13C isotope 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
The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.
One of the challenges the microbial ecologists are facing is the identification of microorganisms that are responsible for particular biochemical processes in the environment. Although small- subunit rRNA gene sequencing is a robust technique whereby the phylogenetic diversity of microbial communities can be described, it provides few direct links between the identity of microorganisms and their metabolic capabilities and function in the environment. In contrast, when a microorganism is cultivable, it can be identified, after isolation, and characterized at the physiological, biochemical and genetic level. However, only a small fraction of microorganisms present in the environment have been successfully cultivated. Stable isotope probing (SIP) techniques are based on analyzing biomarkers (nucleic acids or phospholipid-derived fatty acids) after microbial consumption of 13C- or 15N-labelled substrate added to an environmental sample. The use of SIP allows the direct detection of microorganisms that are truly active in the degradation and assimilation of a particular substrate within a complex microbial community. The main advantages of this method are its cultivation independence and the fact that it links the identity of microorganisms with their function in the environment.
BACKGROUND: Ectomycorrhizal fungi (ECM) play a central role in nutrient cycling in boreal and temperate forests, but their role in the soil food web remains little understood. One of the groups assumed to live as specialised mycorrhizal feeders are Protura, but experimental and field evidence is lacking. We used a combination of three methods to test if Protura are specialized mycorrhizal feeders and compared their trophic niche with other soil invertebrates. Using pulse labelling of young beech and ash seedlings we analysed the incorporation of 13C and 15N into Acerentomon gallicum. In addition, individuals of Protura from temperate forests were collected for the analysis of neutral lipid fatty acids and natural variations in stable isotope ratios. RESULTS: Pulse labelling showed rapid incorporation of root-derived 13C, but no incorporation of root-derived 15N into A. gallicum. The transfer of 13C from lateral roots to ectomycorrhizal root tips was high, while it was low for 15N. Neutral lipid fatty acid (NLFA) analysis showed high amounts of bacterial marker (16:1ω7) and plant marker (16:0 and 18:1ω9) fatty acids but not of the fungal membrane lipid 18:2ω6,9 in A. gallicum. Natural variations in stable isotope ratios in Protura from a number of temperate forests were distinct from those of the great majority of other soil invertebrates, but remarkably similar to those of sporocarps of ECM fungi. CONCLUSIONS: Using three in situ methods, stable isotope labelling, neutral lipid fatty acid analysis and natural variations of stable isotope ratios, we showed that Protura predominantly feed on mycorrhizal hyphae via sucking up hyphal cytoplasm. Predominant feeding on ectomycorrhizal mycelia by Protura is an exception; the limited consumption of ECM by other soil invertebrates may contribute to carbon sequestration in temperate and boreal forests.
- MeSH
- členovci fyziologie MeSH
- izotopy dusíku analýza MeSH
- izotopy uhlíku analýza MeSH
- lesy MeSH
- mykorhiza * chemie MeSH
- potravní řetězec * MeSH
- půdní mikrobiologie MeSH
- stravovací zvyklosti MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Německo MeSH
Helicobacter pylori je vyvolavatelem jedné z nejčastějších infekcí u člověka a je t.č. kauzálně spojován s řadou onemocnění. V jeho průkazu se používá neinvazivní, globální test s ureou značenou izotopem uhlíku 13C (13C-UBT). Test je založen na poznatku, že Helicobacter pylori produkuje ureázu, která štěpí (hydrolyzuje) dodanou s izotopem 13C značenou ureu na amoniak a oxid uhličitý (13CO2). Ten se vstřebává a posléze vydechuje. Ve vydechovaném vzduchu je pak stanovován. V současné době se tento test považuje za zlatý standard průkazu infekce způsobené Helicobacter pylori. Od doby prvého popisu metody (1987) byla popsána řada modifikací a metodik testu. Bohužel však ani European Standard Protocol ani Standard US Protocol nebyly akceptovány. Práce předkládá vlastní metodiku vyšetření 13C-UBT vyvinutou na základě vlastních zkušeností i literárních údajů.
Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, and it is associated with many diseases of the upper gastrointestinal tract. The 13C urea breath test (13C-UBT) is a simple, non-invasive and global test for Helicobacter pylori detection. The test reflects the hydrolysis of 13C-labelled urea by Helicobacter pylori urease. The 13C-UBT is the gold standard test for Helicobacter pylori infection. Since the original description (in 1987) several modifications of 13C-UBT have been published to simplify and optimise the test. However, neither Standardised European Protocol nor Standard US Protocol were accepted. This paper gives the methodology of the 13C-UBT based on our own study and on the review of the literature.
Autoři v článku upozorňují širokou lékařskou veřejnost na možnosti funkční diagnostiky poruch trávicího ústrojí pomocí neinvazivních dechových testů se substráty značenými 13C.
The authors draw attention of the wide medical public to the possible functional diagnosis of digestive tract disorders by means of noninvasive breath analysis test with substrates labelled with 13C.
- MeSH
- dechové testy metody normy přístrojové vybavení MeSH
- diagnostické techniky gastrointestinální metody normy přístrojové vybavení MeSH
- izotopy uhlíku analýza chemie metabolismus MeSH
- lidé MeSH
- nemoci trávicího systému diagnóza metabolismus MeSH
- oxid uhličitý analýza chemie metabolismus MeSH
- spektrální analýza metody normy přístrojové vybavení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Neinvazivní metody s analýzou vydechovaného vzduchu nabízejí v klinické biochemii velmi široká uplatnění, která nejsou v současné době dostatečně využívána. V NLM Pubmed databázi je zařazeno více než 8500 publikací a pro metody se stabilním izotopem uhlíku 13C je popsáno více než 40 13C-značených substrátů. V naší klinické praxi jsou indikovány vodíkové dechové testy, v případě 13C-uhlíku nabízejí naše laboratoře detekci Helicobacter pylori s 13C-močovinou a test exokrinního pankreatu s 13C-mixed triglyceridy. Výjimečně jsou používány testy s 13C-oktanoátem nebo 13C-methioninem. V žádné laboratoři není nabízen 13C-glukózový test pro screening prediabetu, diabetu 2. typu a inzulínové rezistence. Analýza těkavých organických látek (VOC) je zatím předmětem výzkumných projektů. S rozvojem nových technologií, analýzy hmotnostní spektrometrií se selektivními, specifickými senzory na bázi nanočipů lze snad v blízké budoucnosti očekávat vyšší využití neinvazivních dechových testů. Klíčová slova: Dechové testy, analýza vodíku a metanu, detekce izotopu 13C, malabsorbce, syndrom bakteriálního přerůstání, exokrinní funkce pankreatu, detekce Helicobacter pylori, jaterní funkce, rychlost evakuace žaludku, screening diabetu, analýza těkavých organických látek.
Non-invasive methods with the analysis of exhaled air offer a very wide application in clinical biochemistry, which are currently not sufficiently used. More than 8,500 publications are listed in the NLM Pubmed database, and more than 40 13C-labeled substrates are described for stable 13C carbon isotope methods. In our clinical practice, hydrogen breath tests are indicated, in the case of 13C-carbon, our laboratories offer the detection of Helicobacter pylori with 13C-urea and the exocrine pancreas test with 13C-mixed triglycerides. Exceptionally, tests with 13C-octanoate or 13C-methionine are used. No laboratory offers a 13C-glucose test for screening for prediabetes, type 2 diabetes and insulin resistance. The analysis of volatile organic compounds (VOC) is currently the subject of research projects. With the development of new technologies, mass spectrometry analysis with selective, specific nanochip-based sensors, higher use of non-invasive breath tests can be expected in the near future. Keywords: Breath tests, hydrogen and methane analysis, 13C isotope detection, malabsorption, bacterial overgrowth syndrome, pancreatic exocrine function, detection of Helicobacter pylori, liver function, gastric evacuation rate, diabetes screening, analysis of volatile organic compounds.
