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Effect of the 90 kDa heat shock protein, HSP90, on glucocorticoid receptor binding to DNA-cellulose 总被引:1,自引:0,他引:1
K J Howard C W Distelhorst 《Biochemical and biophysical research communications》1988,151(3):1226-1232
Glucocorticoid receptors in the IM-9 human lymphoblastoid cell line were affinity labeled with [3H]dexamethasone 21-mesylate and activated to a DNA-binding form by filtration through a Bio-Gel A-1.5m column. The 90 kDa heat shock protein, HSP90, was identified by labeling IM-9 cells with 35S-methionine at both 37 degrees C and 42 degrees C and purified to near homogeneity by sequential chromatography through DE52 and hydroxyapatite. Addition of purified HSP90 to activated, affinity labeled glucocorticoid receptors in a molecular ratio of 16 to 1 inhibited the binding of the receptors to DNA-cellulose. HSP90 did not affect the binding of other proteins to DNA-cellulose, indicating that the inhibitory effect of HSP90 was specific for the glucocorticoid receptor. These results suggest that HSP90 may associate with the glucocorticoid receptor, masking its DNA-binding site and thereby inhibiting receptor interaction with DNA. 相似文献
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A circannual analysis was made of serum cortisol, luteinizing hormone (LH), and testosterone concentrations in the male clouded leopard (Neofelis nebulosa). Group I males (n = 4), maintained in a standardized environment, were bled serially during a regimented anesthesia/electroejaculation episode occurring monthly (beginning in January, ending in December). Additional sampling intervals were conducted under anesthesia only (control, n = 8), anesthesia plus a single adrenocorticotropin hormone challenge (ACTH, Cortrosyn, n = 4), or anesthesia plus a single 25 micrograms injection of gonadotropin-releasing hormone (GnRH, Gonadorelin, n = 4). Group II males (n = 6) from various zoological collections were sampled serially under the same semen collection conditions on one random occasion within the year. Serum cortisol levels were 2 times greater than values measured in comparable studies involving other felid species. Cortisol concentrations were similar during electroejaculation and control (anesthesia only) episodes, and mean levels did not rise as a result of semen collection. Adrenocorticotropin caused an immediate rise in cortisol to levels at least 1.5 times greater than electroejaculated or control counterparts. Mean concentrations of basal cortisol in individual males gradually increased as the year progressed, possibly as a consequence of repeated psychogenic stress. Between seasons, there were no differences in mean LH; however, testosterone levels were greater (p less than 0.05) in the winter compared to all other seasons. There were no differences (p greater than 0.05) between individual males in secretory patterns or mean concentrations of cortisol, LH, or testosterone. Within males, distinct temporal fluctuations were observed in both LH and testosterone during the approximately 80-min sampling interval. Neither LH nor testosterone profiles appeared affected by cortisol patterns during electroejaculation or after an ACTH challenge. A bolus of GnRH induced a marked rise in serum LH and testosterone within 15 and 30 min respectively, indicating that these two hormones were coupled. Both LH and testosterone profiles in Group II males mimicked those in Group I; concentrations of cortisol in Group II males immobilized on one occasion were similar to those of Group I animals sampled from January-May but appeared to be less than values measured from June-December. These data demonstrate that the clouded leopard, compared to other felids, produces markedly elevated concentrations of cortisol, which are likely related to an aggressive behavioral temperament.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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The myositis-specific anti-Jo-1 autoantibody, which is directed against histidyl-tRNA-synthetase, is found in 30% of polymyositis patients. The Jo-1 antigen has been reported to be a nuclear antigen by some authors. On the contrary we show that less than 2% of the total histidyl-tRNA and lysyl-tRNA synthetase activities are associated with purified rat liver nuclei or the hepatocyte intermediate filament-nuclear fraction. In the presence of polyethylene glycol, in which the high Mr multi-enzyme complex containing lysyl-tRNA synthetase is insoluble, 65% of the lysyl-tRNA synthetase and only 15% of histidyl-tRNA synthetase activities remained associated with the cytoskeletal framework. The Jo-1 antigen exhibited a diffuse granular cytoplasmic distribution in cultured rat hepatocytes as determined by indirect immunofluorescent microscopy. Hence, the Jo-1 antigen is cytoplasmic and unassociated with the cytoskeletal framework or high Mr synthetase complex in situ. 相似文献
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The crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form has been refined at 2.20-A resolution and compared to the substrate-bound form of the enzyme. In the absence of the substrate camphor, the P-450cam heme iron atom is hexacoordinate with the sulfur atom of Cys-357 providing one axial heme ligand and a water molecule or hydroxide ion providing the other axial ligand. A network of hydrogen-bonded solvent molecules occupies the substrate pocket in addition to the iron-linked aqua ligand. When a camphor molecule binds, the active site waters including the aqua ligand are displaced, resulting in a pentacoordinate high-spin heme iron atom. Analysis of the Fno camphor - F camphor difference Fourier and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule. However, large decreases in the mean temperature factors of three separate segments of the protein centered on Tyr-96, Thr-185, and Asp-251 result from camphor binding. This indicates that camphor binding decreases the flexibility in these three regions of the P-450cam molecule without altering the mean position of the atoms involved. 相似文献
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Human LFA-1 is a widely expressed leukocyte antigen present on cells of myeloid and lymphoid lineage. Monoclonal antibodies to LFA-1 have been shown to inhibit in vitro T cell immune functions. However, a role for LFA-1 in B cell activation has not been documented. To investigate this possibility, we examined the distribution of LFA-1 on normal, neoplastic, and EBV-transformed B cells as well as the ability of a monoclonal anti-LFA-1 antibody (NB-107) to inhibit B cell mitogenesis. NB-107 immunoprecipitates a noncovalently linked heterodimer of approximately 170,000 and 95,000 daltons. Sequential immunoprecipitation and cross-blocking studies showed that NB-107 identified a distinct epitope on the LFA-1 molecule. NB-107-defined LFA-1 was present on peripheral blood mononuclear cells (PBMC) from all normal individuals (N = 27) and on EBV-transformed cell lines (N = 9), but was absent from four of seven neoplastic B lymphoma lines. NB-107 was observed to profoundly inhibit the response of PBMC to the B cell mitogens anti-IgM (mean 71% inhibition) and lipopolysaccharide (mean 80% inhibition). In order to investigate the mechanism of inhibition, B cells were sequentially purified from PBMC by using a combination of E rosette depletion of T cells, monocyte removal by glass adherence, and finally cell sorting. These extensively enriched populations of B cells, although still responding to anti-mu, showed no evidence of inhibition by NB-107. Growth of EBV-transformed cell lines, cultured in the presence of NB-107, also was not inhibited by this antibody. When tested in assays for T cell function, NB-107 was shown to inhibit the mixed lymphocyte response, but had no effect on phytohemagglutinin stimulation of PBMC nor on the clonal growth and differentiation of granulopoietic, erythropoietic, and pluripotent progenitor cells. We conclude that anti-LFA-1 monoclonal antibody inhibits B cell mitogens via indirect effects on monocytes and/or T cells, rather than by a direct antiproliferative effect on B cells. 相似文献