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Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity 总被引:2,自引:0,他引:2
B S Katzenellenbogen J F Elliston F J Monsma P A Springer Y S Ziegler G L Greene 《Biochemistry》1987,26(8):2364-2373
We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region. 相似文献
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Isoproterenol-stimulated cyclic AMP (cAMP) accumulation in lymphocytes is an index of beta-adrenergic receptor functioning. It is not known if a delay between blood collection and subsequent cell preparation influences cAMP accumulation. We compared cAMP levels in lymphocytes processed from whole blood immediately after collection and following a 3.5 hour delay. There were no significant changes in either basal or stimulated levels of cAMP. This information may alleviate time constraints in certain experimental designs where immediate isolation of lymphocytes from whole blood may not be practical. 相似文献
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Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb+(86Rb+) and Ca2+(45Ca2+) and contents of K+ and Ca2+ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K+ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K+. Reabsorption of K+ in roots of plants exposed to -4°C was greatly impaired. Rb+ influx decreased and Ca2+ influx increased after sub-zero temperature treatments of the plants. Active Rb+ influx mechanisms and active extrusion of Ca2+ were impaired or irreversibly damaged by the exposure. While Rb+ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca2+ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K+ and to repair Rb+ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C. 相似文献
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Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate. 相似文献
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Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem. 相似文献
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Anna M. Kauppi Alicia Edin Ingrid Ziegler Paula M?lling Anders Sj?stedt ?sa Gylfe Kristoffer Str?lin Anders Johansson 《PloS one》2016,11(1)
A metabolomics approach for prediction of bacteremic sepsis in patients in the emergency room (ER) was investigated. In a prospective study, whole blood samples from 65 patients with bacteremic sepsis and 49 ER controls were compared. The blood samples were analyzed using gas chromatography coupled to time-of-flight mass spectrometry. Multivariate and logistic regression modeling using metabolites identified by chromatography or using conventional laboratory parameters and clinical scores of infection were employed. A predictive model of bacteremic sepsis with 107 metabolites was developed and validated. The number of metabolites was reduced stepwise until identifying a set of 6 predictive metabolites. A 6-metabolite predictive logistic regression model showed a sensitivity of 0.91(95% CI 0.69–0.99) and a specificity 0.84 (95% CI 0.58–0.94) with an AUC of 0.93 (95% CI 0.89–1.01). Myristic acid was the single most predictive metabolite, with a sensitivity of 1.00 (95% CI 0.85–1.00) and specificity of 0.95 (95% CI 0.74–0.99), and performed better than various combinations of conventional laboratory and clinical parameters. We found that a metabolomics approach for analysis of acute blood samples was useful for identification of patients with bacteremic sepsis. Metabolomics should be further evaluated as a new tool for infection diagnostics. 相似文献
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M. von Balthazar K. Raunsgaard Pedersen E. M. Friis 《Plant Systematics and Evolution》2005,255(1-2):55-75
A charcoalified fossil flower bud of a new genus and species (Teixeiria lusitanica) is described from the Early Cretaceous of Portugal. The flower is actinomorphic and unisexually male. At the base of the
bud there are several bracts of different sizes, which are followed by sepal-like and petal-like tepals. Bracts and perianth
organs seem to be arranged spirally and to exhibit transitions between different organ categories. The androecium has numerous
stamens in two sizes, but with unclear arrangement. Pollen is small and tricolpate with a perforate tectum and a densely columellate
infratectal layer. No carpels or remains of carpels could be observed on the floral axis. Teixeiria lusitanica shows most affinities to members of Ranunculales. There are also some similarities with Berberidopsis (Berberidopsidaceae, Berberidopsidales) and members of the Saxifragales (Hamamelidaceae and Daphniphyllaceae). 相似文献
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