatible with its ubiquitous expression in human tissues. Three different cleavage/polyadenylation sites were identified in the 3'-flanking region, leading to the production of multiple DNase II mRNA species. However, a comparison of the entire translated sequences of the gene from a pair of subjects with homozygous DNase II phenotypes H and L revealed no differences in the nucleotide sequences.

Base Sequence↗
PubMed1998

Abrogation of autoimmune disease in Lyn-deficient mice by the mutation of the Btk gene.

Lyn and Btk play a critical role in B cell development and intracellular signaling. Lyn-deficient mice exhibit splenomegaly, elevated serum levels of IgM, production of autoantibody and glomerulonephritis with age. On the other hand, xid mice, which carry a point mutation in the btk gene, show a decrease in numbers of peripheral mature B cells, reduced serum levels of IgM and IgG3, disappearance of CD5+ B-1 cells, and low proliferative response to anti-IgM or LPS stimulation in vitro. In order to investigate the interaction between Lyn and Btk during B cell development, we established lyn-deficient xid mice. Lyn-deficient xid mice exhibited greatly reduced numbers of peripheral mature B cells, disappearance of CD5+ B-1 cells, markedly reduced serum levels of IgM and IgG3, low proliferative response to anti-IgM or lipopolysaccharide stimulation and no evidence for autoimmune disease. In addition, splenomegaly in lyn-deficient mice, which was mainly due to the accumulation of Mac-1+, cytoplasmic IgM+ lymphoblast-like cells, was also diminished in lyn-deficient xid mice. Thus, immunological abnormalities found in lyn-deficient mice were strongly affected by the absence of Btk. The present results suggest that the autoimmune symptoms in lyn-deficient mice may be caused by not only the abnormal response of B-2 cells but also that of B-1 cells, and that the interaction between Lyn and Btk is partly in tandem at the signaling pathway in B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Two novel screening methods for selecting monoclonal antibodies which specifically inhibit DNase I enzyme activity.

Two novel screening methods, single radial enzyme diffusion and the DNA-cast polyacrylamide gel electrophoresis, for selecting monoclonal antibodies which detect human deoxyribonuclease I (DNase I) enzyme activity are described. The former was adopted for initial screening to select potential objective antibodies from numerous hybridoma culture supernatants, because it was easy to perform and a powerful mass-screening tool. The latter was utilized for the subsequent precise selection of the antibodies in the supernatants selected after preliminary screening by the former, because it was clearly more accurate and sensitive, although the procedure was slightly more complicated. The consecutive use of these two methods resulted in the isolation of 25 anti-human DNase I antibodies, all of which specifically inhibited the activity of human DNase I.

Antibodies, Monoclonal↗
PubMed1998

Detection of the two short tandem repeat loci (HumTPO and HumLPL) in Japanese populations using discontinuous polyacrylamide gel electrophoresis.

Two short tandem repeat (STR) systems, HumTPO and HumLPL, were investigated in 218 unrelated Japanese living in Gunma and Fukui Prefectures by means of discontinuous, horizontal polyacrylamide gel electrophoresis and silver staining. Five and six alleles were identified at the HumTPO and HumLPL loci, respectively. No deviations from the Hardy-Weinberg equilibrium or differences in genotype frequency were found between the two Japanese populations in the two systems. Our detection methods offered several advantages including time-saving and safe electrophoretic procedures, and fast and easy silver-staining, in comparison with standard and conventional STR analysis using vertical polyacrylamide gel electrophoresis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis.

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA-cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.

Amino Acid Sequence↗
PubMed1997

Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.

In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

Animals↗
PubMed1997

Redox regulation of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) gene expression mediated by NF kappa B and AP-1 in human astrocytoma U373 cells.

LPS-induced expression of the IL-8 gene was markedly enhanced by H2O2 or by deprivation of the cellular antioxidant glutathione by L-buthionine-(S,R)-sulfoximine (BSO) in human astrocytoma U373 cells. In contrast, it was markedly suppressed by the reductant N-acetyl-L-cysteine (NAC) and other antioxidants. Transient expression analysis using the chloramphenicol acetyltransferase assay revealed that activation of the IL-8 promoter by LPS was stimulated by BSO and was suppressed by NAC; likewise LPS-induced activation of both NF kappa B and AP-1 was enhanced by BSO and inhibited by NAC. These results suggest that LPS-induced IL-8 gene expression is regulated by cellular redox via modulation of these transcription factors.

Acetylcysteine↗