13C labelling Dotaz Zobrazit nápovědu
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.
- MeSH
- chromatografie afinitní MeSH
- dynamický rozptyl světla MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- izotopové značení MeSH
- izotopy dusíku chemie izolace a purifikace MeSH
- izotopy uhlíku chemie izolace a purifikace MeSH
- lidé MeSH
- nádorové proteiny chemie izolace a purifikace MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- protein STIM1 chemie izolace a purifikace MeSH
- proteinové domény MeSH
- rekombinantní proteiny chemie izolace a purifikace MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.
- MeSH
- cesty eliminace léčiva MeSH
- chrupavka metabolismus MeSH
- cyklická ADP-ribosa metabolismus MeSH
- intravenózní podání MeSH
- izotopové značení metody MeSH
- izotopy uhlíku chemie metabolismus farmakokinetika MeSH
- kosti a kostní tkáň metabolismus MeSH
- kyselina hyaluronová chemie metabolismus farmakokinetika MeSH
- molekulová hmotnost MeSH
- myši inbrední BALB C MeSH
- tkáňová distribuce MeSH
- uridindifosfát-N-acetylglukosamin metabolismus MeSH
- uridindifosfátglukosa metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Glucose tolerance represents a complex phenotype in which many tissues play important roles and interact to regulate metabolic homeostasis. Here, we perform an analysis of 13C6-glucose tissue distribution, which maps the metabolome and lipidome across 12 metabolically relevant mouse organs and plasma, with integrated 13C6-glucose-derived carbon tracing during oral glucose tolerance test (OGTT). We measure time profiles of water-soluble metabolites and lipids and integrate the global metabolite response into metabolic pathways. During the OGTT, glucose use is turned on with specific kinetics at the organ level, but fasting substrates like β-hydroxybutyrate are switched off in all organs simultaneously. Timeline profiling of 13C-labeled fatty acids and triacylglycerols across tissues suggests that brown adipose tissue may contribute to the circulating fatty acid pool at maximal plasma glucose levels. The GTTAtlas interactive web application serves as a unique resource for the exploration of whole-body glucose metabolism and time profiles of tissue and plasma metabolites during the OGTT.
- MeSH
- biologické markery krev MeSH
- časové faktory MeSH
- chromatografie kapalinová MeSH
- energetický metabolismus * MeSH
- glukózový toleranční test * MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- krevní glukóza metabolismus MeSH
- lipidomika MeSH
- lipidy krev MeSH
- metabolom * MeSH
- metabolomika * MeSH
- myši inbrední C57BL MeSH
- tandemová hmotnostní spektrometrie MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DCL-1 (CD302) is a single-pass type one transmembrane protein which is predominantly expressed on myeloid cell lines. It possess the ability of endocytosis and is assumed to play a role in cell adhesion and migration. It has been also connected to several illnesses but more on the level of mRNA than on the protein expression level. More interestingly it is alternatively expressed in the form of a fusion protein with another single-pass type one transmembrane protein DEC205 (CD205) which is normally involved in antigen-uptake and endocytosis. The fusion protein has been assigned to have altered function compared to the wild type proteins. We have performed NMR structural analysis of the 16.2 kDa extracellular domain of DCL-1 to get a better insight onto this molecule. We have been able to assign nearly 97 % of resonance frequencies for the (15)N and (13)C labeled recombinant protein. The assignments have been deposited into Biological Magnetic Resonance Data Bank under the accession number 25802.
- MeSH
- extracelulární prostor * MeSH
- lektiny typu C chemie MeSH
- lidé MeSH
- nukleární magnetická rezonance biomolekulární * MeSH
- proteinové domény MeSH
- receptory buněčného povrchu chemie MeSH
- sekundární struktura proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Standard spectral density mapping protocols, well suited for the analysis of (15)N relaxation rates, introduce significant systematic errors when applied to (13)C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested. Applicability of the proposed protocols is documented in two case studies, spectral density mapping of a uniformly labeled RNA hairpin and of a selectively labeled disaccharide exhibiting highly anisotropic tumbling. Combination of auto- and cross-correlated relaxation data acquired at three magnetic fields was applied in the former case in order to separate effects of fast motions and conformational or chemical exchange. An approach using auto-correlated relaxation rates acquired at five magnetic fields, applicable to anisotropically moving molecules, was used in the latter case. The results were compared with a more advanced analysis of data obtained by interpolation of auto-correlated relaxation rates measured at seven magnetic fields, and with the spectral density mapping of cross-correlated relaxation rates. The results showed that sufficiently accurate values of auto- and cross-correlated spectral density functions at zero and (13)C frequencies can be obtained from data acquired at three magnetic fields for uniformly (13)C-labeled molecules with a moderate anisotropy of the rotational diffusion tensor. Analysis of auto-correlated relaxation rates at five magnetic fields represents an alternative for molecules undergoing highly anisotropic motions.
- MeSH
- algoritmy * MeSH
- interpretace statistických dat * MeSH
- magnetická rezonanční spektroskopie s uhlíkem 13C metody MeSH
- magnetické pole MeSH
- malá interferující RNA analýza chemie MeSH
- počítačové zpracování signálu * MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
