In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
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
To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure-sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer.
- MeSH
- cirkulární dichroismus * metody MeSH
- diskriminační analýza MeSH
- karnitin analogy a deriváty krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- lysofosfatidylcholiny krev MeSH
- metabolomika * metody MeSH
- nádorové biomarkery krev MeSH
- nádory slinivky břišní * krev MeSH
- pilotní projekty MeSH
- Ramanova spektroskopie * metody MeSH
- senioři MeSH
- spektroskopie infračervená s Fourierovou transformací * metody MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
The Oct4 gene codes for a transcription factor that plays a critical role in the maintenance of pluripotency in embryonic and cancer stem cells. Its expression thus has to be tightly regulated. We performed biophysical characterization of the promoter region using a combination of UV absorption, CD, and NMR spectroscopies, native PAGE and chemical probing, which was followed by functional studies involving luciferase reporter assays performed in osteosarcoma and human embryonic stem cell lines. We have shown that the evolutionarily conserved G-rich region close to the Oct4 transcription start site in the non-template strand forms a parallel G-quadruplex structure. We characterized its structure and stability upon point mutations in its primary structure. Functional studies then revealed that whereas the wild type quadruplex sequence ensures high reporter gene expression, the expression of mutated variants is significantly decreased proportionally to the destabilizing effect of the mutations on the quadruplex. A ligand, N-methyl mesoporphyrin IX that increases the stability of formed quadruplex rescued the reporter expression of single-mutated variants to the level of wild-type, but it has no effect on a mutated variant that cannot form quadruplex. These data indicate that the quadruplex acts as a strong, positive regulator of Oct4 expression and as such it might serve as a potential target for therapeutic intervention.
- MeSH
- cirkulární dichroismus metody MeSH
- embryonální kmenové buňky účinky léků metabolismus MeSH
- G-kvadruplexy účinky léků MeSH
- lidé MeSH
- magnetická rezonanční tomografie metody MeSH
- mesoporfyriny farmakologie MeSH
- mutace genetika MeSH
- nádorové buněčné linie MeSH
- oktamerní transkripční faktor 3 genetika MeSH
- osteosarkom genetika MeSH
- počátek transkripce účinky léků fyziologie MeSH
- promotorové oblasti (genetika) účinky léků genetika MeSH
- reportérové geny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We report the first vibrational circular dichroism (VCD) measurement of spatial heterogeneity in a sample using infrared (IR) microsampling. Vibrational circular dichroism spectra are typically measured using a standard IR cell with an IR beam diameter of 10 mm or greater making it impossible to investigate the spatial heterogeneity of a solid film sample. We have constructed a VCD sampling assembly with either 3 mm or 1 mm spatial resolution. An XY-translation stage was used to measure spectra at different spatial locations producing IR and VCD maps of the sample. In addition, a rotating sample stage was employed using a dual photoelastic modulator (PEM) setup to suppress artifacts due to linear birefringence in solid-phase or film samples. Infrared and VCD mapping of an insulin fibril film has been carried out at both 3 and 1 mm spatial resolution, and lysozyme films were mapped at 1 mm resolution. The IR spectra of different spots vary in intensity due primarily to sample thickness. The changes in the VCD intensity across the map largely correlate to corresponding changes in the IR map. Closer inspection of the insulin map revealed changes in the relative intensities of the VCD spectra not present in the parent IR spectra, which indicated differences in the degree of supramolecular chirality of the fibrils in the various spatial regions. For lysozyme films, in addition to different degrees of supramolecular chirality, reversal of the net fibril chirality was observed. The large signal-to-noise ratio observed at 1 mm resolution implies the feasibility of further increasing the spatial resolution by one or two orders of magnitude for protein fibril film samples.
- MeSH
- amyloid analýza chemie MeSH
- artefakty MeSH
- cirkulární dichroismus metody MeSH
- inzulin analýza chemie MeSH
- muramidasa analýza chemie MeSH
- počítačové zpracování signálu MeSH
- skot MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- vibrace MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In this study we have chosen a new approach and characterized three miRNAs (miR-23a, miR-34a and miR-320a) related to prostate cancer and head and neck cancer by spectral (circular dichroic and UV-absorption spectra) and electrochemical (voltammetry at graphite and mercury electrodes) methods. The spectral and voltammetric results, reflecting different nucleotide sequences of miRNAs, were complemented by the results of DNAs(U) having the same oligonucleotide sequences as miRNAs. The effect of the substitution of ribose for deoxyribose was shown and structural diversity was confirmed. The stability of RNA and DNA(U) was studied using CD and UV-absorption spectroscopy and melting points were calculated. MiRNA-320a with the highest content of guanine provided the highest melting point. With respect to the rapid progress of miRNA electrochemical sensors, our results will be useful for the research and development of sensitive, portable and time-efficient miRNA sensors, which will be able to diagnose cancer and other diseases.
- MeSH
- cirkulární dichroismus metody MeSH
- elektrochemie metody MeSH
- fotoelektronová spektroskopie metody MeSH
- lidé MeSH
- mikro RNA krev MeSH
- nádorové biomarkery krev MeSH
- nádory hlavy a krku krev MeSH
- nádory prostaty krev MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1×10(-4)M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes-a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15-70°C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.
- MeSH
- bilirubin chemie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- cholesterol metabolismus MeSH
- cirkulární dichroismus metody MeSH
- fluorescenční spektrometrie MeSH
- fosfatidylcholiny metabolismus MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- liposomy MeSH
- micely MeSH
- molekulární modely MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Témou práce je separácia enantiomérov aminokyselín metódou vysokoúčinnej kvapalinovej chromatografie. Enantiomérne formy cieľovej skupiny analytov je možné separovať použitím chirálnych stacionárnych fáz na základe cyklodextrínov, makrocyklických polyéterov a antibiotík, polysacharidov a derivatizovaných cyklofruktánov. Pre chromatografickú separáciu racemických zmesí aminokyselín, v závislosti od typu chirálnej stacionárnej fázy, možno využiť polárno-organický, reverzno-fázový aj konvenčný separačný systém. Popri priamom spôsobe separácie sa využíva aj možnosť derivatizácie chirálnym derivatizačným činidlom. V prípade HPLC analýzy sa využívajú bežné detekčné techniky, ako aj detekcia na základe cirkulárneho dichroizmu a laserovej polarimetrie.
Enantiomeric forms of the target analytes can be separated using chiral stationary phases based on cyclodextrins, macrocyclic polyethers and antibiotics, and derivatized polysaccharides and cyclofructans. The polar-organic, reverse-phase and normal-phase separation system of the mobile phase can be used for chromatographic separation of racemic mixtures of amino acids, depending on the type of the chiral stationary phase. In addition to the direct method of separation, the possibility of derivatization with a chiral derivatization agent is used. The conventional detection techniques, circular dichroism and laser polarimetry detection can be used to detect amino acids after HPLC separation.
- MeSH
- aminokyseliny * fyziologie chemická syntéza izolace a purifikace MeSH
- chemie analytická MeSH
- chemie farmaceutická MeSH
- cirkulární dichroismus metody využití MeSH
- farmaceutická technologie MeSH
- skenovací laserová polarimetrie metody využití MeSH
- stereoizomerie MeSH
- vysokoúčinná kapalinová chromatografie * metody přístrojové vybavení využití MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Magnetic circular dichroism (MCD) spectroscopy has been established as a convenient method to study electronic structure, in particular for small symmetric organic molecules. Newer applications on more complex systems are additionally stimulated by the latest availability of precise quantum-chemical techniques for the spectral simulations. In this work, a sum over states (SOS) summation is reexamined as an alternative to the derivative techniques for the MCD modeling. Unlike in previous works, the excited electronic states are calculated by the time-dependent density functional theory (TDDFT). A gradient formulation of the MCD intensities is also proposed, less dependent on the origin choice than the standard expressions. The dependencies of the results on the basis set, number of electronic states, and coordinate origin are tested on model examples, including large symmetric molecules with degenerate electronic states. The results suggest that the SOS/TDDFT approach is a viable and accurate technique for spectral simulation. It may even considerably reduce the computational time, if compared with the traditional MCD computational procedures based on the response theory.
- Klíčová slova
- dynamika vysychání, FTIR,
- MeSH
- antimikrobiální peptidy MeSH
- cirkulární dichroismus metody využití MeSH
- dehydratace MeSH
- kationické antimikrobiální peptidy * MeSH
- lidé MeSH
- liposomy aplikace a dávkování MeSH
- permeabilita buněčné membrány * fyziologie MeSH
- prospektivní studie MeSH
- spektroskopie infračervená s Fourierovou transformací metody využití MeSH
- struktury buněčné membrány klasifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- grafy a diagramy MeSH
