Ceramides are key components of the skin's permeability barrier. In atopic dermatitis, pathological hydrolysis of ceramide precursors - glucosylceramides and sphingomyelin - into lysosphingolipids, specifically glucosylsphingosine (GS) and sphingosine-phosphorylcholine (SPC), and free fatty acids (FFAs) has been proposed to contribute to impaired skin barrier function. This study investigated whether replacing ceramides with lysosphingolipids and FFAs in skin lipid barrier models would exacerbate barrier dysfunction. When applied topically to human stratum corneum sheets, SPC and GS increased water loss, decreased electrical impedance, and slightly disordered lipid chains. In lipid models containing isolated human stratum corneum ceramides, reducing ceramides by ≥ 30% significantly increased permeability to four markers, likely due to loss of long-periodicity phase (LPP) lamellae and phase separation within the lipid matrix, as revealed by X-ray diffraction and infrared spectroscopy. However, when the missing ceramides were replaced by lysosphingolipids and FFAs, no further increase in permeability was observed. Conversely, these molecules partially mitigated the negative effects of ceramide deficiency, particularly with 5%-10% SPC, which reduced permeability even compared to control with "healthy" lipid composition. These findings suggest that while ceramide deficiency is a key factor in skin barrier dysfunction, the presence of lysosphingolipids and FFAs does not aggravate lipid structural or functional damage, but may provide partial compensation, raising further questions about the behavior of lyso(sphingo)lipids in rigid multilamellar lipid environments, such as the stratum corneum, that warrant further investigation.
- MeSH
- biologické modely MeSH
- ceramidy * metabolismus MeSH
- fosforylcholin analogy a deriváty MeSH
- kůže * metabolismus MeSH
- kyseliny mastné neesterifikované metabolismus MeSH
- lidé MeSH
- lysofosfolipidy metabolismus MeSH
- permeabilita účinky léků MeSH
- sfingosin analogy a deriváty metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common in colon cancer. Numerous Akt-targeted therapies are being developed, the activity of Akt-inhibitors is, however, strongly pH-dependent. Combination therapy thus represents an opportunity to increase their efficacy. In this study, the cytotoxicity of the Akt inhibitor perifosine and the Bcl-2/Bcl-xL inhibitor ABT-737 was tested in colon cancer HT-29 and HCT-116 cells cultured in monolayer or in the form of spheroids. The efficacy of single drugs and their combination was analysed in different tumour-specific environments including acidosis and hypoxia using a series of viability assays. Changes in protein content and distribution were determined by immunoblotting and a "peeling analysis" of immunohistochemical signals. While the cytotoxicity of single agents was influenced by the tumour-specific microenvironment, perifosine and ABT-737 in combination synergistically induced apoptosis in cells cultured in both 2D and 3D independently on pH and oxygen level. Thus, the combined therapy of perifosine and ABT-737 could be considered as a potential treatment strategy for colon cancer.
- MeSH
- apoptóza MeSH
- fosfatidylinositol-3-kinasy MeSH
- fosforylcholin * analogy a deriváty farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie účinky léků MeSH
- nádorové mikroprostředí MeSH
- nádory tračníku * farmakoterapie MeSH
- protinádorové látky * farmakologie MeSH
- protoonkogenní proteiny c-akt metabolismus MeSH
- synergismus léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. METHODS: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. RESULTS: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. CONCLUSION: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.
- MeSH
- antiprotozoální látky farmakologie MeSH
- fosforylcholin analogy a deriváty farmakologie MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania infantum * účinky léků genetika patogenita MeSH
- leishmanióza viscerální farmakoterapie patologie MeSH
- léková rezistence genetika MeSH
- membránové transportní proteiny genetika MeSH
- myši inbrední BALB C parazitologie MeSH
- myši MeSH
- Phlebotomus parazitologie MeSH
- protozoální geny MeSH
- Psychodidae parazitologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVES: To gain insight into the propagation of miltefosine (MIL) resistance in visceral leishmaniasis, this laboratory study explored development of resistant parasites with a defective miltefosine transporter (MT) in sand flies. METHODS: Infectivity, colonization of stomodeal valve and metacyclogenesis of a MIL-resistant (MIL-R) Leishmania infantum LEM3323 line with a defective MT were assessed in the natural sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis. Given our recent description of partial drug dependency of the MT-deficient line, the impact of MIL pre-exposure on sand fly infectivity was explored as well. RESULTS: A significant reduction in sand fly infection, stomodeal valve colonization and differentiation into metacyclics (determined by a lower flagellum/cell body length ratio) was observed in both vectors for MIL-R as compared to the isogenic parent MIL-susceptible line. Re-introduction of the wildtype MT gene into MIL-R tended to partially rescue the capacity to infect sand flies. Pre-exposure to MIL did not alter infectivity of the MIL-R line. CONCLUSION: The MIL resistant L. infantum LEM3323 line is significantly hampered in its development and transmissibility potential in two sand fly vector species. Additional studies are warranted to evaluate whether this applies to other visceral Leishmania parasites with acquired MIL-resistance.
- MeSH
- analýza rozptylu MeSH
- antiprotozoální látky farmakologie MeSH
- fosforylcholin analogy a deriváty farmakologie MeSH
- hmyz - vektory parazitologie MeSH
- inhibiční koncentrace 50 MeSH
- králíci MeSH
- Leishmania infantum účinky léků růst a vývoj MeSH
- leishmanióza viscerální farmakoterapie parazitologie přenos MeSH
- léková rezistence MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- Phlebotomus parazitologie MeSH
- Psychodidae parazitologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In an effort to delineate how cholesterol protects membrane structure under oxidative stress conditions, we monitored the changes to the structure of lipid bilayers comprising 30 mol% cholesterol and an increasing concentration of Class B oxidized 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) glycerophospholipids, namely, 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), using atomistic molecular dynamics simulations. Increasing the content of oxidized phospholipids (oxPLs) from 0 to 60 mol% oxPL resulted in a characteristic reduction in bilayer thickness and increase in area per lipid, thereby increasing the exposure of the membrane hydrophobic region to water. However, cholesterol was observed to help reduce water injury by moving into the bilayer core and forming more hydrogen bonds with the oxPLs. Cholesterol also resists altering its tilt angle, helping to maintain membrane integrity. Water that enters the 1-nm-thick core region remains part of the bulk water on either side of the bilayer, with relatively few water molecules able to traverse through the bilayer. In cholesterol-rich membranes, the bilayer does not form pores at concentrations of 60 mol% oxPL as was shown in previous simulations in the absence of cholesterol.
- MeSH
- cholesterol chemie metabolismus MeSH
- fosfatidylcholiny chemie metabolismus MeSH
- fosfolipidy chemie metabolismus MeSH
- fosforylcholin analogy a deriváty chemie metabolismus MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The formation and properties of supported lipid bilayers (SLB) containing hydrophobic nanoparticles (NP) was studied in relation to underlying cushion obtained from selected polyelectrolyte multilayers. Lipid vesicles were formed from zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in phosphate buffer (PBS). As hydrophobic nanoparticles - quantum dots (QD) with size of 3.8nm (emission wavelength of 420nm) were used. Polyelectrolyte multilayers (PEM) were constructed by the sequential, i.e., layer-by-layer (LbL) adsorption of alternately charged polyelectrolytes from their solutions. Liposomes and Liposome-QDs complexes were studied with Transmission Cryo-Electron Microscopy (Cryo-TEM) to verify the quality of vesicles and the position of QD within lipid bilayer. Deposition of liposomes and liposomes with quantum dots on polyelectrolyte films was studied in situ using quartz crystal microbalance with dissipation (QCM-D) technique. The fluorescence emission spectra were analyzed for both: suspension of liposomes with nanoparticles and for supported lipid bilayers containing QD on PEM. It was demonstrated that quantum dots are located in the hydrophobic part of lipid bilayer. Moreover, we proved that such QD-modified liposomes formed supported lipid bilayers and their final structure depended on the type of underlying cushion.
Acid sphingomyelinase deficiency (ASMd, Niemann-Pick disease A/B) and Niemann-Pick type C disease (NPC) share core clinical symptoms. Initial diagnostic discrimination of these two rare lysosomal storage diseases is thus difficult. As sphingomyelin accumulates in ASMd as well as NPC, lysosphingomyelin (sphingosylphosphorylcholine) and its m/z 509 analog were suggested as biomarkers for both diseases. Herein we present results of simultaneous LC-ESI-MS/MS measurements of lysosphingomyelin and lysosphingomyelin 509 in plasma and dried blood spots (DBS) collected from ASMd and NPC patients and suggest that the plasma but not DBS levels of the two analytes allow differential biochemical screening of ASMd and NPC.
- MeSH
- biologické markery krev MeSH
- chromatografie kapalinová metody MeSH
- fosforylcholin analogy a deriváty krev MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lidé MeSH
- Niemannova-Pickova nemoc typu A krev diagnóza MeSH
- Niemannova-Pickova nemoc typu B krev diagnóza MeSH
- Niemannova-Pickova nemoc typu C krev diagnóza MeSH
- sfingosin analogy a deriváty krev MeSH
- studie případů a kontrol MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- test suché kapky krve metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.
- MeSH
- diferenciální skenovací kalorimetrie MeSH
- fosforylcholin analogy a deriváty metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie s uhlíkem 13C MeSH
- mitochondriální membrány metabolismus MeSH
- oxidace-redukce MeSH
- permeabilita MeSH
- protein X asociovaný s bcl-2 metabolismus MeSH
- protonová magnetická rezonanční spektroskopie MeSH
- unilamelární lipozómy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The infection of mice with the wild-type (WT) strain of Y. pseudotuberculosis did not induce polyclonal activation of B lymphocytes. Suppression in the production of certain isotypes of Ig was observed, provoked mainly by YopH, YopJ and YpkA. The WT strain induced a progressive increase in the serum-specific IgG, which peaked after 4 weeks after infection, IgM being produced only after 1 week. Autoantibodies against phosphorylcholine, myelin, thyroglobulin and cardiolipin could be detected in the serum of mice infected with the WT strain. The infection of mice provoked suppression in the production of immunoglobulins by splenic B cells and that YopH, YopJ and YpkA must be involved here.
- MeSH
- autoprotilátky imunologie krev MeSH
- bakteriální proteiny imunologie MeSH
- financování organizované MeSH
- fosforylcholin imunologie MeSH
- imunoglobulin G imunologie krev MeSH
- imunoglobulin M imunologie krev MeSH
- infekce vyvolané Yersinia pseudotuberculosis imunologie MeSH
- kardiolipiny imunologie MeSH
- myelinová pochva imunologie MeSH
- myši MeSH
- protein-serin-threoninkinasy imunologie MeSH
- proteiny vnější bakteriální membrány imunologie MeSH
- protilátky bakteriální imunologie krev MeSH
- specificita protilátek MeSH
- thyreoglobulin imunologie MeSH
- tyrosinfosfatasy imunologie MeSH
- Yersinia pseudotuberculosis imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.
- MeSH
- chromatografie afinitní metody MeSH
- chromatografie kapalinová metody MeSH
- elektroforéza kapilární metody MeSH
- financování organizované MeSH
- fosforylcholin chemie MeSH
- heparin chemie MeSH
- lidé MeSH
- protein - isoformy analýza MeSH
- proteiny semenné plazmy analýza MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
