Giant unilamellar vesicles (GUV) are widely used cell membrane models. GUVs have a cell-like diameter and contain the same phospholipids that constitute cell membranes. The most frequently used protocol to obtain these vesicles is termed electroformation, since key steps of this protocol consist in the application of an electric field to a phospholipid deposit. The potential oxidation of unsaturated phospholipids due to the application of an electric field has not yet been considered even though the presence of oxidized lipids in the membrane of GUVs could impact their permeability and their mechanical properties. Thanks to mass spectrometry analyses, we demonstrated that the electroformation technique can cause the oxidation of polyunsaturated phospholipids constituting the vesicles. Then, using flow cytometry, we showed that the amplitude and the duration of the electric field impact the number and the size of the vesicles. According to our results, the oxidation level of the phospholipids increases with their level of unsaturation as well as with the amplitude and the duration of the electric field. However, when the level of lipid oxidation exceeds 25 %, the diameter of the vesicles is decreased and when the level of lipid oxidation reaches 40 %, the vesicles burst or reorganize and their rate of production is reduced. In conclusion, the classical electroformation method should always be optimized, as a function of the phospholipid used, especially for producing giant liposomes of polyunsaturated phospholipids to be used as a cell membrane model.
Abstract. Mercury, Lead and Cadmium were determined in 100 samples of human breast milk samples from urban and rural mothers in Isfahan (IRAN). A questionnaire about area of residence, nutrition, smoking habits, and dental fillings was filled out by the lactating mothers. The combination of nitric acid, hydrogen peroxide and perchloric acid was found to be one of the most suitable acids in wet digestion of milk. Cold vapor atomic absorption was used to determine the mercury content in milk after wet digestion. The effect of concentration of nitric acid, influence of flow rate and tin(П) chloride were investigated. The mean concentration of mercury in human breast milk samples was 0.96 ppb. Extraction of Pb and Cd were performed with ammonium pyrrolidine dithiocarbamate (APDC) to methyl isobutyl ketone (MIBK) and were determined by Flame Atomic Absorption Spectrometry. The factors influencing, the complex formation, pH, time and buffer were optimized. The mean concentration of Pb and Cd in human breast milk was 0.0147 and 0.0121 ppm, respectively. The maximum concentrations were found in breast milk of rural mothers.
The induction of heat shock response in the macula has been proposed as a useful therapeutic strategy for retinal neurodegenerative diseases by promoting proteostasis and enhancing protective chaperone mechanisms. We applied transpupillary 1064 nm long-duration laser heating to the mouse (C57Bl/6J) fundus to examine the heat shock response in vivo. The intensity and spatial distribution of heat shock protein (HSP) 70 expression along with the concomitant probability for damage were measured 24 h after laser irradiation in the mouse retinal pigment epithelium (RPE) as a function of laser power. Our results show that the range of heating powers for producing heat shock response while avoiding damage in the mouse RPE is narrow. At powers of 64 and 70 mW, HSP70 immunostaining indicates 90 and 100% probability for clearly elevated HSP expression while the corresponding probability for damage is 20 and 33%, respectively. Tunel staining identified the apoptotic regions, and the estimated 50% damaging threshold probability for the heating (ED50) was ~72 mW. The staining with Bestrophin1 (BEST1) demonstrated RPE cell atrophy with the most intense powers. Consequently, fundus heating with a long-duration laser provides an approachable method to develop heat shock-based therapies for the RPE of retinal disease model mice.
To maintain homeostasis, cells have evolved stress-response pathways to cope with exogenous and endogenous stress factors. Diverse stresses at high doses may be detrimental, albeit low doses of stress, known as hormesis, can be beneficial. Upon exposure to stress, such as temperature rise, the conventional heat shock response (HSR) regulated by the heat shock transcription factor 1 (HSF1) facilitates refolding of misfolded proteins with the help of heat shock proteins (HSPs). However, the role and molecular mechanisms underlying the beneficial effects of HSR with other clearance processes, such as autophagy, remain poorly understood. In this study, human ARPE-19 cells, an in vitro model of retinal pigment epithelium, were treated with hormetic heat shock (HHS) and the autophagy expression profile was examined using quantitative PCR (qPCR), immunoblotting, immunoprecipitation, and immunofluorescence. We demonstrate that HHS enhances the expression of fundamental autophagy-associated genes in ARPE-19 cells through the activation of HSF1. HHS transiently increases the level of SQSTM1 and LC3B-II and activates autophagy. These findings reveal a role for autophagic HSF1-regulated functions and demonstrate the contribution of autophagy to hormesis in the HSR by improving proteostasis.
In order to obtain molecular level insight into the biophysics of the apoptosis promoting phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) we studied its partitioning into different lipid phases by isothermal titration calorimetry (ITC). To aid the interpretation of these data for PazePC, we additionally characterized by both ITC and fluorescence spectroscopy the fluorescent phospholipid analog 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-C6-PC), which similarly to PazePC can adopt extended conformation in lipid bilayers. With the NBD-hexanoyl chain reversing its direction and extending into the aqueous space out of the bilayer, 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) becomes accessible to the water soluble dithionite, which reduces to non-fluorescent product. Our results suggest that these phospholipid derivatives first partition and penetrate into the outer bilayer leaflet of liquid disordered phase liposomes composed of unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Upon increase up to 2 mol% PazePC and NBD-C6-PC of the overall content, flip-flop from the outer into the inner bilayer leaflet commences. Interestingly, the presence of 40 mol% cholesterol in POPC liposomes did not abrogate the partitioning of PazePC into the liquid ordered phase. In contrast, only insignificant partitioning of PazePC and NBD-C6-PC into sphingomyelin/cholesterol liposomes was evident, highlighting a specific membrane permeability barrier function of this particular lipid composition against oxidatively truncated PazePC, thus emphasizing the importance of detailed characterization of the biophysical properties of membranes found in different cellular organelles, in terms of providing barriers for lipid-mediated cellular signals in processes such as apoptosis. Our data suggest NBD-C6-PC to represent useful fluorescent probe to study the cellular dynamics of oxidized phospholipid species, such as PazePC.
Boosting natural mechanisms to retain cellular homeostasis and combat oxidative stress by inducing a hormetic heat shock to retinal pigment epithelium with non-damaging transpupillary laser heating, i.e., with subthreshold laser treatment (SLT), has been suggested as a promising therapeutic target for many retinal diseases, including age-related macular degeneration. However, the therapeutic temperature window for the treatments is narrow and thermal dosimetry is unavailable. Here we introduce an SLT modality where the retinal temperature is monitored with electroretinography (ERG)-based thermal dosimetry and demonstrate its feasibility with anesthetized pigs. In 60-second treatments with 810 nm laser, the ED50 peak temperature for visible lesion generation was 48 °C and the relative temperature determination error was below 10% from the temperature increase. Heat shock protein expression increased, and autophagy was activated at 44.2 °C and no signs of oxidative stress or apoptosis emerged at 44.2 °C or 46.5 °C. The demonstrated method permits a controlled activation of intracellular chaperones and waste clearance in RPE cell with a clear temperature margin for adverse events. In the clinical setting, ERG-based dosimetry would allow safe and personalized SLTs for retinal diseases currently lacking effective treatments.
Purpose Impaired protein degradation and increased protein aggregation in retinal pigment epithelium (RPE) cells associate with the age‐related macular degeneration (AMD) pathology. Heat shock proteins (Hsps) refold misfolded proteins and attempt to prevent the accumulation of detrimental cytoplasmic protein aggregates. Once Hsps capacity is exceeded the protein aggregates are secondarily degraded by autophagy or proteasomes. The Hsps and clearance mechanisms are important to prevent RPE damage and AMD development. We showed recently that stilbenoid polyphenol, pinosylvin has strong cytoprotective power against oxidative stress in ARPE‐19 cells. Here, we investigated the effects of pinosylvin and/or heat shock on the regulation of autophagy in ARPE‐19 cells in the protein aggregation model. Methods ARPE‐19 cells were solely or simultaneously treated with 15 µM pinosylvin and/or heat shock at 42˚C for 30 min with or without proteasome inhibitor MG‐132 (1 µM) or autophagy inhibitor bafilomycin A1 (50 nM) and followed up to 48 h. Autophagy markers p62/SQSTM1 (sequestosome 1), LC3 (microtubule‐associated protein 1A/1B‐light chain 3) and Hsp70 (heat shock protein 70) were analyzed by Western blotting (WB). Cytotoxicity was analysed by LDH assay. Results Inhibition of proteasomes with MG‐132 or heat shock highly upregulated Hsp70, p62 and LC3 detected in WB. Simultaneous treatment with pinosylvin and heat shock showed stronger autophagy induction and cytoprotection rather than theirs single treatments during proteasome inhibition. Conclusions Combination of polyphenolic pinosylvin and thermotherapy may provide improved autophagy and cytoprotective response in RPE cells.
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