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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
2.
Sylvanne M Daniels Carlos E Melendez-Peña Robert J Scarborough Aïcha Daher Helen S Christensen Mohamed El Far Damian FJ Purcell Sébastien Lainé Anne Gatignol 《BMC molecular biology》2009,10(1):38-13
Background
Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. 相似文献3.
Determining the quality and complexity of next-generation sequencing data without a reference genome
Seyed Yahya Anvar Lusine Khachatryan Martijn Vermaat Michiel van Galen Irina Pulyakhina Yavuz Ariyurek Ken Kraaijeveld Johan T den Dunnen Peter de Knijff Peter AC ’t Hoen Jeroen FJ Laros 《Genome biology》2014,15(12)
We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users. 相似文献4.
D Taruscio C Morciano P Laricchiuta P Mincarone F Palazzo CG Leo S Sabina R Guarino J Auld T Sejersen D Gavhed K Ritchie M Hilton-Boon J Manson PG Kanavos D Tordrup V Tzouma Y Le Cam J Senecat G Filippini S Minozzi C Del Giovane H Schünemann JJ Meerpohl B Prediger L Schell R Stefanov G Iskrov T Miteva-Katrandzhieva P Serrano-Aguilar L Perestelo-Perez MM Trujillo-Martín J Pérez-Ramos A Rivero-Santana A Brand H van Kranen K Bushby A Atalaia J Ramet L Siderius M Posada I Abaitua-Borda V Alonso Ferreira M Hens-Pérez FJ Manzanares 《Orphanet journal of rare diseases》2014,9(Z1):O14
5.
The cellular prion protein (PrPC) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrPSc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrPC is needed for the establishment and further evolution of prions.The present work compares the expression and localization of PrPC between healthy human brains and those suffering from Alzheimer disease (AD).In both situations we have observed a rostrocaudal decrease in the amount of PrPC within the CNS, both by immunoblotting and immunohistochemistry techniques. PrPC is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrPC in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands.With regards to amyloid plaques, those present in AD cases were positively labeled for PrPC, a result which is further supported by the presence of PrPC in the amyloid plaques of a transgenic line of mice mimicking AD.The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre.Key words: cellular prion protein, Alzheimer disease, transgenic mice 相似文献
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7.
The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system 总被引:20,自引:0,他引:20
Funes S Hedrick JA Vassileva G Markowitz L Abbondanzo S Golovko A Yang S Monsma FJ Gustafson EL 《Biochemical and biophysical research communications》2003,312(4):1357-1363
GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes. 相似文献
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9.
The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. 总被引:19,自引:9,他引:10 下载免费PDF全文
To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (SfpOP64-6) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc64z) contained OpMNPV GP64 EFP supplied by the Sf9OP64-6 cells. Virions propagated in Sf9OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc64z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Using cytochemical staining to detect vAc64z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc64z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc64z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc64z did not proceed into the hemocoel. Analysis of vAc64z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc64z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64 EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect. 相似文献
10.
Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene 总被引:8,自引:1,他引:7
We have analyzed the conserved regions of the gene coding for the
circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria
parasite. The closest evolutionary relative of P. falciparum, the agent of
malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is
consistent with the hypothesis that P. falciparum is an ancient human
parasite, associated with humans since the divergence of the hominids from
their closest hominoid relatives. Three other human Plasmodium species are
each genetically indistinguishable from species parasitic to nonhuman
primates; that is, for the DNA sequences included in our analysis, the
differences between species are not greater than the differences between
strains of the human species. The human P. malariae is indistinguishable
from P. brasilianum, and P. vivax is indistinguishable from P. simium; P.
brasilianum and P. simium are parasitic to New World monkeys. The human P.
vivax-like is indistinguishable from P. simiovale, a parasite of Old World
macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are
evolutionarily recent human parasites, the first two at least acquired only
within the last several thousand years, and perhaps within the last few
hundred years, after the expansion of human populations in South America
following the European colonizations. We estimate the rate of evolution of
the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year.
The divergence between the P. falciparum and P. reichenowi lineages is
accordingly dated 8.9 Myr ago. The divergence between the three lineages
leading to the human parasites is very ancient, about 100 Myr old between
P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between
P. falciparum and the other two.
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