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21.
A R Rezaie M M Fiore P F Neuenschwander C T Esmon J H Morrissey 《Protein expression and purification》1992,3(6):453-460
The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor. 相似文献
22.
Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. 总被引:29,自引:3,他引:26 下载免费PDF全文
M Disdier J H Morrissey R D Fugate D F Bainton R P McEver 《Molecular biology of the cell》1992,3(3):309-321
P-selectin (CD62), formerly called GMP-140 or PADGEM, is a membrane protein located in secretory storage granules of platelets and endothelial cells. To study the mechanisms responsible for the targeting of P-selectin to storage granules, we transfected its cDNA into COS-7 and CHO-K1 cells, which lack a regulated exocytic pathway, or into AtT20 cells, which are capable of regulated secretion. P-selectin was expressed on the plasma membrane of COS-7 and CHO-K1 cells but was concentrated in storage granules of AtT20 cells. Immunogold electron microscopy indicated that the electron-dense granules containing P-selectin in AtT20 cells also stored the endogenous soluble hormone ACTH. Activation of AtT20 cells with 8-Br-cAMP increased the surface expression of P-selectin, consistent with agonist-induced fusion of granule membranes with the plasma membrane. Deletion of the last 23 amino acids of the 35-residue cytoplasmic domain resulted in delivery of P-selectin to the plasma membrane of AtT20 cells. Replacement of the cytoplasmic tail of tissue factor, a plasma membrane protein, with the cytoplasmic domain of P-selectin redirected the chimeric molecule to granules. We conclude that the cytoplasmic domain of P-selectin is both necessary and sufficient for sorting of membrane proteins into the regulated pathway of secretion. 相似文献
23.
Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP 总被引:8,自引:5,他引:3 下载免费PDF全文
We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase. 相似文献
24.
The lower lethal temperature of many insects indicates an overwintering flexibility as a result of either extensive supercooling or production of cryoprotectants. Ontogenetically, the gall fly (Eurosta solidagensis) utilizes both means of seasonal cryoprotection. All stages except third instar larvae demonstrate supercooling points well below the lowest temperature normally experienced by that particular stage. The third instar larvae exhibit a high supercooling point but are well protected by a cryoprotectant system consisting of glycerol, sorbitol, and trehalose. Glycerol is accumulated, possibly from triglyceride sources, during early autumn and reaches plateau levels (0·6 M) by early winter. Sorbitol synthesis is delayed until freezing exposures and reaches a plateau with glycerol at 0·3 M. It is not until mid-winter that peak trehalose levels are reached (300 mg %). All cryoprotectant levels are a reflection of haemolymph concentrations.Laboratory acclimation experiments further quantify these results. Trehalose synthesis is time and temperature dependent and appears to be affected by developmental processes. 相似文献
25.
High-quality early care and education (ECE) programs promote positive child outcomes, allow parents to work, and contribute to the local economy. Although extant research takes into account the ECE sector in its entirety, recent economic and policy interest has centered on part-day prekindergarten for 3- and 4-year-olds only. Using an ecological framework, we review and synthesize the research literature to examine whether the emphasis on pre-k is justified as economically superior to a comprehensive approach. We compare impacts on the macrosystem (regional economy), exosystem (parents), and microsystem (children's long-term human development) and argue that a holistic approach that includes comprehensive ECE services has economic returns as great as or greater than pre-k alone. Finally, we explore the conceptual barriers that have contributed to the narrow focus on pre-k and the policy implications of ignoring the broader ecological context. 相似文献
26.
Adam M. Siepielski Kiyoko M. Gotanda Michael B. Morrissey Sarah E. Diamond Joseph D. DiBattista Stephanie M. Carlson 《Ecology letters》2013,16(11):1382-1392
Local adaptation, adaptive population divergence and speciation are often expected to result from populations evolving in response to spatial variation in selection. Yet, we lack a comprehensive understanding of the major features that characterise the spatial patterns of selection, namely the extent of variation among populations in the strength and direction of selection. Here, we analyse a data set of spatially replicated studies of directional phenotypic selection from natural populations. The data set includes 60 studies, consisting of 3937 estimates of selection across an average of five populations. We performed meta‐analyses to explore features characterising spatial variation in directional selection. We found that selection tends to vary mainly in strength and less in direction among populations. Although differences in the direction of selection occur among populations they do so where selection is often weakest, which may limit the potential for ongoing adaptive population divergence. Overall, we also found that spatial variation in selection appears comparable to temporal (annual) variation in selection within populations; however, several deficiencies in available data currently complicate this comparison. We discuss future research needs to further advance our understanding of spatial variation in selection. 相似文献
27.
Holly M. Nguyen Nazanin Ruppender Xiaotun Zhang Lisha G. Brown Ted S. Gross Colm Morrissey Roman Gulati Robert L. Vessella Frauke Schimmoller Dana T. Aftab Eva Corey 《PloS one》2013,8(10)
Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease. 相似文献
28.
Jessica E. Howell Ann E. McKellar Richard H. M. Espie Christy A. Morrissey 《Ibis》2020,162(2):535-547
High-quality staging sites are critical for long-distance migratory shorebirds to rest and refuel but are under threat from human development, including expansion of wind energy projects. However, predicting migration timing and movements in relation to weather conditions at staging sites can increase our understanding and mitigate effects of wind turbine collisions. Here we assessed northward migration timing and orientation in relation to environmental conditions at an inland staging area in Saskatchewan, Canada, with active and proposed wind energy developments. The area is known to host ~25% of North America's Sanderling Calidris alba population and 16 other Arctic-breeding migrant shorebird species. We quantified arrival and departure time of day in relation to weather using data from 140 of 237 Sanderlings radiotagged locally and at a southern staging site in the Gulf of Mexico with the Motus Wildlife Tracking System (April–June, 2015–2017). Although Sanderling arrival times were not related to time of day or weather, departures were more likely at sunset in winds blowing towards the northwest at intermediate speeds (<22 km/h). Departure flights were also primarily oriented north-northwest in the direction of a proposed wind energy development site at a mean ground speed of 21.4 m/s. Based on published climb rates and flight speed data, we estimated that shorebirds needed between 2 and 14 km setback distance to clear maximum turbine heights of 165 m. Given that departure events were predictable in time and space, adaptive mitigation may be useful for planning wind energy developments while reducing risk for staging Arctic-breeding shorebirds. 相似文献
29.
J. Poissant M. B. Morrissey A. G. Gosler J. Slate B. C. Sheldon 《Journal of evolutionary biology》2016,29(10):2022-2035
When selection differs between the sexes for traits that are genetically correlated between the sexes, there is potential for the effect of selection in one sex to be altered by indirect selection in the other sex, a situation commonly referred to as intralocus sexual conflict (ISC). While potentially common, ISC has rarely been studied in wild populations. Here, we studied ISC over a set of morphological traits (wing length, tarsus length, bill depth and bill length) in a wild population of great tits (Parus major) from Wytham Woods, UK. Specifically, we quantified the microevolutionary impacts of ISC by combining intra‐ and intersex additive genetic (co)variances and sex‐specific selection estimates in a multivariate framework. Large genetic correlations between homologous male and female traits combined with evidence for sex‐specific multivariate survival selection suggested that ISC could play an appreciable role in the evolution of this population. Together, multivariate sex‐specific selection and additive genetic (co)variance for the traits considered accounted for additive genetic variance in fitness that was uncorrelated between the sexes (cross‐sex genetic correlation = ?0.003, 95% CI = ?0.83, 0.83). Gender load, defined as the reduction in a population's rate of adaptation due to sex‐specific effects, was estimated at 50% (95% CI = 13%, 86%). This study provides novel insights into the evolution of sexual dimorphism in wild populations and illustrates how quantitative genetics and selection analyses can be combined in a multivariate framework to quantify the microevolutionary impacts of ISC. 相似文献
30.