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Acrosin was extracted from turkey spermatozoa by use of urea together with sonication and freezing, and purified approximately 18-fold by sequential use of chromatofocusing and affinity chromatography. The use of chromatofocusing for the initial purification step proved to be superior to preparative isoelectric focusing. Similar to acrosin from many mammalian species, turkey acrosin was found to be a glycoprotein possessing characteristics of serine proteases. Polyacrylamide gel electrophoresis (PAGE) of the enzyme indicated the presence of two isozymes. Sodium-dodecyl sulfate PAGE under reducing conditions revealed three subunits with approximate molecular weights of 11,700, 13,900, and 15,900. 相似文献
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Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible 总被引:2,自引:0,他引:2
The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible. 相似文献
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The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice 总被引:18,自引:0,他引:18
Bodine PV Zhao W Kharode YP Bex FJ Lambert AJ Goad MB Gaur T Stein GS Lian JB Komm BS 《Molecular endocrinology (Baltimore, Md.)》2004,18(5):1222-1237
Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults. 相似文献
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Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins 总被引:2,自引:0,他引:2
Boulanger L Sabatino DE Wong EY Cline AP Garrett LJ Garbarz M Dhermy D Bodine DM Gallagher PG 《The Journal of biological chemistry》2002,277(44):41563-41570
alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells. 相似文献
9.
Proteomic analysis of rat soleus and tibialis anterior muscle following immobilization 总被引:4,自引:0,他引:4
Isfort RJ Wang F Greis KD Sun Y Keough TW Bodine SC Anderson NL 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,769(2):323-332
A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle. 相似文献
10.
Isfort RJ Wang F Greis KD Sun Y Keough TW Farrar RP Bodine SC Anderson NL 《Proteomics》2002,2(5):543-550
A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy. 相似文献