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Biomedical subjects

A Shinohara

Publications and source records attributed to A Shinohara.

At least 55 records · Page 3Linked to original sources
PubMed1998

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Amino Acid Sequence↗
PubMed1998

Xrcc3 is required for assembly of Rad51 complexes in vivo.

Rad51 is a member of a family of eukaryotic proteins related to the bacterial recombinational repair protein RecA. Rad51 protein localizes to multiple subnuclear foci in Chinese hamster ovary cells. Subnuclear Rad51 foci are induced by ionizing radiation or the DNA cross-linking agent cisplatin. Formation of these foci is likely to reflect assembly of a multimeric form of Rad51 that promotes DNA repair. Formation of damage-induced Rad51 foci does not occur in the Chinese hamster ovary cell line irs1SF, which is sensitive to DNA damaging agents. The Rad51 focus formation defect of irs1SF cells is corrected by a construct that encodes the repair protein Xrcc3. Xrcc3 is a human homolog of Rad51 previously isolated by virtue of its ability to correct the radiation sensitivity of irs1SF cells. Changes in the steady state level of Rad51 protein do not account for the irs1SF defect nor do they account for the appearance of foci following DNA damage. These results suggest that Xrcc3 is required for the assembly or stabilization of a multimeric form of Rad51 during DNA repair. Cell lines defective in two different components of DNA protein kinase formed Rad51 foci in response to damage, indicating DNA protein kinase is not required for damaged-induced mobilization of Rad51.

Animals↗
PubMed1998

Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes.

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.

Adenosine Triphosphatases↗
PubMed1998

Isotope dilution analysis of Se in human blood serum by using high-power nitrogen microwave-induced plasma mass spectrometry coupled with a hydride generation technique.

To establish a method for sensitive, accurate, and precise determination of Se in real samples, isotope dilution analysis using high-power nitrogen microwave-induced plasma mass spectrometry (N2 MIP-IDMS) was conducted. In this study, freeze-dried human blood serum (Standard Reference Material, NIES No. 4) provided by NIES (National Institute for Environmental Studies) was used as a real sample. The measured isotopes of Se were 78Se and 80Se which are the major isotopes of Se. The appropriate amount of a Se spike solution was theoretically calculated by using an error multiplication factor (F) and was confirmed experimentally for the isotope dilution analysis. The mass discrimination effect was corrected for by using a standard Se solution for the measurement of Se isotope ratios in the spiked sample. However, the sensitivity for the detection of Se was not so good and the precision of the determination was not improved (2-3%) by N2 MIP-IDMS with use of the conventional nebulizer. Therefore, a hydride generation system was connected to N2 MIP-IDMS as a sample introduction system (HG-N2 MIP-IDMS) in order to establish a more sensitive detection and a more precise determination of Se. A detection limit (3 sigma) of 10 pg mL-1 could be achieved, and the RSD was less than 1% at the concentration level of 5.0-10.0 ng mL-1 by HG-N2 MIP-IDMS. The analytical results were found to be in a good agreement with those obtained by the standard addition method using conventional Ar ICPMS.

Freeze Drying↗
PubMed1998

Regulation of Rad51 function by c-Abl in response to DNA damage.

The Rad51 protein, a homolog of bacterial RecA, functions in DNA double-strand break repair and genetic recombination. Whereas Rad51 catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules, regulation of this function is unknown. The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. Here we demonstrate that c-Abl interacts constitutively with Rad51. We show that c-Abl phosphorylates Rad51 on Tyr-54 in vitro. The results also show that treatment of cells with ionizing radiation induces c-Abl-dependent phosphorylation of Rad51. Phosphorylation of Rad51 by c-Abl inhibits the binding of Rad51 to DNA and the function of Rad51 in ATP-dependent DNA strand exchange reactions. These findings represent the first demonstration that Rad51 is regulated by phosphorylation and support a functional role for c-Abl in regulating Rad51-dependent recombination in the response to DNA damage.

Cell Line↗
PubMed1998

Stimulation by Rad52 of yeast Rad51-mediated recombination.

In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homologue of the recA gene and the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologues of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Here we show that Rad52 protein stimulates Rad51 reactions and that binding to Rad51 is necessary for this stimulatory effect. We conclude that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments.

Adenosine Triphosphatases↗
PubMed1998

Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death.

Yeast rad51 mutants are viable, but extremely sensitive to gamma-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51- cells accumulated in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51- cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.

Animals↗
PubMed1998

Telomerase activity in the female reproductive tract and neoplasms.

OBJECTIVE: To study a possible utility of telomerase determination for cancer diagnosis. METHODS: In a total of 227 tissue samples comprising 114 normal tissues of the reproductive age, 10 fallopian tubes of the postmenopausal age, and 103 neoplastic tissues from female reproductive tracts, telomerase activity was determined. Using densitometrical analysis, telomerase activity was compared between carcinoma tissues and normal counterparts. RESULTS: A total of 97.3% (71/73) of cancer samples comprising ovarian carcinoma, endometrial carcinoma, and epidermoid carcinoma of the cervix and 89.5% (77/86) of the epithelia of the reproductive-aged uterus and fallopian tube showed telomerase activity. The epithelia of the fallopian tube of reproductive age showed significantly higher frequency of positivity (16/18) than the postmenopausal epithelia of the tube (3/10). No difference in telomerase activity was found between endometrial carcinomas and normal proliferative endometria. A significantly higher activity was found in ovarian epithelial carcinoma and epidermoid carcinoma of the cervix than in normal counterparts, although 92% (11/12) of the normal exocervix and 30% (3/10) of the normal ovary showed telomerase activity. CONCLUSIONS: Most epithelia of the female reproductive tract maintain telomerase activity during the reproductive age. Therefore, the detection of malignancies by telomerase determination may be feasible in ovarian carcinoma and epidermoid carcinoma of the cervix, but requires accurate quantification of telomerase activity.

Adult↗
PubMed1998

Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing.

BACKGROUND: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein. RESULTS: Purified Rad52 protein binds to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Electron microscope observations revealed that Rad52 molecules form multimeric rings. An increase in the intensity of fluorescence when Rad52 is bound to epsilonDNA showed an alteration of the structure of ssDNA. RPA was binding to Rad52 and enhanced the annealing of complementary ssDNA molecules. This enhancement was not observed in Escherichia coli SSB protein or T4 phage gp32 protein. CONCLUSION: Rad52 forms a ring-like structure and binds to ssDNA. Its structure and DNA binding properties are different from those of Rad51. The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs. We therefore propose that Rad52 mediates the RAD51-independent recombination through an ssDNA annealing, assisted by RPA.

DNA Replication↗
PubMed1998

Telomerase activity is found in the epithelial cells but not in the stromal cells in human endometrial cell culture.

Telomerase activity is associated with the proliferative activity of cells. In the endometrium, telomerase activity is higher in the proliferative phase than in the secretory phase of the menstrual cycle, suggesting that telomerase activity may occur primarily in the glandular epithelial cells. To test this, a dissociated cell culture of the endometrium was performed, and the telomerase activity in each cell fraction was analysed. Telomerase activity was found in all 10 endometrial tissues of the proliferative phase of the menstrual cycle. Both the fragments of epithelial glands and single cells, which were prepared by enzymatic dissociation, showed telomerase activity. In the 7 day cell culture, it was found in nine out of 10 epithelial cell enriched fractions, but in none of the stromal cell enriched fractions. Flow cytometric analysis showed that the epithelial enriched fraction was contaminated with a predominant number of stromal cells, while the stromal cell enriched fraction was comprised mostly of stromal cells with apparent proliferative activity. Our results suggest that telomerase activity of the endometrium occurs primarily in the epithelial cells in the endometrium and that the stromal cells do not express telomerase activity regardless of their potent proliferative activity.

Adult↗
PubMed1998

Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52.

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.

Animals↗
PubMed1998

Studies on lead-binding protein and interaction between lead and selenium in the human erythrocytes.

To clarify the function of Pb-binding protein and the interaction between lead and selenium in human erythrocytes, the following experiments were performed. (a) blood from a man who has not been exposed to lead occupationally, (b) blood from lead-exposed workers and (c) blood from control subjects working in the same plant were used. (a) was incubated with Pb and/or Se. Hemolysates of rinsed erythrocytes were applied onto a Bio-gel A 1.5 m column and eluted by 40 mM Tris-HCl buffer (pH 7.0). As the results, (1) Pb in erythrocytes was found in ALAD fraction. When Pb was added in vitro at 1.25 microM of final concentration, Pb content of ALAD fraction increased and the other Pb-containing fraction appeared at the position of a smaller molecular size than that of Hb fraction. When Pb dose added was increased to 2.5 or 10 microM, Pb contents in the newly appeared fraction was larger than that in ALAD fraction. The affinity of Pb with the protein in the new fraction was weak, and Pb was released from the protein by ultrafiltration. (2) There were main and sub-peaks containing Pb in erythrocytes from Pb-workers; the former was ALAD fraction and the others were smaller molecules than ALAD. The peak of Pb observed with the blood from a worker who has worked at the same plant for more than 20 years (chronic exposure) corresponded with the new peak which appeared in vitro Pb exposure. (3) As to the interaction between Pb and Se, there was no effect of Se added in vitro on the position of Pb-containing fraction and on Pb amounts in the chromatographic profile. When Se was incubated with blood, however, coexistence of Pb made Se concentration in erythrocytes high compared with the case of Se alone.

Carrier Proteins↗
PubMed1998

Effects of sex steroids on silver stained proteins of nucleolar organizer regions (Ag-NOR) in the rabbit uterus.

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17beta (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.

Animals↗
PubMed1998

Expression level of G1-cyclins and cell proliferation in human cultured leukemia/lymphoma cell lines.

We observed the expression of wild-type retinoblastoma protein (RB) in all 17 hematologic cultured cell lines tested. However, no p16INK4 expression was detected in any cell line among 16 leukemia/lymphoma cell lines, although an EBV-transformed cell line expressed p16INK4. The expression levels of cyclin D1 and CDK4 varied widely among the cell lines. The correlation coefficient (r2) between doubling time (DT) and cyclin D1 in the 14 cell lines that doubled within 47.2 h was 0.4856, while the r2 between DT and cyclin dependent kinase 4 (CDK4) and that between DT and RB among those cell lines were 0.3761 and 0.0874, respectively. The levels of protein expression in vincristine (VCR)-resistant cell lines was not different from those in corresponding wild-type cell lines. Thus, we concluded that the loss of p16INK4 protein and inactivation of RB protein could be an essential step for oncogenesis of leukemia/ lymphoma, and that cyclin D1 may possibly be a target protein to control cell growth of hematologic cell lines which lack the expression of p16INK4.

Cell Division↗
PubMed1998

Finding Genetic Network from Experiments by Weighted Network Model.

We study the problem of finding a genetic network from data obtained by multiple gene disruptions and overexpressions. We define a genetic network as a weighted graph, and analyze the computational complexity of the problem. We show that if there exists a weighted network which is consistent with given data, we can find it in polynomial time. Moreover, we also consider the optimization problem, where we try to find an optimally consistent weighted network with given data. We show that the problem is NP-hard. On the other hand, we give a polynomial-time approximation algorithm to solve it with approximation ratio 2. We report some simulation results on experiments.

Journal Article↗
PubMed1998

Multiple possible sites of BRCA2 interacting with DNA repair protein RAD51.

To investigate the biological consequences of aberrant BRCA2 protein during mammary carcinogenesis, we attempted to identify proteins that normally interact with BRCA2. By using a yeast two-hybrid system with a hybrid protein that contained residues 639-1,508 of BRCA2 protein fused to the GAL4 DNA-binding domain, we isolated five independent cDNA clones that encoded parts of RAD51 protein, a human homolog of bacterial RecA. In vitro experiments using anti-RAD51 antibody confirmed interaction of BRCA2 with RAD51. The RAD51-binding region of BRCA2 detected in the present study was distinct from the region reported recently. Further studies using smaller portions of BRCA2 defined at least two additional RAD51-binding domains, residues 982-1,066 and 1,139-1,266. Our results suggest that BRCA2 can interact with RAD51 through multiple sites of BRCA2 and that control of mitotic and meiotic recombination and/or of genomic integrity through binding to RAD51 may be a crucial mechanism by which BRCA2 suppresses abnormal proliferation of mammary cells.

Amino Acid Sequence↗
PubMed1998

[Application of hammerhead ribozyme targeting telomerase molecule for cancer therapy].

Because telomerase is expressed in most cancer cells, but not in most normal somatic cells, telomerase is a promising target molecule of human cancer therapy. To reduce telomerase activity in cancer cells, a hammerhead ribozyme is a choice. The RNA component of telomerase (hTR) is an essential molecule for telomerase, therefore hammerhead ribozyme against it can directly interfere with the telomerase activity. The expression level of hEST2, a catalytic subunit of telomerase correlates well to telomerase activity. Hammerhead ribozyme against hEST2 mRNA, therefore, will reduce telomerase activity by breaking its transcript down. Although hammerhead ribozyme can be designed to target any particular three-base sequences; it is still difficult to predict its efficiency in vivo. Application of hammerhead ribozyme targeting hTR is discussed.

Humans↗