排序方式: 共有38条查询结果,搜索用时 433 毫秒
1.
Kristin Stamyr Olavi Vaittinen Janne Jaakola Joseph Guss Markus Metsälä Gunnar Johanson 《Biomarkers》2013,18(5):285-291
Hydrogen cyanide (HCN) in breath has been suggested as a diagnostic tool for cyanide poisoning and for cyanide-producing bacterial infections. To distinguish elevated levels of breath HCN, baseline data are needed. Background levels of HCN were measured in mixed exhaled air from 40 healthy subjects (26 men, 14 women, age 21–61 years; detection limit: 1.5?ppb; median: 4.4?ppb; range <1.5–14?ppb) by near-infrared cavity ring down spectroscopy (CRDS). No correlation was observed with smoking habits, recent meals or age. However, female subjects had slightly higher breath levels of HCN than male subjects. CRDS has not previously been used for this purpose. 相似文献
2.
Chen Wu Cecilia Deng Elena Hilario Nick W. Albert Declan Lafferty Ella R. P. Grierson Blue J. Plunkett Caitlin Elborough Ali Saei Catrin S. Günther Hilary Ireland Alan Yocca Patrick P. Edger Laura Jaakola Katja Karppinen Adrian Grande Ritva Kylli Veli-Pekka Lehtola Andrew C. Allan Richard V. Espley David Chagn 《Molecular ecology resources》2022,22(1):345-360
Bilberry (Vaccinium myrtillus L.) belongs to the Vaccinium genus, which includes blueberries (Vaccinium spp.) and cranberry (V. macrocarpon). Unlike its cultivated relatives, bilberry remains largely undomesticated, with berry harvesting almost entirely from the wild. As such, it represents an ideal target for genomic analysis, providing comparisons with the domesticated Vaccinium species. Bilberry is prized for its taste and health properties and has provided essential nutrition for Northern European indigenous populations. It contains high concentrations of phytonutrients, with perhaps the most important being the purple colored anthocyanins, found in both skin and flesh. Here, we present the first bilberry genome assembly, comprising 12 pseudochromosomes assembled using Oxford Nanopore (ONT) and Hi-C Technologies. The pseudochromosomes represent 96.6% complete BUSCO genes with an assessed LAI score of 16.3, showing a high conservation of synteny against the blueberry genome. Kmer analysis showed an unusual third peak, indicating the sequenced samples may have been from two individuals. The alternate alleles were purged so that the final assembly represents only one haplotype. A total of 36,404 genes were annotated after nearly 48% of the assembly was masked to remove repeats. To illustrate the genome quality, we describe the complex MYBA locus, and identify the key regulating MYB genes that determine anthocyanin production. The new bilberry genome builds on the genomic resources and knowledge of Vaccinium species, to help understand the genetics underpinning some of the quality attributes that breeding programs aspire to improve. The high conservation of synteny between bilberry and blueberry genomes means that comparative genome mapping can be applied to transfer knowledge about marker-trait association between these two species, as the loci involved in key characters are orthologous. 相似文献
3.
Sen S Jaakola VP Heimo H Engström M Larjomaa P Scheinin M Lundstrom K Goldman A 《Protein expression and purification》2003,32(2):265-275
The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology. 相似文献
4.
5.
Hanson MA Brooun A Baker KA Jaakola VP Roth C Chien EY Alexandrov A Velasquez J Davis L Griffith M Moy K Ganser-Pornillos BK Hua Y Kuhn P Ellis S Yeager M Stevens RC 《Protein expression and purification》2007,56(1):85-92
Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants. 相似文献
6.
Henni Piirainen Maarit Hellman Helena Tossavainen Perttu Permi Petri Kursula Veli-Pekka Jaakola 《Biophysical journal》2015,108(4):903-917
Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors. 相似文献
7.
A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads. 相似文献
8.
Laura Jaakola Anna Maria Pirttilä Minna Halonen Anja Hohtola 《Molecular biotechnology》2001,19(2):201-203
A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the
use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer
in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of
the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable
for a variety of plant tissues. 相似文献
9.
A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.We have expressed the three subtypes of α2-adrenergic receptor (α2-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered Kd values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α2-AR is active even in a lipid-free environment. The Kd values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α2-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads. 相似文献
10.
A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor 总被引:2,自引:0,他引:2
Hanson MA Cherezov V Griffith MT Roth CB Jaakola VP Chien EY Velasquez J Kuhn P Stevens RC 《Structure (London, England : 1993)》2008,16(6):897-905
The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery. 相似文献