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排序方式: 共有862条查询结果,搜索用时 15 毫秒
101.
Ueda K Saichi N Takami S Kang D Toyama A Daigo Y Ishikawa N Kohno N Tamura K Shuin T Nakayama M Sato TA Nakamura Y Nakagawa H 《PloS one》2011,6(4):e18567
The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens. 相似文献
102.
Ishigamori H Hosokawa M Kohno H Tanaka T Miyashita K Takahashi K 《Molecular and cellular biochemistry》2005,275(1-2):127-133
18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products. 相似文献
103.
Sawafuji M Ishizaka A Kohno M Koh H Tasaka S Ishii Y Kobayashi K 《American journal of physiology. Lung cellular and molecular physiology》2005,289(6):L946-L953
Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema. 相似文献
104.
Wheat omega-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because omega-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the omega-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length omega-5 gliadin genes, designated omega-5 and omega-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by omega-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the omega-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that omega-5b is pseudogene. The C-terminal half (178 amino acids) of the omega-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native omega-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro. 相似文献
105.
OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes. 相似文献
106.
Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway. 相似文献
107.
Nomura M Kobayashi T Kohno T Fujiwara K Tenno T Shirakawa M Ishizaki I Yamamoto K Matsuyama T Mishima M Kojima C 《FEBS letters》2004,566(1-3):157-161
The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information. 相似文献
108.
Kimata Y Oikawa D Shimizu Y Ishiwata-Kimata Y Kohno K 《The Journal of cell biology》2004,167(3):445-456
In the unfolded protein response, the type I transmembrane protein Ire1 transmits an endoplasmic reticulum (ER) stress signal to the cytoplasm. We previously reported that under nonstressed conditions, the ER chaperone BiP binds and represses Ire1. It is still unclear how this event contributes to the overall regulation of Ire1. The present Ire1 mutation study shows that the luminal domain possesses two subregions that seem indispensable for activity. The BiP-binding site was assigned not to these subregions, but to a region neighboring the transmembrane domain. Phenotypic comparison of several Ire1 mutants carrying deletions in the indispensable subregions suggests these subregions are responsible for multiple events that are prerequisites for activation of the overall Ire1 proteins. Unexpectedly, deletion of the BiP-binding site rendered Ire1 unaltered in ER stress inducibility, but hypersensitive to ethanol and high temperature. We conclude that in the ER stress-sensory system BiP is not the principal determinant of Ire1 activity, but an adjustor for sensitivity to various stresses. 相似文献
109.
The 70-kDa heat shock protein (Hsp70) family comprises the most abundant and important group of molecular chaperones. Hsp70s cooperate with a number of cofactors, which define their functions. We recently reported that a yeast protein, Rot1, is a putative cofactor of BiP, an endoplasmic reticulum (ER)-localized Hsp70. Rot1 is an essential ER membrane protein and may be involved in protein folding. Mutation of the ROT1 gene caused defects in cell wall synthesis and lysis of autophagic bodies. We suggest that Rot1 is required for folding of proteins engaged in these cellular processes. 相似文献
110.