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A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor. 相似文献
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In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1. 相似文献
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p53 mutations, a tumor suppressor gene located on chromosome 17p, are the most common genetic alterations found in human cancers. Although the p53 expression or mutation has been investigated in a variety of cancers there have been very few studies in extrahepatic bile duct cancers. In this study, we investigated the immunohistochemical expression of p53 in formalin fixed paraffin embedded archival specimens of 36 extrahepatic bile duct cancers in which p53 expression was found in eighteen (50%) cases. There was no significant difference in age, gender, size of tumor, histologic grade, extent of tumor involvement, lymph node metastasis and tumor resectability according to p53 immunoreactivity. Comparison of survival duration according to p53 expression showed no significant difference. In conclusion, we reported 50 percent of p53 expression in extrahepatic bile duct cancers by immunohistochemical staining and we found no prognostic significance of p53 expression in dinicopathologic parameters. 相似文献
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FY Ghauri JK Nicholson BC Sweatman J Wood CR Beddell JC Lindon NJ Cairns 《Canadian Metallurgical Quarterly》1993,6(2):163-167
Mutations of ras oncogenes in 37 human stomach cancers and 13 adenomas were investigated with regard to the histological phenotypes using polymerase chain reaction (PCR), allele-specific oligonucleotide hybridization and/or direct sequencing of the PCR products. The ras mutation was found only in one case (2.7%), the histology of which was poorly differentiated adenocarcinoma. We found no mutation in stomach adenomas. The mutation consisted of a guanine-to-adenine transition in the first base of codon 13 of c-Ki-ras which replaced wild-type glycine with serine, indicating that a putative glycine-to-aspartic acid change is not necessarily the critical event for c-Ki-ras gene activation in codon 13. These results further confirm the infrequency of ras mutation in stomach tumors and also suggest that ras mutations are not specific to the differentiated type of stomach cancer. 相似文献
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The number of patients requiring revision total hip arthroplasty continues to increase each year. Accurate preoperative planning is a key factor in obtaining a good result. Radiographs provide little information concerning the actual extent of the acetabular defects. Computed tomography-generated models of the acetabulum can provide the surgeon with accurate information concerning the size and location of the defects. Evaluation of radiographs and models in 24 cases showed that radiographs alone failed to detect all 13 anterior wall defects (P < .001), 8 of 18 posterior wall defects (44.4%, P < .001), and 8 of 19 segmental central defects (42%, P < .001), all of which were easily identified with the models. This study showed that preoperative planning based on the foam models accurately predicted the actual implant used in 22 of 24 cases (92%). 相似文献
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Neoxanthin, a major carotenoid pigment of spinach, is found in the Chloroplast membrane and has an unknown function in plants. Neoxanthin inhibited the production of superoxide anions in an artificial xanthine and xanthine oxidase system and depressed DNA synthesis in methylcholanthrene (MCA)-initiated C3H10T1/2 fibroblasts. in two-stage carcinogenesis experiments, neoxanthin at 0.2 micrograms/0.2 ml inhibited the formation of tumors that were induced sequentially by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) in the buccal pouch of Syrian Golden hamsters. To assess the ongoing process of carcinogenesis, the activity of ornithine decarboxylase (ODC), required for cell proliferation, was analyzed. Neoxanthin inhibited the activity of ODC when animals were treated with neoxanthin one hour before the application of TPA in two-stage carcinogenesis. However, neoxanthin did not inhibit ODC activity when animals were treated with neoxanthin one hour before the application of DMBA in two-stage carcinogenesis, and there was no subsequent tumor formation. In a short-term anti-initiation experiment, neoxanthin inhibited the covalent binding of isotope-labeled DMBA to DNA by 53%. These results indicate that neoxanthin inhibits the initiation stage and the promotion stage in two-stage carcinogenesis. This suggests that neoxanthin may act as a potential chemopreventive agent. 相似文献