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H Takeshita

Publications and source records attributed to H Takeshita.

At least 19 recordsLinked to original sources
PubMed1997

HAX-1, a novel intracellular protein, localized on mitochondria, directly associates with HS1, a substrate of Src family tyrosine kinases.

Cross-linking of the Ag receptors on lymphocytes initiates activation of the receptor-coupled tyrosine kinases. HS1 is one of the substrates of these kinases and has been shown to transduce the signals for both clonal expansion and deletion in lymphoid cells. To gain further insight into the mechanism of action of HS1, we have tried to identify a protein that interacts with HS1 by yeast two-hybrid screening. The isolated cDNA, designated HAX-1, encodes a novel 35-kDa protein. The HAX-1 gene is expressed ubiquitously among tissues, and its protein is localized mainly in mitochondria, but also in endoplasmic reticulum and nuclear envelope in the cell. HS1/HAX-1 association is confirmed by coimmunoprecipitation of these proteins in the lysates of B lymphoma cells and COS-7 cells transfected with the corresponding cDNA expression vectors. Colocalization of these proteins in the cell is evident under confocal laser scanning microscope. Deletion mutant analysis of these proteins reveals that the association is mediated by the amino terminal region of HS1 and the carboxyl-terminal half of HAX-1. The potential role of the HAX-1/HS1 complex is discussed.

Adaptor Proteins, Signal Transducing↗
PubMed1997

[Detection of Epstein-Barr virus genomes in nasopharyngeal carcinoma].

In situ hybridization using EBV encoded small RNAs (EBERs) probe and polymerase chain reaction (PCR) were performed to detect the presence of Epstein-Barr virus (EBV) genomes in 56 primary nasopharyngeal carcinomas. EBERs was detected in 46 cases(82%); 30 of 32 cases (94%) in differentiated non-keratinizing carcinoma (NKC, WHO-2) and 16 of 17 cases (94%) in undifferentiated nasopharyngeal carcinoma (UNPC, WHO-3). But none of 5 cases of squamous cell carcinoma (SCC, WHO-1) nor 2 cases of adenocarcinoma showed EBERs. EBV-DNA was also detected in 22 of 24 cases of NKC and in all 13 cases of UNPC by PCR but not in 3 cases of SCC or 2 cases of adenocarcinoma. This findings shows that the examination of EBERs and EBV-DNA is very useful, sensitive method in the detection of EBV.

Adenocarcinoma↗
PubMed1996

pH gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: detection and inhibition of enzyme activity in the gel.

A simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8-10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory-type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as substrate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory-type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental-type RNase inhibitor, can be performed.

Alkalies↗
PubMed1996

Alpha-2-HS-glycoprotein (AHSG) polymorphism in semen and saliva.

Polymorphism of alpha-2-HS-glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin-layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P-40 into the IEF gel (pH 4.2-4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2-5 microL semen and 50-100 microL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.

Blood Proteins↗
PubMed1996

Successful deoxyribonuclease I (DNase I) phenotyping from a small piece of used sock.

The forensic application of the DNase I polymorphism for individualization of used socks is described. We devised a technique that combines a simple extraction method, including a partial purification step, using a Phenyl-Sepharose CL-4B column, isoelectric focusing and activity staining, for DNase I typing from socks. As a result, DNase I types were determined from socks kept at room temperature for a month or more in all of the samples tested. The DNase I types determined from socks exactly matched those determined from corresponding sweat and urine samples. By contrast, AB0 groupings could not be determined from these samples, using the conventional serological method. Therefore, we have shown that the DNase I polymorphism is the first genetic marker found to be well suited for individualization of used socks.

ABO Blood-Group System↗
PubMed1996

Purification of mammalian ribonuclease using immobilized human ribonuclease inhibitor.

Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNase inhibitor was conjugated to CNBr-activated Sepharose in the presence of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was eluted with 3 M NaCl at pH 5.0. Two 25-kDa and 18-kDa RNases, which were obtained from human liver using a cellulose phosphate column, were bound to the immobilized RNase inhibitor and recovered in a pure and active form after treatment of the resin with p-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with different sugar contents.

Amidohydrolases↗
PubMed1996

Identification of the nucleotide substitution that generates the fourth polymorphic site in human deoxyribonuclease I (DNase I).

In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1-2, and 2.

Base Sequence↗
PubMed1996

Experimental models for the study of drug resistance in osteosarcoma: P-glycoprotein-positive, murine osteosarcoma cell lines.

P-glycoprotein is an adenosine triphosphate-dependent drug-efflux pump that extrudes drugs from cells and causes drug-resistance. P-glycoprotein is believed to mediate drug-resistance in a wide variety of tumors. In this study, we developed two P-glycoprotein-positive, murine osteosarcoma cell lines that were resistant to Adriamycin (doxorubicin) (MOS/ADR1 and MOS/ADR2). We created the cell lines by short-term pulse exposures of the parent cell line to Adriamycin followed by single-cell cloning. The MOS/ADR1 and MOS/ADR2 cells were sevenfold and eighteenfold more resistant to Adriamycin than the cells from the parent line. Expression of P-glycoprotein, as examined with an immunofluorescence method, was detected in most of the MOS/ADR1 and MOS/ADR2 cells but not in the parent cells. After the cells had been incubated with Adriamycin for one hour, there was less accumulation of the drug in the resistant cell lines than in the parent cell line. The reduced accumulation was due to the increased efflux of Adriamycin. The Adriamycin-resistant cell lines demonstrated greater alkaline phosphatase activity than the parent cell line and produced more differentiated osteoblastic sarcomas in mice. Dose survival studies with use of a tetrazolium colorimetric assay showed that the MOS/ADR1 cells were cross-resistant to vincristine, vinblastine, etoposide, bleomycin, mitomycin C, and actinomycin D but not to dacarbazine, cisplatin, carboplatin, cytosine arabinoside, carmustine, cyclophosphamide, ifosfamide, methotrexate, and 5-fluorouracil. Although the MOS/ADR2 cells exhibited a similar spectrum of cross-resistance, they were more resistant than the MOS/ADR1 cells. We also tested the effect of three different resistance-modifying agents on the reversal of resistance to Adriamycin. We found that verapamil and trifluoperazine substantially reversed resistance to Adriamycin in the P-glycoprotein positive cell lines, whereas cyclosporin A was relatively ineffective. Because these cell lines retain the histological and biochemical features of bone-producing sarcomas and display the multidrug-resistant phenotype, they may be useful models for additional investigations of drug resistance in osteosarcoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

A new individualization marker of sweat: deoxyribonuclease I (DNase I) polymorphism.

We confirmed for the first time, both biochemically and immunologically, the existence of deoxyribonuclease I (DNase I) in human liquid sweat. Isoelectric focusing of sweat samples on polyacrylamide gels (pH 3.5 to 5), followed by dried agarose film overlay detection, was used to determine the phenotypes of sweat DNase I. Because this detection method not only had high sensitivity, but also high band resolution, it was possible to determine DNase I types from sweat samples of 50 to 100 microL. Pretreatment of sweat samples with sialidase was essential for typing to enhance markedly the sensitivity accompanied by simplification of the isozyme pattern. The DNase I types in all sweat samples were consistently related to the types found in corresponding blood, urine, and semen samples. DNase I typing could, therefore, provide a novel discriminant characteristic in the forensic examination of sweat.

Biomarkers↗
PubMed1996

[Interaction between Epstein-Barr virus (EBV) gene expression and antibodies to EBV in nasopharyngeal carcinomas].

In situ hybridization using EBERs and BHLF oligonucleotide probes and immunohistochemistry using monoclonal antibodies against LMP1, EBNA2, BZLF1 protein, p53 protein and bcl-2 protein were performed on 56 primary nasopharyngeal carcinomas. EBERs was detected in 46 cases (82%), and LMP in 17 cases (30%), but EBNA2 was not detected. While 30 of 32 cases (94%) in differentiated non-keratinizing carcinoma (NKC) and 16 of 17 cases (94%) in undifferentiated carcinoma (UNPC) showed EBERs, neither 5 cases of squamous cell carcinoma (SCC) nor 2 cases of adenocarcinoma showed EBERs. This finding confirms latent infection of EBV, especially phenotypical latency II, in NKC and UNPC but not in SCC. Bcl-2 protein was positive in 50 cases (89%), but its expression did not depend on expression of LMP1, which did not demonstrate induction of bcl-2 by LMP1 as seen in vitro. Cytoplasmic BZLF1 expression was detected in 18 cases (32%) whereas BHLF was positive only in 6 cases (11%). This suggests dysfunction of BZLF1, which disrupts viral latency despite its expression. p53 protein was positive in 31 cases (55%), and there was a distinct correlation between expression of BZLF1 and p53 protein (p < 0.001). This suggests that the interaction between BZLF1 protein and p53 protein, which inactivate each other, is one of the tumorigenic factors in NPC.

Adolescent↗
PubMed1995

Activity staining of mammalian ribonuclease inhibitors after electrophoresis in sealed vertical slab polyacrylamide gels.

A method for detecting the activity of ribonuclease inhibitors (RIs) after nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed using vertical slab polyacrylamide gels in the presence of dithiothreitol and in a sealed system. In each system, purified 50 kDa human RI was visualized as a single band by immunoblotting with a specific antibody. RI activity in the polyacrylamide gel slab was detected by sandwiching the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose film sheet containing substrate yeast RNA plus ethidium bromide and incubating at 37 degrees C. The ribonuclease penetrated the polyacrylamide gel and digested the substrate RNA in the agarose film. However, if an RI was present in the gel, the enzyme was inactivated by complex formation. Fluorescent bands corresponding to RIs were observed on a dark background under ultraviolet light. This activity staining had a high sensitivity allowing detection of less than 0.6 units of mammalian RIs (corresponding to 5 ng of purified human RI) and produced a sharp band which compared favorably with that obtained on immunoblotting. These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples.

Animals↗
PubMed1995

Cell proliferation and differentiation of cultured chondrocytes isolated from growth plate cartilage of rat rib.

The present study was undertaken to investigate the relationship among cell morphology, proliferation, and maturation of chondrocytes in primary cultures. Chondrocytes were isolated from the growth cartilages of the rat ribs and cultured for 6 days. In situ DNA cytofluorometry using an inverted epi-illumination cytofluorometer (Nikon P1-I) and 3H-thymidine autoradiography were carried out for the correlated analysis of cell morphology and proliferation. Cytoskeletal staining with fluorescent phalloidin and 35S-sulphate autoradiography were also performed. In addition, in situ hybridization to c-myc mRNA was carried out using DNA probe. According to the results obtained, the cultured chondrocytes were composed of mixed populations of large, polygonal cells and of small, round cells. The round cells showed a significantly higher 35S uptake than the polygonal cells. The cytoskeletal staining clearly revealed stress fibers in the cytoplasm of the polygonal cells, whereas only a fine filamentous structure was shown in the cytoplasm of the round cells. In situ DNA cytofluorometry clearly demonstrated that cell proliferative activity was high in the polygonal cells and low in the round cells. In addition, 3H-thymidine autoradiography with cumulative labeling method revealed that the polygonal cells were changing into the small, round cells. C-myc mRNA signals were detected in the cytoplasm of over a half of the round cells, whereas no evidence of c-myc expression were found in the polygonal cells. From these results, it appears that as the shape of the cultured chondrocytes shifts from polygonal to round, the cell proliferative activity decreases in association with cell differentiation. It was also suggested that c-myc mRNA is amplified in the well differentiated round chondrocytes, and not in the proliferative polygonal cells.

Animals↗
PubMed1995

The molecular basis for genetic polymorphism of human deoxyribonuclease I: identification of the nucleotide substitution that generates the fourth allele.

In addition to the three alleles commonly responsible for the protein polymorphism of human deoxyribonuclease I, a mutation encoded by a fourth allele, DNASE1*4, was detected by isoelectric focusing. All 8 exons covering the entire open reading frame of the human DNase I gene were amplified by the polymerase chain reaction and subjected to direct sequencing. Only one nucleotide substitution, a C-to-G transition (CAG-->GAG), in the codon for amino acid 9 of the mature enzyme was found. This substitution resulted in the replacement of Gln with Glu (Q9E).

Alleles↗
PubMed1995

Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction.

We recently completely elucidated the molecular basis of genetic polymorphism in human deoxyribonuclease I and found it to be controlled by four codominant alleles, DNASE1*1, *2, *3 and *4. In this paper we describe a novel DNase I-genotyping system that could be used directly on DNA samples using the polymerase chain reaction (PCR) based on the three nucleotide substitutions underlying the protein polymorphism. The system consists of three independent reactions. Since the substitutions neither suppress nor create any known enzyme recognition site in the DNase I gene, two separate mismatched PCR followed by XhoI digestion methods were introduced to discriminate between the DNASE1*1 (or *3) and the DNASE1*2 (or *4) alleles, and to detect the DNASE1*4 allele. An amplification refractory mutation system was employed to detect DNASE1*3. A 100% correlation was found between the results of this genotyping method and those obtained by phenotyping using conventional isoelectric focusing. The high sensitivity and specificity of this genotyping method allows us to survey DNase I-polymorphism in small DNA samples.

Alleles↗
PubMed1995

Ribonuclease inhibitors in human blood: comparative studies on the inhibitors detected in erythrocytes, platelets, mononuclear leukocytes and granulocytes.

The erythrocyte ribonuclease inhibitor (RI) and platelet RI were separately purified to homogeneity and showed similar properties to those of human brain and placental RIs. The same type of RI seemed to be present in mononuclear leukocytes and granulocytes. The RI contents of these cells were detectable by immunoblot analysis using anti-human placental RI antibody. Neither RI activity nor the molecule cross-reactive with the anti-human placental RI antibody was detectable in any plasma sample. It is intriguing to detect active RI in mature erythrocytes, where no nucleus exists and RNA metabolism is unlikely to occur.

Amino Acid Sequence↗
PubMed1995

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies.

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.

Amino Acid Sequence↗
PubMed1995

Molecular analysis of the third allele of human deoxyribonuclease I polymorphism.

In addition to common phenotypes 1, 1-2 and 2 of human deoxyribonuclease I (DNase I), phenotypes 1-3 and 2-3, encoded by a third allele DNASE1*3, have been found by means of isoelectric focusing. The main objective of this study was to identify the mutation site(s) underlying phenotype 3. All eight exons covering the entire open reading frame of the human DNase I structural gene were amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequencing. When the entire 780-bp coding region and exon/intron junctions of the DNase I gene of two individuals with phenotypes 1-3 and 2-3 were sequenced, only one nucleotide substitution, a C-G transition (CCC-->GCC), in the codon for amino acid 132 of the mature enzyme located in exon VI was found that resulted in the replacement of proline with alanine (P132A). The mutation was confirmed by allele-specific amplification of genomic DNA. The replacement of the amino acid residue may reduce the hydrophobicity of the enzyme and thus increase the pI value of the type-3 isozyme compared with that of type 1, as increasing the hydrophobicity of a protein is known to decrease its pI value. The specific PCR-amplifications of exons and alleles developed in this study may provide a new tool suitable for rapid screening of DNase I variants.

Alleles↗