Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using (31)P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.
- MeSH
- apoptóza fyziologie MeSH
- diferenciální skenovací kalorimetrie MeSH
- Escherichia coli MeSH
- fluorescenční barviva MeSH
- fosfolipidy metabolismus MeSH
- kardiolipiny metabolismus MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondrie metabolismus MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- oxidace-redukce MeSH
- oxidační stres fyziologie MeSH
- permeabilita buněčné membrány MeSH
- protein X asociovaný s bcl-2 metabolismus MeSH
- teplota MeSH
- unilamelární lipozómy chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2.
- MeSH
- fibroblastový růstový faktor 2 sekrece MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- heparitinsulfát metabolismus MeSH
- membránové transportní proteiny metabolismus MeSH
- multimerizace proteinu * MeSH
- sekreční vezikuly metabolismus MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cholesterol is important for the formation of microdomains in supported lipid bilayers and is enriched in the liquid-ordered phase. To understand the interactions leading to this enrichment, we developed an AFM-based single-lipid-extraction (SLX) approach that enables us to determine the anchoring strength of cholesterol in the two phases of a phase-separated lipid membrane. As expected, the forces necessary for extracting a single cholesterol molecule from liquid-ordered phases are significantly higher than for extracting it from the liquid-disordered phases. Interestingly, application of the Bell model shows two energy barriers that correlate with the head and full length of the cholesterol molecule. The resulting lifetimes for complete extraction are 90 s and 11 s in the liquid-ordered and liquid-disordered phases, respectively. Molecular dynamics simulations of the very same experiment show similar force profiles and indicate that the stabilization of cholesterol in the liquid-ordered phase is mainly due to nonpolar contacts.
- MeSH
- cholesterol chemie MeSH
- fosfatidylcholiny chemie MeSH
- fosfatidylethanolaminy chemie MeSH
- lipidové dvojvrstvy chemie MeSH
- mikroskopie atomárních sil metody MeSH
- simulace molekulární dynamiky MeSH
- spektrální analýza MeSH
- unilamelární lipozómy chemie MeSH
- vodíková vazba MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An investigation was made of the adhesion, growth and differentiation of osteoblast-like MG-63 and Saos-2 cells on titanium (Ti) and niobium (Nb) supports and on TiNb alloy with surfaces oxidized at 165°C under hydrothermal conditions and at 600°C in a stream of air. The oxidation mode and the chemical composition of the samples tuned the morphology, topography and distribution of the charge on their surfaces, which enabled us to evaluate the importance of these material characteristics in the interaction of the cells with the sample surface. Numbers of adhered MG-63 and Saos-2 cells correlated with the number of positively-charged (related with the Nb2O5 phase) and negatively-charged sites (related with the TiO2 phase) on the alloy surface. Proliferation of these cells is correlated with the presence of positively-charged (i.e. basic) sites of the Nb2O5 alloy phase, while cell differentiation is correlated with negatively-charged (acidic) sites of the TiO2 alloy phase. The number of charged sites and adhered cells was substantially higher on the alloy sample oxidized at 600°C than on the hydrothermally treated sample at 165°C. The expression values of osteoblast differentiation markers (collagen type I and osteocalcin) were higher for cells grown on the Ti samples than for those grown on the TiNb samples. This was more particularly apparent in the samples treated at 165°C. No considerable immune activation of murine macrophage-like RAW 264.7 cells on the tested samples was found. The secretion of TNF-α by these cells into the cell culture media was much lower than for either cells grown in the presence of bacterial lipopolysaccharide, or untreated control samples. Thus, oxidized Ti and TiNb are both promising materials for bone implantation; TiNb for applications where bone cell proliferation is desirable, and Ti for induction of osteogenic cell differentiation.
- MeSH
- biologické markery metabolismus MeSH
- buněčná adheze účinky léků MeSH
- buněčná diferenciace účinky léků MeSH
- buněčné linie MeSH
- kolagen typu I metabolismus MeSH
- lidé MeSH
- lipopolysacharidy farmakologie MeSH
- makrofágy cytologie účinky léků metabolismus MeSH
- myši MeSH
- osteoblasty cytologie účinky léků metabolismus MeSH
- osteokalcin metabolismus MeSH
- oxidace-redukce MeSH
- povrchové vlastnosti MeSH
- proliferace buněk účinky léků MeSH
- slitiny chemie farmakologie MeSH
- statická elektřina MeSH
- tkáňové podpůrné struktury * MeSH
- TNF-alfa farmakologie MeSH
- vysoká teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Large number of extracellular signals is received by plasma membrane receptors which, upon activation, transduce information into the target cell interior via trimeric G-proteins (GPCRs) and induce activation or inhibition of adenylyl cyclase enzyme activity (AC). Receptors for opioid drugs such as morphine (micro-OR, delta-OR and kappa-OR) belong to rhodopsin family of GPCRs. Our recent results indicated a specific up-regulation of AC I (8-fold) and AC II (2.5-fold) in plasma membranes (PM) isolated from rat brain cortex exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. Increase of ACI and ACII represented the specific effect as the amount of ACIII-ACIX, prototypical PM marker Na, K-ATPase and trimeric G-protein alpha and beta subunits was unchanged. The up-regulation of ACI and ACII faded away after 20 days since the last dose of morphine. Proteomic analysis of these PM indicated that the brain cortex of morphine-treated animals cannot be regarded as being adapted to this drug because significant up-regulation of proteins functionally related to oxidative stress and alteration of brain energy metabolism occurred. The number of delta-OR was increased 2-fold and their sensitivity to monovalent cations was altered. Characterization of delta-OR-G-protein coupling in model HEK293 cell line indicated high ability of lithium to support affinity of delta-OR response to agonist stimulation. Our studies of PM structure and function in context with desensitization of GPCRs action were extended by data indicating participation of cholesterol-enriched membrane domains in agonist-specific internalization of delta-OR. In HEK293 cells stably expressing delta-OR-G(i)1alpha fusion protein, depletion of PM cholesterol was associated with the decrease in affinity of G-protein response to agonist stimulation, whereas maximum response was unchanged. Hydrophobic interior of isolated PM became more "fluid", chaotically organized and accessible to water molecules. Validity of this conclusion was supported by the analysis of an immediate PM environment of cholesterol molecules in living delta-OR-G(i)1alpha-HEK293 cells by fluorescent probes 22- and 25-NBD-cholesterol. The alteration of plasma membrane structure by cholesterol depletion made the membrane more hydrated. Understanding of the positive and negative feedback regulatory loops among different OR-initiated signaling cascades (micro-, delta-, and kappa-OR) is crucial for understanding of the long-term mechanisms of drug addiction as the decrease in functional activity of micro-OR may be compensated by increase of delta-OR and/or kappa-OR signaling.
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- fluidita membrány fyziologie MeSH
- HEK293 buňky MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- membránové lipidy metabolismus MeSH
- mozková kůra metabolismus ultrastruktura MeSH
- opioidní analgetika metabolismus MeSH
- receptory opiátové metabolismus MeSH
- signální transdukce fyziologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
PURPOSE OF REVIEW: To summarize the recent progress made in noninvasive monitoring of volatile compounds in exhaled breath and above biological liquids, as they are becoming increasingly important in assessing the nutritional and clinical status and beginning to provide support to conventional clinical diagnostics and therapy. To indicate the potential of these developments in medicine and the specific areas which are currently under investigation. RECENT FINDINGS: The significance of the following breath gases and their concentrations are reported: acetone and the influence of diet; ammonia confirmed as an indicator of dialysis efficacy; hydrogen and the (13)CO(2)/(12)CO(2) ratio (following the ingestion of (13)C-labeled compounds) as related to gastric emptying and bowel transit times; hydrogen cyanide released by Pseudomonas and its detection in breath of children with cystic fibrosis; and multiple trace compounds in breath of patients with specific pathophysiological conditions and 'metabolic profiling'. SUMMARY: Advanced analytical methods, especially exploiting mass spectrometry, are moving breath analysis towards the clinical setting; some trace gas metabolites are already being exploited in diagnosis and therapy. Much effort is being given to the search for biomarkers of tumours in the body. HCN as an indicator of the presence of Pseudomonas in the airways has real potential in therapeutically alleviating the symptoms of cystic fibrosis.
- MeSH
- aceton metabolismus MeSH
- amoniak metabolismus MeSH
- dechové testy metody MeSH
- dýchací soustava metabolismus MeSH
- hmotnostní spektrometrie MeSH
- kyanovodík metabolismus MeSH
- lidé MeSH
- nutriční stav MeSH
- oxid uhličitý metabolismus MeSH
- stopové prvky metabolismus MeSH
- těkavé organické sloučeniny metabolismus MeSH
- vodík metabolismus MeSH
- zdravotní stav MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.
- MeSH
- antibiotika antitumorózní metabolismus farmakologie MeSH
- buněčné jádro metabolismus MeSH
- buňky 3T3 MeSH
- DNA metabolismus MeSH
- doxorubicin analogy a deriváty metabolismus farmakologie MeSH
- fluorescenční spektrometrie MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- lymfom T-buněčný farmakoterapie metabolismus MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- polymery metabolismus MeSH
- průtoková cytometrie MeSH
- systémy cílené aplikace léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.
- MeSH
- biofyzika metody MeSH
- buněčná membrána metabolismus MeSH
- design vybavení MeSH
- fluorescenční barviva MeSH
- konfokální mikroskopie metody přístrojové vybavení MeSH
- lasery MeSH
- membránové transportní proteiny metabolismus MeSH
- normální rozdělení MeSH
- optika a fotonika MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- světlo MeSH
- terciární struktura proteinů MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- práce podpořená grantem MeSH
The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.
BACKGROUND: Fluorescence correlation spectroscopy (FCS) can be used for the determination of diffusion coefficients of single molecules. Since diffusion coefficients are correlated with size and shape of the labeled species, FCS provides information on conformational changes in plasmids aggregates. METHODS: A 10-kbp plasmid stained with PicoGreen was condensed by spermine or liposomes formulated from cationic lipid and egg phosphatidylcholine. RESULTS: The diffusion coefficient of DNA increases from 1.0 x 10(-12) m2/s to 3.2 x 10(-12) m2/s by the addition of spermine, whereas the addition of cationic liposomes leads to complexes characterized by diffusion coefficients with values ranging from 1.7 to 1.9 x 10(-12) m2/s. CONCLUSIONS: FCS experiments allow determining the diffusion coefficients of DNA-containing aggregates which provide information regarding the topology and homogeneity of the aggregate.
