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181.
182.
Chaudhary S Pak JE Pedersen BP Bang LJ Zhang LB Ngaw SM Green RG Sharma V Stroud RM 《Methods (San Diego, Calif.)》2011,55(4):273-280
It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks. 相似文献
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Eric Hanssen Christian Knoechel Nectarios Klonis Nurhidanatasha Abu-Bakar Samantha Deed Mark LeGros Carolyn Larabell Leann Tilley 《Journal of structural biology》2011,173(1):161-168
Cryo transmission X-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin. 相似文献
185.
Jacobsson JA Almén MS Benedict C Hedberg LA Michaëlsson K Brooks S Kullberg J Axelsson T Johansson L Ahlström H Fredriksson R Lind L Schiöth HB 《PloS one》2011,6(5):e20158
Background
The rs9939609 single-nucleotide polymorphism (SNP) in the fat mass and obesity (FTO) gene has previously been associated with higher BMI levels in children and young adults. In contrast, this association was not found in elderly men. BMI is a measure of overweight in relation to the individuals'' height, but offers no insight into the regional body fat composition or distribution.Objective
To examine whether the FTO gene is associated with overweight and body composition-related phenotypes rather than BMI, we measured waist circumference, total fat mass, trunk fat mass, leg fat mass, visceral and subcutaneous adipose tissue, and daily energy intake in 985 humans (493 women) at the age of 70 years. In total, 733 SNPs located in the FTO gene were genotyped in order to examine whether rs9939609 alone or the other SNPs, or their combinations, are linked to obesity-related measures in elderly humans.Design
Cross-sectional analysis of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) cohort.Results
Neither a single SNP, such as rs9939609, nor a SNP combination was significantly linked to overweight, body composition-related measures, or daily energy intake in elderly humans. Of note, these observations hold both among men and women.Conclusions
Due to the diversity of measurements included in the study, our findings strengthen the view that the effect of FTO on body composition appears to be less profound in later life compared to younger ages and that this is seemingly independent of gender. 相似文献186.
Grantham HS Game ET Lombard AT Hobday AJ Richardson AJ Beckley LE Pressey RL Huggett JA Coetzee JC van der Lingen CD Petersen SL Merkle D Possingham HP 《PloS one》2011,6(2):e16552
Pelagic ecosystems support a significant and vital component of the ocean's productivity and biodiversity. They are also heavily exploited and, as a result, are the focus of numerous spatial planning initiatives. Over the past decade, there has been increasing enthusiasm for protected areas as a tool for pelagic conservation, however, few have been implemented. Here we demonstrate an approach to plan protected areas that address the physical and biological dynamics typical of the pelagic realm. Specifically, we provide an example of an approach to planning protected areas that integrates pelagic and benthic conservation in the southern Benguela and Agulhas Bank ecosystems off South Africa. Our aim was to represent species of importance to fisheries and species of conservation concern within protected areas. In addition to representation, we ensured that protected areas were designed to consider pelagic dynamics, characterized from time-series data on key oceanographic processes, together with data on the abundance of small pelagic fishes. We found that, to have the highest likelihood of reaching conservation targets, protected area selection should be based on time-specific data rather than data averaged across time. More generally, we argue that innovative methods are needed to conserve ephemeral and dynamic pelagic biodiversity. 相似文献
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188.
Plasmodium falciparum causes the most virulent form of malaria and encodes a large number of molecular chaperones. Because the parasite encounters radically different environments during its lifecycle, many members of this chaperone ensemble may be essential for P. falciparum survival. Therefore, Plasmodium chaperones represent novel therapeutic targets, but to establish the mechanism of action of any developed therapeutics, it is critical to ascertain the functions of these chaperones. To this end, we report the development of a yeast expression system for PfHsp70-1, a P. falciparum cytoplasmic chaperone. We found that PfHsp70-1 repairs mutant growth phenotypes in yeast strains lacking the two primary cytosolic Hsp70s, SSA1 and SSA2, and in strains harboring a temperature sensitive SSA1 allele. PfHsp70-1 also supported chaperone-dependent processes such as protein translocation and ER associated degradation, and ameliorated the toxic effects of oxidative stress. By introducing engineered forms of PfHsp70-1 into the mutant strains, we discovered that rescue requires PfHsp70-1 ATPase activity. Together, we conclude that yeast can be co-opted to rapidly uncover specific cellular activities mediated by malarial chaperones. 相似文献
189.
Oligonucleotides containing CpG motifs (cytosine-phosphate-guanosine oligodeoxynucleotide [CpG ODN]) display strong immunostimulatory effects, and polycations have been previously reported as cellular delivery system. In the present study, we investigated the adjuvant properties of combinations of a CpG ODN with various polycations (poly-arginine, poly-lysine, poly-histidine, or chitosan) in an ovalbumin vaccination model. We showed that, when combined to CpG ODN, poly-arginine and poly-histidine, but not poly-lysine or chitosan, enhanced efficiently both the IgG antibody production and the number of splenocytes secreting interferon-gamma after stimulation with a CD8+ T cell-restricted peptide. Interestingly, CpG ODN-poly-arginine, which was the most efficient, compared favorably to the complete Freund's adjuvant and aluminium salts and induced no local toxicity, making this combination a very attractive adjuvant for vaccines. 相似文献
190.