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Pérodin J Deraët M Auger-Messier M Boucard AA Rihakova L Beaulieu ME Lavigne P Parent JL Guillemette G Leduc R Escher E 《Biochemistry》2002,41(48):14348-14356
The human angiotensin II type 1 receptor (hAT(1)) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, (125)I-[Sar(1), Val(5), p-Benzoyl-L-phenylalanine(8)]AngII ((125)I-[Sar(1),Bpa(8)]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT(1). Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe(293) and Asn(294) within the seventh transmembrane domain of the hAT(1). Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N- and C-terminal proximity of those residues 293 and 294 of the hAT(1) produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT(1) receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (Boucard, A. A., Wilkes, B. C., Laporte, S. A., Escher, E., Guillemette, G., and Leduc, R. (2000) Biochemistry 39, 9662-70) the Ang II molecule must adopt an extended structure in the AngII binding pocket. 相似文献
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Marjorie Maillet Jennifer Davis Mannix Auger-Messier Allen York Hanna Osinska Jér?me Piquereau John N. Lorenz Jeffrey Robbins Renée Ventura-Clapier Jeffery D. Molkentin 《The Journal of biological chemistry》2010,285(9):6716-6724
Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca2+ following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca2+ handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca2+-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the α-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca2+-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility. 相似文献
4.
Rihakova L Deraët M Auger-Messier M Pérodin J Boucard AA Guillemette G Leduc R Lavigne P Escher E 《Journal of receptor and signal transduction research》2002,22(1-4):297-313
Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors. 相似文献
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Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier. 相似文献
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Justin Werfel Silva Krause Ashley G. Bischof Robert J. Mannix Heather Tobin Yaneer Bar-Yam Robert M. Bellin Donald E. Ingber 《PloS one》2013,8(10)
Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation. 相似文献
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The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome. 相似文献
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Roberts MA O'Dea J Boyce A Mannix ET 《Journal of strength and conditioning research / National Strength & Conditioning Association》2002,16(2):271-277
Federal law prohibits pre-employment physical examination of firefighter recruits, but these workers must perform intense exercise in arduous environments. Components of physical fitness of rookie firefighters (n = 115; 104 men, mean +/- SD: age = 28.3 +/- 4.3 years; height = 1.76 +/- 0.07 m; weight = 83.2 +/- 13.9 kg; percent body fat = 17 +/- 8%) were measured upon being hired and following a 16-week exercise training program (1 h.d(-1), 3 d.wk(-1)) designed to improve physical fitness. Maximum aerobic capacity (VO2max) was estimated from submaximal cycle ergometry, body composition from skinfold tests, flexibility from a sit and reach test, strength by hand grip dynamometry, and muscle endurance by a push-up test. The results are as follows (*, p 相似文献
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L F Lloyd T Skarzyński A J Wonacott T E Cawston I M Clark C J Mannix G P Harper 《Journal of molecular biology》1989,210(1):237-238
Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A. 相似文献
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Gautam Kao Daniel G. Mannix Brian L. Holaway Mary C. Finn Andrea E. Bonny Mary J. Clancy 《Molecular & general genetics : MGG》1989,215(3):490-500
Summary
SPR3 is one of at least nine genes which are expressed in sporulating Saccharomyces cerevisiae cells at the time of meiosis I. We show below that strains homozygous for null alleles of SPR3 are capable of normal meiosis and the production of viable ascospores. We have also monitored SPR3 expression in a series of strains that are defective in meiotic development, using an SPR3: lacZ fusion carried on a single copy plasmid. -Galactosidase activity occurred at wild-type levels in diploid strains homozygous for mutations in spo13, rad50, rad57 and cdc9, but was greatly reduced in strains carrying cdc8 or spo7 defects. We conclude that SPR3 expression is a valid monitor of early meiotic development, even though the gene is inessential for the sporulation process. 相似文献