Spectroscopic methods
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Spectroscopic imaging (SI) is a method that enables the measurement of the spatial distribution of metabolite concentrations in tissue. In this paper, an overview of measurement and processing techniques for SI is given. First, the basic structure of SI pulse sequences is introduced and the concepts of k-space, point spread function and spatial resolution are described. Then, special techniques are presented for the purpose of eliminating spurious signals and reducing measurement time. Finally, basic post-processing of SI data and the methods for viewing the results of SI measurement are summarized.
Oxidative stress can lead to various derivatives of the tyrosine residue in peptides and proteins. A typical product is 3-nitro-L-tyrosine residue (Nit), which can affect protein behavior during neurodegenerative processes, such as those associated with Alzheimer's and Parkinson's diseases. Surface enhanced Raman spectroscopy (SERS) is a technique with potential for detecting peptides and their metabolic products at very low concentrations. To explore the applicability to Nit, we use SERS to monitor tyrosine nitration in Met-Enkephalin, rev-Prion protein, and α-synuclein models. Useful nitration indicators were the intensity ratio of two tyrosine marker bands at 825 and 870 cm-1 and a bending vibration of the nitro group. During the SERS measurement, a conversion of nitrotyrosine to azobenzene containing peptides was observed. The interpretation of the spectra has been based on density functional theory (DFT) simulations. The CAM-B3LYP and ωB97XD functionals were found to be most suitable for modeling the measured data. The secondary structure of the α-synuclein models was monitored by electronic and vibrational circular dichroism (ECD and VCD) spectroscopies and modeled by molecular dynamics (MD) simulations. The results suggest that the nitration in these peptides has a limited effect on the secondary structure, but may trigger their aggregation.
- MeSH
- azosloučeniny chemie MeSH
- cirkulární dichroismus MeSH
- peptidy chemická syntéza chemie MeSH
- Ramanova spektroskopie metody MeSH
- sekundární struktura proteinů MeSH
- simulace molekulární dynamiky MeSH
- teorie funkcionálu hustoty MeSH
- tyrosin analogy a deriváty analýza MeSH
- Publikační typ
- časopisecké články MeSH
The present study evaluates purified aspartate transaminase (AST, EC 2.6.1.1) preparations from three commercial sources. The enzyme molecule contains pyridoxal-5'-phosphate coenzyme (PLP), which provides AST characteristic absorption spectra in the wavelength range of 300-500 nm. The coenzyme bound in the active site also shows circular dichroism (CD) spectra in the same range. Besides, AST like other proteins may be modified in vitro or in vivo by reactions with other molecules, e.g. reactive sugars, and may form fluorescent products (advanced glycation end products, AGE). Spectroscopic methods were used to assess the quality of AST preparations from three different sources, Serva, Roche, and Sigma. Absorption spectra showed that the peak 360 nm characteristic of the active PLP form of AST prevailed in the Serva and Sigma preparations, while 330 nm was the major peak in the Roche preparation. CD spectra demonstrated the major maximum at 360 nm in the Serva and Roche samples, thus suggesting the predominance of the active PLP form in both preparations. The Sigma sample showed a CD profile less characteristic of AST. Fluorescence measurements revealed formation of AGE in the case of the Roche preparation, while fluorescence of the other two preparations was low. In general, the Serva sample presented the most convenient properties of purified AST among the preparations tested. The results will be used for the selection of a commercial enzyme preparation applicable in our future spectroscopic studies of glycation of AST as a model protein and in our research of the influence of antioxidants on this process.
- MeSH
- financování organizované MeSH
- fluorescenční spektrometrie metody MeSH
- prasata MeSH
- pyridoxalfosfát analýza chemická syntéza normy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
The interactions of epoxiconazole and prothioconazole with human serum albumin and bovine serum albumin were investigated using spectroscopic methods complemented with molecular modeling. Spectroscopic techniques showed the formation of pesticide/serum albumin complexes with the static type as the dominant mechanism. The association constants ranged from 3.80 × 104-6.45 × 105 L/mol depending on the pesticide molecule (epoxiconazole, prothioconazole) and albumin type (human or bovine serum albumin). The calculated thermodynamic parameters revealed that the binding of pesticides into serum albumin macromolecules mainly depended on hydrogen bonds and van der Waals interactions. Synchronous fluorescence spectroscopy and the competitive experiments method showed that pesticides bind to subdomain IIA, near tryptophan; in the case of bovine serum albumin also on the macromolecule surface. Concerning prothioconazole, we observed the existence of an additional binding site at the junction of domains I and III of serum albumin macromolecules. These observations were corroborated well by molecular modeling predictions. The conformation changes in secondary structure were characterized by circular dichroism, three-dimensional fluorescence, and UV/VIS absorption methods.
- MeSH
- cirkulární dichroismus metody MeSH
- epoxidové sloučeniny chemie MeSH
- fluorescenční spektrometrie metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- lidský sérový albumin chemie MeSH
- pesticidy chemie MeSH
- sekundární struktura proteinů MeSH
- sérový albumin hovězí chemie MeSH
- simulace molekulového dockingu metody MeSH
- skot MeSH
- statická elektřina MeSH
- teplota MeSH
- triazoly chemie MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cíl: Porovnat spektroskopické metody stanovení steatózy sekvencemi PRESS, STEAM a HISTO na 3T tomografech TRIO a VIDA. Metodika: Byla naměřena 1H MR spektra jater dobrovolníků na 3T tomografech Siemens TRIO a VIDA (45/25 subjektů) sekvencemi PRESS s TE = 30/33 ms, STEAM s TE = 20/33 ms a metodou HISTO s TE = 12-72 ms. Stejné sekvence byly použity pro stanovení T2 lipidů a vody. Spektra byla zpracována programem LCModel. Frakční (FF) a objemové (HFC) zlomky (%) byly korigovány individuálními a průměrnými hodnotami relaxačních časů T2. Analýza výsledků byla provedena korelační, regresní a Bland-Altmanovou metodou. Výsledky: Relaxační časy T2 snižují hodnoty parametrů FF a HFC až o cca 47 % při použití nominálních T2. Regresní analýza a Bland-Altmanovy grafy ukazují, že parametry získané technikami PRESS, STEAM a HISTO s TE 20 a 33 ms na VIDA jsou srovnatelné. Byly změřeny hodnoty T2 lipidů mezi 45-53 ms a T2 vody mezi 24-31 ms. Byly stanoveny limitní hodnoty FF pro určení stupně steatózy u transplantovaných pacientů v těchto rozsazích: S0: < 0,8 %; S1: 0,81-6,2 %; S2: 6,21-16,5 %; S3: > 16,5 %. Závěr: Získané hodnoty FF a HFC jsou citlivé na T2 korekce a použití průměrných hodnot T2 dává vyšší hodnoty parametrů FF a HFC. Bland-Altmanovy grafy ukazují na dobrou shodu metod STEAM, PRESS a HISTO. Limitní hodnoty pro stanovení stupně steatózy u transplantovaných pacientů z výsledků HISTO metody jsou v rozmezí literárních hodnot.
Aim: To compare MR spectroscopic methods for steatosis determination by PRESS, STEAM and HISTO sequences on 3T TRIO and VIDA tomographs. Method: 1H MR spectra of the liver of volunteers were measured on 3T tomographs Siemens TRIO and VIDA (45/25 subjects). The sequences PRESS with TE = 30/33 ms, STEAM with TE = 20/33 ms and the HISTO method with TE = 12-72 ms were used. The same sequences were used to determine T2 of lipids and water. The spectra were processed using the LCModel method. Fractional (FF) and volume (HFC) fractions (%) were corrected for individual and mean T2 values. Correlation, regression and Bland-Altman data analyses were performed. Results: Relaxation times T2 reduce FF and HFC values by up to approx. 47%. Regression analysis and Bland-Altman graphs show that the parameters obtained by PRESS, STEAM and HISTO techniques with short TE are comparable on VIDA. Mean T2 values of lipids between 45 and 53 ms and T2 of water between 24 and 31 ms were measured. HISTO FF cut-off values for the degree of steatosis in transplant patients were set in the following ranges: S0: < 0.8%; S1: 0.81-6.2%; S2: 6.21-16.5%; S3: > 16.5%. Conclusion: The obtained FF and HFC values are sensitive to T2 corrections. Bland-Altman´s graphs indicate good agreement among STEAM, PRESS and HISTO methods. Limit values for the steatosis degree in transplant patients from the result of the HISTO method agree with literature values.
New approaches to the synthesis of 4,7-dichloro-1,10-phenanthrolines and their corresponding 9H-carbazol-9-yl-, 10H-phenothiazin-10-yl- and pyrrolidin-1-yl derivatives were developed. Their properties have been characterized by a combination of several techniques: MS, HRMS, GC-MS, electronic absorption spectroscopy and multinuclear NMR in both solution and solid state including 15N CP/MAS NMR. The structures of 5-fluoro-2,9-dimethyl-4,7-di(pyrrolidin-1-yl)-1,10-phenanthroline (5d), 4,7-di(9H-carbazol-9-yl)-9-oxo-9,10-dihydro-1,10-phenanthroline-5-carbonitrile (6a) and 4,7-di(10H-phenothiazin-10-yl)-1,10-phenanthroline-5-carbonitrile (6b) were determined by single-crystal X-ray diffraction measurements. The nucleophilic substitutions of hydrogen followed by oxidation produced compounds 6a and 6b. The electrochemical properties of selected 1,10-phenanthrolines were investigated using cyclic voltammetry and compared with commercially available reference 1,10-phenanthrolin-5-amine (5l). The spatial distribution of frontier molecular orbitals of the selected compounds has been calculated by density functional theory (DFT). It was shown that potentials of reduction and oxidation were in consistence with the level of HOMO and LUMO energies.
Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.
- MeSH
- cytochrom P-450 CYP2E1 chemie metabolismus MeSH
- financování organizované MeSH
- lidé MeSH
- prasata MeSH
- Ramanova spektroskopie metody MeSH
- spektrofotometrie metody MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
Chiroptical methods are widely used in structural and conformational analyses of biopolymers. The application of these methods to investigations of biofluids would provide new avenues for the molecular diagnosis of protein-misfolding diseases. In this work, samples of human blood plasma and hen egg white were analyzed using a combination of conventional and chiroptical methods: ultraviolet absorption/electronic circular dichroism (UV/ECD), Fourier transform infrared absorption/vibrational circular dichroism (FTIR/VCD), and Raman scattering/Raman optical activity (Raman/ROA). For comparison, the main components of these substances--human serum albumin (HSA) and ovalbumin (Ova)--were also analyzed by these methods. The ultraviolet region of the ECD spectrum was analyzed using the CDNN CD software package to evaluate the secondary structures of the proteins. The UV/ECD, FTIR/VCD, and Raman/ROA spectra of the substances were quite similar to those of the corresponding major proteins, while some differences were also detected and explained. The conclusions drawn from the FTIR/VCD and Raman/ROA data were in good agreement with the secondary structures calculated from ECD. The results obtained in this work demonstrate that the chiroptical methods used here can be applied to analyze not only pure protein solutions but also more complex systems, such as biological fluids.
- MeSH
- aminokyseliny chemie MeSH
- cirkulární dichroismus metody MeSH
- krevní proteiny analýza chemie MeSH
- lidé MeSH
- Ramanova spektroskopie metody MeSH
- sekundární struktura proteinů * MeSH
- sérový albumin analýza MeSH
- software MeSH
- spektrofotometrie ultrafialová metody MeSH
- spektroskopie infračervená s Fourierovou transformací metody MeSH
- stereoizomerie MeSH
- vaječný bílek analýza MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
V období let 1988 -1994 byla zavedena metoda 31 MR spektroskopie kombinovaná s MR zobrazením kosterního svalu u pacientů s neuromuskulámím onemocněním do klinické praxe. Cílem zavedení této metody bylo odpovědět na otázku, zda relativní koncentrace anorganického fosfátu (Pi), fosfomonoesterů (Pmc), fosfodiesterů (Pdb), fosfokreatinu (Pcr) a adenosintrifosfátu (Patp) u pacientů se liší od kontrolních vyšetření. Celkově bylo vyšetřeno 680 pacientů a různých věkově odpovídajících kontrol a bylo získáno více než 1000 spekter. Podle očekávání největší rozdíly vzhledem ke kontrolám byly zjištěny u pacientů s Duchennovou/Beckerovou svalovou dystrofií. Velmi často a úspěšně se P MRS ukázala jako rychlá a neinvazivní metoda objektivizace svalových slabostí souvisejících s nervosvalovými onemocněními. Metoda je vhodná pro sledování změn Pcr/Pi V kosterním svalu při podávání některých léčiv (kamitin, prednison). S instalací tomografu nové generace a tím i nové verze 31P MR spektroskopického vybavení vzniká potřeba porovnat hodnoty kontrolních dat z nového zařízení s daty získanými na původním MR tomografu Magnetom SP. Tato studie shrnuje základní poznatky nezbytné pro porovnání obou zařízení.
During 1988 -1994 the method of 31P MR spectroscopy combined with MR visualization of skeletal muscles in patients with neuromuscular diseases was introduced into clinical practice. The objective of this method was to find an answer to the question whether the relative concentration of inorganic phosphate (Pi), phosphomonoesters (Pwe,) phosphodiesters (Poe), phosphocreatine (Pcr) and adenosine triphosphate (Patp) in patients differs from control examinations. A total of 680 patients were examined and age matched controls, and more than 1000 spectra were obtained. As ker's muscular dystrophy. Very freauentlv 31P MRS proved a rapid and non-invasive method of objectivization of muscular weakness associated with neuromuscular disease. The method is suitable for investigating Pcr/Pi in skeletal muscle when administering some drugs (Karniten, Prednisone). After installation of a new generation tomograph and thus a new version of 31P MR of spectroscopic equipment it is necessary to compare the values of control data from the new equipment with data assembled on the original MR tomograph Magnetom SP. The present study summarizes basic findings essential for comparison of the two sets of equipment.
- MeSH
- adenosintrifosfát metabolismus MeSH
- dítě MeSH
- dospělí MeSH
- fosfokreatin metabolismus MeSH
- fosfor metabolismus MeSH
- kosterní svaly metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie metody přístrojové vybavení MeSH
- neuromuskulární nemoci metabolismus MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
