[Roles of BRCA1 and BRCA2 in DNA repair and tumor suppression].
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Animals↗
Biomedical subjects
Y Miki
Publications and source records attributed to Y Miki.
New thiocholine ester substrates for the assay of human serum cholinesterase.
BACKGROUND: Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after being subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443-50 and Arch Biochem Biophys 1959;82:70-7). METHODS: We synthesized thiocholine ester iodides by the method of Renshow et al. (J Am Chem Soc 1938;60:1765-70). We examined solubility in H(2)O, substrate specificity serum for cholinesterase, (spontaneous) self-hydrolysis, storage stability, and reaction conditions for measurement of the activity of the enzyme. RESULTS: Isobutyryl and cyclohexane-carboxyl esters showed the best efficiency for the specific and stable assay of human serum cholinesterase. Aqueous solubility of each was >10 mmol/L, and the reactivity with acetylcholinesterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, respectively, spontaneous hydrolysis in the aqueous phase was approximately 1/25 and approximately 1/175 slower than the enzymatic hydrolysis, and assays with these substrates were linear to 1800 and 3000 U/L, respectively. The K(m) values of these acylthiocholines with human cholinesterase were almost equivalent (6.9 x 10(-3) mmol/L). The substrates were stable in aqueous solution and in the solid state as the iodides for at least 5 years at 5 degrees C. CONCLUSIONS: The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase.
Cholinesterases↗
Lipase-catalyzed transesterification of 2-hydroxy-2-(pentafluorophenyl)acetonitrile leading to (1R,2R)- and (1S,2S)-bis(pentafluorophenyl)ethane-1,2-diol
Optically pure (1R,2R)- and (1S,2S)-1,2-bis(pentafluorophenyl)ethane-1,2-diol (1) were synthesized from key intermediates (R)- and (S)-2-hydroxy-2-(pentafluorophenyl)acetonitrile (2), both of which were prepared by the lipase LIP-catalyzed transesterification (E = 465). The absolute configuration of (S)-2 was determined by X-ray structural analysis after transformation into (S)-alpha-cyano-2,3,4,5,6-pentafluorobenzyl (S)-6-methoxy-alpha-methyl-2-naphthaleneacetate (S,S)-9. In addition, the crystal structure of (S,S)-9 has an interesting well-ordered packing pattern which shows face-to-face stacking interactions and end-to-end parallel contacts between the pentafluorophenyl and 6-methoxynaphthyl groups of the adjacent molecules.
Journal Article↗
Nuclear localization signals of the BRCA2 protein.
BRCA2 is a tumor suppressor gene whose germline mutations increase the lifetime risk of breast cancer. BRCA2 encodes a large nuclear protein involved in DNA repair, but the location of its functional domain has been unclear. Here, we report nuclear localization signals (NLSs) of the BRCA2 protein. By expressing various portions of the BRCA2 protein tagged with enhanced green fluorescent protein in HeLa cells, we show that the C-terminal domain is necessary for nuclear localization. Two regions in the C-terminal domain were identified with functional NLSs by site-directed mutagenesis analyses. The NLSs locate between the germline mutation found in the most downstream position and the polymorphic stop codon, suggesting that defects in the proper nuclear transport of the BRCA2 protein are causative of carcinogenesis. Our data thus provide a possible explanation for the high frequency of frame-shift and nonsense mutations in BRCA2 of hereditary breast cancer patients.
BRCA2 Protein↗
Potential role of BRCA2 in a mitotic checkpoint after phosphorylation by hBUBR1.
BRCA2, a gene responsible for inherited susceptibility to breast cancer in a number of families, is thought to be critical for replication and repair of DNA during S-phase. To elucidate the physiological functions of BRCA2, we used a yeast two-hybrid system to screen for proteins that could associate with BRCA2. Here we report interaction of BRCA2 with a mitotic checkpoint protein, hBUBR1, and its phosphorylation by hBUBR1 in vitro. After cotransfection of BRCA2 and hBUBR1 expression vectors into the COS7 cell line, both proteins were stained together in the nuclei of cells whose spindle fibers were disrupted, but not in undamaged cells. Treatment with vincristine, which disrupts microtubules, significantly increased expression of both hBUBR1 and BRCA2 in the MCF7 cells. The results suggest that BRCA2 protein might be involved in a mitotic checkpoint in vivo after it has been phosphorylated by hBUBR1.
Animals↗
Parathyroid hormone-activated volume-sensitive calcium influx pathways in mechanically loaded osteocytes.
This paper documents for the first time a volume-sensitive Ca(2+) influx pathway in osteocytes, which transmits loading-induced signals into bone formation. Stretch loading by swelling rat and chicken osteocytes in hypo-osmotic solution induced a rapid and progressive increase of cytosolic calcium concentration, [Ca(2+)](i). The influx of extracellular Ca(2+) explains the increased [Ca(2+)](i) that paralleled the increase in the mean cell volume. Gadolinium chloride (Gd(3+)), an inhibitor of stretch- activated cation channels, blocked the [Ca(2+)](i) increase caused by hypotonic solutions. Also, the expression of alpha1C subunit of voltage-operated L-type Ca(2+) channels (alpha1C) is required for the hypotonicity-induced [Ca(2+)](i) increase judging from the effect of alpha1C antisense oligodeoxynucleotides. Parathyroid hormone (PTH) specifically potentiated the hypotonicity-induced [Ca(2+)](i) increase in a dose-dependent manner through the activation of adenyl cyclase. The increases induced by both PTH and hypotonicity were observed primarily in the processes of the osteocytes. In cyclically stretched osteocytes on flexible-bottomed plates, PTH also synergistically elevated the insulin-like growth factor-1 mRNA level. Furthermore, Gd(3+) and alpha1C antisense significantly inhibited the stretch-induced insulin-like growth factor-1 mRNA elevation. The volume-sensitive calcium influx pathways of osteocytes represent a mechanism by which PTH potentiates mechanical responsiveness, an important aspect of bone formation.
Animals↗
Comparison of short inversion time inversion recovery (STIR) and fat-saturated (chemsat) techniques for background fat intensity suppression in cervical and thoracic MR imaging.
The purpose of this study was to compare short inversion time inversion recovery (STIR) fast spin-echo (FSE), and fat-saturated T2-weighted FSE sequences in terms of uniformity of fat suppression and lesion conspicuity for magnetic resonance (MR) imaging of the neck and thorax. STIR FSE and fat-saturated T2-weighted FSE images were scored for uniformity of fat suppression (n = 40) and lesion conspicuity (n = 35). Five-point rank score analyses were utilized by three experienced radiologists. The mean scores of STIR and fat-saturated FSE techniques for uniformity of fat suppression were 4.3 and 2.3, respectively (P < 0.0001). The mean scores of STIR and fat-saturated FSE techniques for lesion conspicuity were 4.2 and 3.5, respectively (P < 0.0001). Insufficient fat suppression was prominent in the mandible, supraclavicular region, anterior mediastinum, epipericardial fat, and subdiaphragmatic fat. In addition, fat-saturated T2-weighted FSE showed inadvertent water suppression in 25%. The STIR FSE technique was superior to the fat-saturated FSE technique for cervical and thoracic MR imaging.
Adolescent↗
Identification of Rad51 alteration in patients with bilateral breast cancer.
The human Rad51 gene, HsRAD51, is a homolog of RecA of Escherichia coli and functions in recombination and DNA repair. BRCA1 and BRCA2 proteins form a complex with Rad51, and these genes are thought to participate in a common DNA damage response pathway associated with the activation of homologous recombination and double-strand break repair. Additionally, we have shown that the pattern of northern blot analysis of the RadS gene is closely similar to those of the BRCA1 and BRCA2 genes. It is therefore possible that alterations of the Rad51 gene may be involved in the development of hereditary breast cancer. To investigate this possibility, we screened Japanese patients with hereditary breast cancer for Rad51 mutations and found a single alteration in exon 6. This was determined to be present in the germline in two patients with bilateral breast cancer, one with synchronous bilateral breast cancer and the other with synchronous bilateral multiple breast cancer. In both patients, blood DNAs showed a G-to-A transition in the second nucleotide of codon 150, which results in the substitution of glutamine for arginine. As this alteration was not present in any patients with breast or colon cancer examined, we assume that this missense alteration is likely to be a disease-causing mutation.
Blotting, Northern↗
Novel missense mutations in the glutamate dehydrogenase gene in the congenital hyperinsulinism-hyperammonemia syndrome.
OBJECTIVES: The objectives of this study were to clarify the involvement of the glutamate dehydrogenase gene in congenital hyperinsulinemia-hyperammonemia syndrome (CHHS) and the relationships between the mutation of the gene and clinical severity. STUDY DESIGN: Five unrelated Japanese patients (3 girls and 2 boys) with CHHS were investigated. All patients had convulsions or loss of consciousness resulting from hypoglycemia at less than 1 year of age. We examined mutations of the glutamate dehydrogenase gene using genomic or reverse-transcriptase polymerase chain reactions, followed by direct sequencing. RESULTS: We identified heterozygous missense mutations in all patients. Three patients had a previously identified mutation (C-->T at nt 1506) at codon 445 in the allosteric domain. Two novel missense mutations were identified in the other patients. These mutations included a change of A-->C at nt 1059 and a change of G-->A at nt 966, within the catalytic domain of the glutamate dehydrogenase gene. The locus of the mutations was not associated with the severity of hypoglycemia. CONCLUSIONS: Our results suggest that structural aberrations of not only the allosteric domain but also the catalytic domain of the glutamate dehydrogenase protein, caused by missense mutations, can result in the development of CHHS.
Adenine↗
Marriage rate and number of children among young adults with insulin-dependent diabetes mellitus in Japan.
The main purpose of our study was to identify the social circumstances and lifestyle of IDDM patients in Japan. The present study focused on the marriage status of both men and women with IDDM as well as the number of children of women with IDDM. A questionnaire was sent to hospitals across the country. Doctors handed it or mailed it to IDDM patients aged 18 years or older. Unsigned answer sheets were returned directly by the patients. Data on the marriage rate and number of children were obtained, and possible factors affecting these indices were assessed. One thousand and thirteen patients (354 men and 659 women) answered the questionnaire. Both men and women with IDDM were less likely to be married in comparison with age-matched Japanese. The number of children of married IDDM women in various age groups was also lower in comparison with the general Japanese female population. Several factors other than diabetes complications including job discrimination, high medical costs, and psychological pressures, were thought to be responsible for these results.
Adult↗
A novel mutation in the glucose-6-phosphatase gene in Korean twins with glycogen storage disease type Ia.
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Child, Preschool↗
Protective effect of human urinary thrombomodulin on ischemia- reperfusion injury in the canine liver.
This study was performed to determine whether human urinary soluble thrombomodulin plays a role in liver ischemia-reperfusion injury. Liver ischemia was induced in two groups of dogs. Group 1 was exposed to 60 min ischemia, and group 2 was exposed to 60 min ischemia after preischemic administration of human urinary soluble thrombomodulin. In group 1, the thrombin-antithrombin complex and hyaluronic acid were significantly elevated after ischemia, compared with the preischemic values. While liver issue blood flow and the plasmin-alpha(2)-plasmin inhibitor complex significantly decreased, AST, ALT and m-AST dramatically increased after reperfusion. In group 2, the increase in the thrombin-antithrombin complex and hyaluronic acid was significantly suppressed, and AST, ALT and liver tissue blood flow significantly improved, compared with group 1. Histologically, in group 2, the hepatic tissue structure, including endothelial cells, was relatively intact. These findings suggest that administration of thrombomodulin inhibits endothelial cell injury and coagulopathy and offers protection from liver ischemia-reperfusion injury.
Animals↗
Biological activity of alkyl 2-(acylthio)benzoates.
Of a series of synthetic alkyl 2-(acylthio)benzoate (1-20), all the derivatives except for n-butyl 2-butyrylthiobenzoate (18) and n-butyl 2-n-valerylthiobenzoate (20) showed clear phytogrowth-inhibitory activity. All the compounds tested except for methyl 2-butyrylthiobenzoate (3) exhibited cytotoxic activity on mouse splenic T cells. Strong phytogrowth-inhibition and cytotoxic activity were found with 1, 6, 11 and 16 with an acetylthio group at C-2, suggesting that the acetyl group seems to play an important role in both activities of alkyl 2-(acylthio)benzoates. Among them, methyl 2-acetylthiobenzoate (1) was the strongest inhibitor. On the other hand, potent inhibition of prolyl endopeptidase was exhibited by 2, 7, 12 and 17 with a propionylthio group at C-2. These findings imply that a propionyl group might be useful for increasing the inhibitory activity against on prolyl endopeptidase.
Animals↗
Arterial dissections of penetrating cerebral arteries causing hypertension-induced cerebral hemorrhage.
OBJECT: For the past 130 years, it has been believed that hypertension-induced cerebral hemorrhages are the result of ruptures of microaneurysms or ruptures of arteries that have degenerative changes. The majority of previous investigations have focused on autopsied brain. In this study, the authors attempted to verify the cause of hypertension-induced cerebral hemorrhage by using surgical specimens of the penetrating arteries responsible for the hemorrhages. METHODS: Between 1997 and 1999, the authors performed pathological studies in surgical specimens of lenticulostriate arteries that had been confirmed during microsurgery to be the cause of hypertension-induced hemorrhage of the putamen. Nineteen lenticulostriate arteries were collected from 12 patients. Fifteen of these arteries were verified as the pathological causes of hemorrhage. They included six arterial dissections, six arterial ruptures with substantial degenerative changes, and three arterial ruptures with few degenerative changes. The pathological findings in the lenticulostriate artery dissections were similar to those of typical arterial dissections in major cerebral arteries. CONCLUSIONS: To the best of the authors' knowledge, arterial dissections of lenticulostriate arteries have not been identified as a cause of hypertension-induced cerebral hemorrhages. When penetrating arteries are included as causative vessels, cerebral arterial dissections may be much more common than previously thought.
Aged↗
[Genetic epidemiology of hereditary tumor].
The last decade has seen many great successes in isolating genes involved in inherited disease, a process that is now scaled up by Human Genome Project. The identification of genes responsible for hereditary tumors has made it possible to do the genetic diagnosis of hereditary tumors. However, the benefits and limits of genetic testing for cancer susceptibility are different in each hereditary tumor. It is important to continue the analysis of significance(frequency and penetrance) of mutations of cancer predisposition genes and to make clear the genotype-phenotype and other correlation. Hereditary breast cancer and hereditary non-polyposis colorectal cancer are reviewed and the significance of genetic epidemiology of hereditary tumor is discussed.
Breast Neoplasms↗
[Genetic diagnosis of hereditary breast cancer].
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BRCA1 Protein↗
DNA replication errors produced by the replicative apparatus of Escherichia coli.
It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high fidelity and a high background level of pre-existing mutations in the single-stranded template DNA used. Using the oriC plasmid DNA replication in vitro system and the rpsL forward mutation assay, we examined the fidelity of DNA replication catalyzed by the replicative apparatus of Escherichia coli. Upon DNA synthesis by the fully reconstituted system, the frequency of rpsL-mutations in the product DNA was increased to 1.9x10(-4), 50-fold higher than the background level of the template DNA. Among the mutations generated in vitro, single-base frameshifts predominated and occurred with a pattern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the same nucleotide. Large deletions and other structural alterations of DNA appeared to be induced also during the action of the replicative apparatus.
Base Pairing↗
Clinicopathologic analysis of BRCA1- or BRCA2-associated hereditary breast carcinoma in Japanese women.
BACKGROUND: The purpose of this investigation was to elucidate the clinicopathologic characteristics of BRCA1- and BRCA2-associated hereditary breast carcinomas (HBCs) in Japanese women. METHODS: Various clinicopathologic characteristics of HBCs arising in patients with BRCA1 or BRCA2 germline mutations were compared with those of the control group (sporadic breast carcinomas). RESULTS: The mean age at the time of diagnosis of BRCA1-associated HBCs and that of BRCA2-associated HBCs (44 years for both) were significantly younger than that of the control group (54 years) and the incidence of bilateral tumors was significantly higher in the BRCA1-associated HBCs (32%) and BRCA2-associated HBCs (29%) than in the control group (6%). BRCA1-associated HBCs showed a tendency (P = 0.06) toward an increase in solid-tubular type tumors and a significant increase in histologic grade 3 tumors (P < 0.01) and lymphatic invasion positive tumors (P < 0.05) compared with the control group. BRCA1-associated HBCs were significantly more estrogen receptor negative (P < 0.01), c-erb B-2 negative (P < 0.05), and p53 positive (P < 0.01), and they also showed a significant increase in MIB-1 staining grades (P < 0.01) as well as microvessel counts (P < 0.05) compared with the control group. However, there was no significant difference in these parameters between the BRCA2-associated HBCs and the control group. CONCLUSIONS: BRCA1-associated HBCs in Japanese women have biologically aggressive phenotypes. However, BRCA2-associated HBCs are without distinctive clinicopathologic features compared with sporadic breast carcinomas.
Adult↗