Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.

• MeSH
• 2-naftylamin analogy a deriváty   chemie   MeSH
• fluorescenční barviva chemie   MeSH
• laurany chemie   MeSH
• lipidové dvojvrstvy chemie   MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Turing's diffusion-driven instability for the standard two species reaction-diffusion system is only achievable under well-known and rather restrictive conditions on both the diffusion rates and the kinetic parameters, which necessitates the pairing of a self-activator with a self-inhibitor. In this study we generalize the standard two-species model by considering the case where the reactants can bind to an immobile substrate, for instance extra-cellular matrix, and investigate the influence of this dynamics on Turing's diffusion-driven instability. Such systems have been previously studied on the grounds that binding of the self-activator to a substrate may effectively reduce its diffusion rate and thus induce a Turing instability for species with equal diffusion coefficients, as originally demonstrated by Lengyel and Epstein (1992) under the assumption that the bound state dynamics occurs on a fast timescale. We, however, analyse the full system without any separation of timescales and demonstrate that the full system also allows a relaxation of the standard constraints on the reaction kinetics for the Turing instability, increasing the type of interactions that could give rise to spatial patterning. In particular, we show that two self-activators can undertake a diffusively driven instability in the presence of a binding immobile substrate, highlighting that the interactions required of a putative biological Turing instability need not be associated with a self-activator-self-inhibitor morphogen pair.

• MeSH
• biologické modely * MeSH
• difuze MeSH
• kinetika MeSH
• numerická analýza pomocí počítače MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH

Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations of protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, in vivo FRET has not been applied to in vivo quantitative conformational analysis of NA thus far. Here we explored parameters critical for characterization of NA structure using single-pair (sp)FRET in the complex cellular environment of a living Escherichia coli cell. Our measurements showed that the fluorophore properties in the cellular environment differed from those acquired under in vitro conditions. The precision for the interprobe distance determination from FRET efficiency values acquired in vivo was found lower (≈ 31%) compared to that acquired in diluted buffers (13%). Our numerical simulations suggest that despite its low precision, the in-cell FRET measurements can be successfully applied to discriminate among various structural models. The main advantage of the in-cell spFRET setup presented here over other established techniques allowing conformational analysis in vivo is that it allows investigation of NA structure in various cell types and in a native cellular environment, which is not disturbed by either introduced bulk NA or by the use of chemical transfectants.

• MeSH
• DNA chemie   MeSH
• Escherichia coli genetika   MeSH
• fluorescenční barviva MeSH
• konformace nukleové kyseliny MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oleaginous microbes, which contain over 20% intracellular lipid, predominantly triacylglycerols (TG), by dry weight, have been discovered to have high oil content by many different protocols, ranging from simple staining to more complex chromatographic methods. In our laboratory, a methodical process was implemented to identify high oil yeasts, designed to minimize labor while optimizing success in identifying high oil strains among thousands of candidates. First, criteria were developed to select candidate yeast strains for analysis. These included observation of buoyancy of the yeast cell mass in 20% glycerol, and phylogenetic placement near known oleaginous species. A low-labor, semiquantitative Nile red staining protocol was implemented to screen numerous yeast cultures for high oil content in 96-well plates. Then, promising candidates were selected for more quantitative analysis. A more labor-intensive and quantitative gravimetric assay was implemented that gave consistent values for intracellular oil content for a broad range of yeast species. Finally, an LC-MS protocol was utilized to quantify and identify yeast triacylglycerols. This progressive approach was successful in identifying 30 new oleaginous yeast species, out of over 1000 species represented in the Phaff Yeast Culture Collection.

• MeSH
• barvení a značení metody   MeSH
• chromatografie kapalinová metody   MeSH
• fluorescenční barviva analýza   MeSH
• fluorescenční mikroskopie metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• kvasinky chemie   MeSH
• lipidomika metody   MeSH
• lipidy analýza   MeSH
• oxaziny analýza   MeSH
• triglyceridy analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The plant hormone auxin is a key player in the regulation of plant growth and development. Despite numerous studies devoted to understanding its role in a wide spectrum of physiological processes, full appreciation of its function is linked to a comprehensive determination of its spatio-temporal distribution, which plays a crucial role in its mode of action. Conjugation of fluorescent tracers to plant hormones enables sensitive and specific visualization of their subcellular and tissue-specific localization and transport in planta, which represents a powerful tool for plant physiology. However, to date, only a few fluorescently labeled auxins have been developed. We report the synthesis of four novel fluorescently labeled derivatives of indole-3-acetic acid (IAA) in the form of a conjugate with a nitrobenzoxadiazole (NBD) fluorophore together with validation of their biological activity. These compounds, unlike other previously reported auxins fluorescently labeled at N1 position (nitrogen of the indole ring), do not possess auxin activity but rather show dose-dependent inhibition of auxin-induced effects, such as primary root growth inhibition, root hair growth and the auxin reporter DR5::GUS expression. Moreover, the study demonstrates the importance of the character of the linker and optimal choice of the labeling site in the preparation of fluorescently labeled auxins as important variables influencing their biological activity and fluorescent properties.

• MeSH
• Arabidopsis účinky léků   genetika   růst a vývoj   MeSH
• fluorescenční barviva chemická syntéza   chemie   MeSH
• fluorescenční spektrometrie MeSH
• geneticky modifikované rostliny MeSH
• kořeny rostlin účinky léků   růst a vývoj   MeSH
• kyseliny indoloctové antagonisté a inhibitory   chemie   farmakologie   MeSH
• molekulární struktura MeSH
• regulátory růstu rostlin antagonisté a inhibitory   chemie   farmakologie   MeSH
• spektrofotometrie MeSH
• Publikační typ
• časopisecké články MeSH

Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8.

• MeSH
• elektroforéza kapilární metody   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• kyselina listová chemie   MeSH
• limita detekce MeSH
• lineární modely MeSH
• nanokonjugáty chemie   MeSH
• protilátky chemie   MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Eugregarines represent a diverse group of Apicomplexa parasitising numerous invertebrates. Their sporozoites generally develop into epicellular trophozoites attached to the host epithelium by a specialised attachment organelle known as an epimerite. They are considered peculiar protists due to their unique cell architecture and dimensions as well as their attachment strategy which is similar to that of cryptosporidia. Using electron and fluorescence microscopy, the fine structure of the epimerite with associated structures and the mechanism of trophozoite detachment from the host epithelium were studied in Gregarina polymorpha parasitising the intestine of Tenebrio molitor larvae. The epimerite appears to be a very dynamic structure whose shape dramatically changes depending on whether or not it is embedded into the host epithelium. The trophozoite's most fragile zone is the area below the membrane fusion site at the epimerite base. The epimerite plasma membrane forms basal radial ribs which are involved in increasing its surface and strengthening the epimerite-host cell junction. FITC-phalloidin labelling demonstrated the presence of filamentous actin in trophozoites along with its accumulation at the epimerite base and in the apical end of the protomerite, as well as a patch accumulation of filamentous actin in the protomerite of maturing and mature trophozoites. Indirect immunofluorescence revealed the presence of myosin in the cortical zone of the epimerite and in the membrane fusion site area. The data obtained strongly suggest that these structures could facilitate the detachment of a mature trophozoite from the host epithelium. Supported by data presented herein and our previous observations, we propose a new hypothesis on the mechanism of trophozoite detachment from the host epithelium based on epimerite retraction into the protomerite. This is contrary to the commonly accepted hypothesis describing gradual epimerite constriction and subsequent separation facilitated by contractility of the membrane fusion site (osmiophilic ring).

• MeSH
• aktiny metabolismus   MeSH
• Apicomplexa fyziologie   ultrastruktura   MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• epitel parazitologie   ultrastruktura   MeSH
• faloidin MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva MeSH
• fúze membrán fyziologie   MeSH
• interakce hostitele a parazita fyziologie   MeSH
• larva parazitologie   MeSH
• mikroskopie elektronová rastrovací MeSH
• organely metabolismus   ultrastruktura   MeSH
• Tenebrio parazitologie   MeSH
• tkáňová distribuce MeSH
• trávicí systém parazitologie   MeSH
• trofozoiti fyziologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.

• MeSH
• bakteriální geny MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• barvení a značení * MeSH
• fluorescenční barviva * MeSH
• fluorescenční mikroskopie * MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• myši MeSH
• průtoková cytometrie MeSH
• Spirochaetales cytologie   genetika   metabolismus   MeSH
• spirochetové infekce mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH

Vulnerability of mitochondrial Complex I to oxidative stress determines an organism's lifespan, pace of aging, susceptibility to numerous diseases originating from oxidative stress and certain mitopathies. The mechanisms involved, however, are largely unknown. We used confocal microscopy and fluorescent probe MitoSOX to monitor superoxide production due to retarded forward electron transport in HEPG2 cell mitochondrial Complex I in situ. Matrix-released superoxide production, the un-dismuted surplus (J(m)) was low in glucose-cultivated cells, where an uncoupler (FCCP) reduced it to half. Rotenone caused a 5-fold J(m) increase (AC(50) 2 microM), which was attenuated by uncoupling, membrane potential (DeltaPsi(m)), and DeltapH-collapse, since addition of FCCP (IC(50) 55 nM), valinomycin, and nigericin prevented this increase. J(m) doubled after cultivation with galactose/glutamine (i.e. at obligatory oxidative phosphorylation). A hydrophobic amiloride that acts on the ND5 subunit and inhibits Complex I H(+) pumping enhanced J(m) and even countered the FCCP effect (AC(50) 0.3 microM). Consequently, we have revealed a new principle predicting that Complex I produces maximum superoxide only when both electron transport and H(+) pumping are retarded. H(+) pumping may be attenuated by high protonmotive force or inhibited by oxidative stress-related mutations of ND5 (ND2, ND4) subunit. We predict that in a vicious cycle, when oxidative stress leads to higher fraction of, e.g. mutated ND5 subunits, it will be accelerated more and more. Thus, inhibition of Complex I H(+) pumping, which leads to oxidative stress, appears to be a missing link in the theory of mitochondrial aging and in the etiology of diseases related to oxidative stress.

• MeSH
• financování organizované MeSH
• fluorescenční barviva metabolismus   MeSH
• fosforylace účinky léků   MeSH
• glukosa chemie   MeSH
• intracelulární prostor metabolismus   účinky léků   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nemoc MeSH
• oxidační stres účinky léků   MeSH
• respirační komplex I antagonisté a inhibitory   genetika   metabolismus   MeSH
• rotenon toxicita   MeSH
• rozpřahující látky toxicita   MeSH
• stárnutí MeSH
• superoxidy metabolismus   MeSH
• transport elektronů MeSH
• Check Tag
• lidé MeSH

Numerous association studies have been involved in studying the angiotensinogen (AGT) variants, AGT plasma levels and relations to cardiovascular diseases, such as hypertension, myocardial infarction, coronary heart disease. To investigate a role of AGT G(-6)A and M235T genetic variants for chronic heart failure (CHF) and advanced atherosclerosis (AA), a total of 240 patients with CHF and 200 patients with AA of the Czech origin were evaluated for the study. The study shows the role of polymorphism AGT G(-6)A in genetic background among advanced atherosclerosis patients and chronic heart failure patients (Pg=0.001). This difference was also observed in comparison of AA patients with subgroup of CHF with dilated cardiomyopathy (Pg=0.02; Pa=0.009), and ischemic heart disease (Pg=0.007). The greatest difference between triple-vessel disease and chronic heart failure groups was observed in frequency of GT haplotype (P<0.001) and GGMT associated genotype (P<0.001). Retrospectively, we found the same trend when the subgroups of CHF were compared to AA group (AA vs. IHD with CHF P<0.001; AA vs. DCM P<0.001). These results suggest AGT genetic variants as a risk factor for chronic heart failure compared to advanced atherosclerosis disease without heart failure, with a strong difference between IHD patients and chronic heart failure patients with ischemic heart disease, especially in haplotypes and associated genotypes. PMID:20945963[PubMed - in process] Free Article LinkOut - more resourcesFull Text SourcesInstitute of Physiology, Academy of Sciences of the Czech Republic, Prague - PDFEBSCO • Supplemental Content Related citations Association of two angiotensinogen gene polymorphisms, M235T and G(-6)A, with chronic heart failure. [Int J Cardiol. 2003] Association of two angiotensinogen gene polymorphisms, M235T and G(-6)A, with chronic heart failure. Goldbergova M, Spinarova L, Spinar J, Toman J, Vasku A, Vacha J. Int J Cardiol. 2003 Jun; 89(2-3):267-72. Association between variants in the genes for leptin, leptin receptor, and proopiomelanocortin with chronic heart failure in the Czech population. [Heart Vessels. 2009] Association between variants in the genes for leptin, leptin receptor, and proopiomelanocortin with chronic heart failure in the Czech population. Bienertová-Vasků JA, Spinarová L, Bienert P, Vasků A. Heart Vessels. 2009 Mar; 24(2):131-7. Epub 2009 Apr 1.Angiotensinogen M235T polymorphism is associated with plasma angiotensinogen and cardiovascular disease. [Am Heart J. 1999] Angiotensinogen M235T polymorphism is associated with plasma angiotensinogen and cardiovascular disease. Winkelmann BR, Russ AP, Nauck M, Klein B, Böhm BO, Maier V, Zotz R, Matheis G, Wolf A, Wieland H, et al. Am Heart J. 1999 Apr; 137(4 Pt 1):698-705. Renin-angiotensin system gene polymorphisms: assessment of the risk of coronary heart disease. [Kardiol Pol. 2003] Renin-angiotensin system gene polymorphisms: assessment of the risk of coronary heart disease. Buraczyńska M, Pijanowski Z, Spasiewicz D, Nowicka T, Sodolski T, Widomska - Czekajska T, Ksiazek A. Kardiol Pol. 2003 Jan; 58(1):1-9. Review A haplotype of the angiotensinogen gene is associated with hypertension in african americans. [Clin Exp Pharmacol Physiol. 2005] Review A haplotype of the angiotensinogen gene is associated with hypertension in african americans. Kumar A, Li Y, Patil S, Jain S. Clin Exp Pharmacol Physiol. 2005 May-Jun; 32(5-6):495-502. See reviews... See all... All links from this record Related Citations Calculated set of PubMed citations closely related to the selected article(s) retrieved using a word weight algorithm. Related articles are displayed in ranked order from most to least relevant, with the “linked from” citation displayed first.Gene Gene records that cite the current articles. Citations in Gene are added manually by NCBI or imported from outside public resources.Gene (GeneRIF) Gene records that have the current articles as Reference into Function citations (GeneRIFs). NLM staff reviewing the literature while indexing MEDLINE add GeneRIFs manually.HomoloGene HomoloGene clusters of homologous genes and sequences that cite the current articles. These are references on the Gene and sequence records in the HomoloGene entry.Nucleotide Primary database (GenBank) nucleotide records reported in the current articles as well as Reference Sequences (RefSeqs) that include the articles as references.

• MeSH
• angiotensinogen genetika   MeSH
• ateroskleróza genetika   MeSH
• dospělí MeSH
• financování organizované MeSH
• frekvence genu genetika   MeSH
• genotyp MeSH
• haplotypy MeSH
• hypertenze MeSH
• koronární nemoc genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• polymorfismus genetický MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• srdeční selhání genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH