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1.
Anti-androgens were designed based on the principle of inhibiting the folding of helix 12 of the nuclear androgen receptor. The prepared anti-androgens exhibited full antagonistic activity toward human prostate tumor LNCaP cells with T877A point-mutated nuclear androgen receptor, as far as examined, towards which other known anti-androgens, including hydroxyflutamide, are inactive or act as androgen agonists.  相似文献   

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Sertoli cells in culture isolated from immature rat testes secrete androgen binding protein (ABP) in the culture medium. Binding activity of ABP in concentrated medium was estimated with equilibrium dialysis against 1 nM dihydrotestosterone at 4 degrees C. The ABP protein activity was inhibited approximately 50% through addition of cytosol preparations from testis or liver, but not from brain tissue, to the concentrated culture medium; this inhibition remained constant for at least two days. The inhibitor is probably a macromolecule, because the activity could not be removed by charcoal treatment and dialysis. The percent inhibition of ABP binding activity was increased when increasing amounts of cytosol were added, it decreased in the presence of increased concentrations of androgens, but it was not influenced by variations of the concentration of ABP. Inhibition of androgen binding to ABP by cytosols in the presence of 1 nM testosterone could be reversed after dialysis in the presence of 10 nM testosterone. These results suggest a reversible competition between testosterone and the testicular macromolecule for ABP. The occurrence of this interaction between ABP and a testicular macromolecule can explain the variable results of estimated ABP binding activity in testis cytosol preparations.  相似文献   

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10 beta-Chloroestradien-3-one and its derivatives with chlorine substitution in ring A have been prepared. Efficient synthetic methods for 2-chloro- and 4-chloroestradiol are described. The binding affinity of these chlorinated estrogens to the uterine estrogen receptor was measured by a competitive binding assay using [3H]estradiol as ligand. 4-Chloroestradiol showed high binding affinity for the receptor (110% of that of estradiol). 2-Chloroestradiol, 10 beta-chloroestradien-3-one and 4,10 beta-dichloroestradien-3-one had moderate binding affinity. The structures of 10 beta-chloroestradien-3-one and androst-1,4-dien-3-one are very similar and can almost be superimposed. However, their binding affinities to the estrogen and androgen receptor were different. Androst-1,4-dien-3-one displayed no measurable affinity for the estrogen receptor and measurable affinity for the androgen receptor whereas 10 beta-chloroestradien-3-one had very low affinity for the androgen receptor.  相似文献   

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LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.  相似文献   

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Various 4-(anilino)pyrrole-2-carboxamides were designed and synthesized as novel androgen receptor (AR) antagonists without steroidal or anilide structure, based on our strategy for developing full antagonists of nuclear receptors. Introduction of a bulky N-alkyl group, such as a cyclohexylmethyl or benzyl group, increased the binding affinity for wild-type AR and the potency for growth inhibition of androgen-dependent SC-3 cells. Among the compounds obtained, N-[4-[(benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-2-carbonyl]pyrrolidine (22) is as potent an AR antagonist as the typical anilide-type AR antagonists hydroxyflutamide and bicalutamide. Further, compound 22 had potent binding affinity for T877A mutated AR, and dose-dependently inhibited the testosterone-induced production of prostate-specific antigen in LNCaP cells bearing T877A AR.  相似文献   

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The human prostate tumor cell line LNCaP containd an abnormal androgen receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor proteon and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induced reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.  相似文献   

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Nucleotide sequence analysis of mini-Rts1 and its copy mutant disclosed the presence of two clusters of direct-repeat sequences flanking the coding region for the 33,000-dalton RepA protein and two base substitutions on the mini-Rts1cop1 genome (Kamio et al., J. Bacteriol. 158:307-312, 1984). On subcloning of the left cluster (incI) that is located downstream from repA, the five 24-base-pair repeats expressed a stronger incompatibility toward mini-Rts1 than did the four repeats. The right cluster (incII) that contains three 21-base-pair repeats also exhibited strong incompatibility toward mini-Rts1. By separating the two base substitutions of mini-Rts1cop1, the mutation that is responsible for the copy increase was determined to be a single base change in the RepA coding region. Both clusters of the repeats, cloned separately into the vector plasmid, showed a weaker incompatibility toward mini-Rts1cop1 than to the wild-type mini-Rts1. These findings suggest a lowered binding affinity of the mutated RepA protein to the direct repeats.  相似文献   

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M Liao  Z x Zhou  E M Wilson 《Biochemistry》1999,38(30):9718-9727
Full-length histidine-tagged, dihydrotestosterone-bound human androgen receptor (AR) was purified to homogeneity by affinity and gel-filtration chromatography for antibody production and analysis of AR dimerization and DNA binding properties. A monoclonal antibody was raised that recognized human and rat AR epitope (360)ArgAspTyrTyrAsnPheProLeuAla(368) in the NH(2)-terminal domain and slowed migration of AR-DNA complexes in mobility shift assays. AR binding to androgen response element DNA had a K(d) of 2.0 nM and a Hill coefficient of 2.1, indicating high-affinity, cooperative binding. AR solution dimerization was detected only at >/=0.2 microM AR, and DNA binding increased dimerization up to 30-fold. Slow- and fast-migrating AR-DNA complexes were detected under different reducing conditions that differed 5-fold in their dissociation rates from DNA. Treatment with the sulfhydryl oxidizing reagent diamide formed the faster migrating, slower dissociating complex, indicating it represents disulfide-linked AR dimers bound to DNA. The results indicate that high concentrations of purified AR are required for solution dimerization and that cooperative DNA binding stabilizes two dimer forms that differ in redox state.  相似文献   

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HFE is a class I major histocompatibility complex (MHC)-related protein that is mutated in patients with the iron storage disease hereditary hemochromatosis. HFE binds tightly to transferrin receptor (TfR), the receptor that mediates uptake of iron-loaded transferrin. The binding affinities for TfR of HFE mutants, designed using the HFE crystal structure, were measured using biosensor assays. The results allow localization of the TfR binding site on HFE to the C-terminal portion of the alpha1 domain helix and an adjacent loop, a region distinct from the ligand binding sites on class I MHC and related proteins. A biosensor-derived pH-dependent affinity profile for the HFE-TfR interaction is discussed in terms of HFE's hypothesized role in intracellular trafficking.  相似文献   

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We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.  相似文献   

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The mouse germ cell nuclear factor (mGCNF) is an orphan nuclear receptor implicated in diverse biological processes, including gametogenesis, embryonic development and embryonal carcinoma cell differentiation. We have examined the binding and regulation of the human orthologue, hGCNF, expressed in the teratocarcinoma-derived cell line NTera-2/clone D1 (NT2/D1). Binding of GCNF to the direct repeat of the sequence -AGGTCA- (DR-0) is conserved in mammalia. The formation of interspecies dimers of the in vitro synthesized proteins suggests that cellular GCNF binding is mediated by homodimers. Both the mouse and the human protein bind in concert with cellular factors to DNA. Treatment of NT2/D1 cells with all-trans retinoic acid (atRA) is accompanied first by an up-regulation followed later by a down-regulation of hGCNF and its mRNA. Temporary up-regulation in NT2/D1 cells after treatment with atRA suggests that hGCNF is important for human neural determination and differentiation.  相似文献   

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Androgen binding was studied in cytosol of human fibroblasts at 4 degrees C. When 5 alpha-dihydrotestosterone (DHT) was the ligand, a curvilinear Scatchard plot was seen, which was resolved into two components: I the androgen receptor (AR), Kd = 0.12-0.44 nM, and II a low affinity species, Kd = 6.3-28 nM. The same cytosol demonstrated only type I binding for 3H-methyltrienolone (MTr), Kd = 0.10-0.40 nM. The AR, i.e., 3H-MTr binding activity, eluted at 440,000 d by gel filtration chromatography in pre-labeling and post-labeling experiments. When the ligand was 3H-DHT, binding activity in the 10,000-45,000 d range was seen in addition to AR. Thus, saturable nonreceptor steroid binding was seen for DHT but not for MTr. The latter is the preferred ligand for the study of the AR in this system.  相似文献   

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