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1.
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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Sandra L. Almeyda Zambrano Eben N. Broadbent Sam Shanee Noga Shanee Anneke Deluycker Michael Steinberg Scott A. Ford Alma Hernndez Jaramillo Robin Fernandez‐Hilario Carolina Lagos Castillo Angelica M. Almeyda Zambrano 《American journal of primatology》2019,81(8)
Habitat loss is one of the main threats to wildlife. Therefore, knowledge of habitat use and preference is essential for the design of conservation strategies and identification of priority sites for the protection of endangered species. The yellow‐tailed woolly monkey (Lagothrix flavicauda Humboldt, 1812), categorized as Critically Endangered on the IUCN Red List, is endemic to montane forests in northern Peru where its habitat is greatly threatened. We assessed how habitat use and preference in L. flavicauda are linked to forest structure and composition. The study took place near La Esperanza, in the Amazonas region, Peru. Our objective was to identify characteristics of habitat most utilized by L. flavicauda to provide information that will be useful for the selection of priority sites for conservation measures. Using presence records collected from May 2013 to February 2014 for one group of L. flavicauda, we classified the study site into three different use zones: low‐use, medium‐use, and high‐use. We assessed forest structure and composition for all use zones using 0.1 ha Gentry vegetation transects. Results show high levels of variation in plant species composition across the three use zones. Plants used as food resources had considerably greater density, dominance, and ecological importance in high‐use zones. High‐use zones presented similar structure to medium‐ and low‐use zones; thus it remains difficult to assess the influence of forest structure on habitat preference. We recommend focusing conservation efforts on areas with a similar floristic composition to the high‐use zones recorded in this study and suggest utilizing key alimentation species for reforestation efforts. 相似文献
4.
The natural aversion of rats to ethanol was overcome by subjecting rats to immobilization stress for a two-week period during which increasing concentrations of ethanol were offered in the drinking water. The rats subjected to this regimen consumed 47% of total calories as ethanol, indefinitely, following removal of the stress. Ethanol was consumed at a rate of 17.1 g/kg body weight along with sufficient stock diet to assure adequate nutrition in the absence of ethanol. 相似文献
5.
Vincent Anquetil Caroline Le Sommer Agn��s M��reau Sandra Hamon Hubert Lerivray Serge Hardy 《The Journal of biological chemistry》2009,284(47):32370-32383
Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA. 相似文献
6.
Amanda McGrosky Carlo Meloro Ana Navarrete Sandra A. Heldstab Andrew C. Kitchener Karin Isler Marcus Clauss 《American journal of primatology》2019,81(8)
Although it is generally assumed that among mammals and within mammal groups, those species that rely on diets consisting of greater amounts of plant fiber have larger gastrointestinal tracts (GIT), statistical evidence for this simple claim is largely lacking. We compiled a dataset on the length of the small intestine, caecum, and colon in 42 strepsirrhine, platyrrhine, and catarrhine primate species, using specimens with known body mass (BM). We tested the scaling of intestine length with BM, and whether dietary proxies (percentage of leaves and a diet quality index) were significant covariates in these scaling relationships, using two sets of models: one that did not account for the phylogenetic structure of the data, and one that did. Intestine length mainly scaled geometrically at exponents that included 0.33 in the confidence interval; Strepsirrhini exhibited particularly long caeca, while those of Catarrhini were comparatively short. Diet proxies were only significant for the colon and the total large intestine (but not for the small intestine or the caecum), and only in conventional statistics (but not when accounting for phylogeny), indicating the pattern occurred across but not within clades. Compared to terrestrial Carnivora, primates have similar small intestine lengths, but longer large intestines. The data on intestine lengths presented here corroborate recent results on GIT complexity, suggesting that diet, as currently described, does not exhaustively explain GIT anatomy within primate clades. 相似文献
7.
Reverse-phase high-performance liquid chromatography was used to analyse the products of treatment of mouse nerve growth factor with cyanogen bromide followed by trypsin as described by Mercanti et al. All the biological activity was found to be due to incompletely cleaved starting material. Total digestion with trypsin led to complete loss of activity. 相似文献
8.
Sandra Danner Christiane Kirchhoff Richard Ivell 《Reproductive biology and endocrinology : RB&E》2009,7(1):67-14
Background
Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. 相似文献9.
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