RESUMEN
Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts.
Asunto(s)
Membrana Celular/química , Colesterol/química , Células Epiteliales/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , Cromatografía Liquida , Células Epiteliales/metabolismo , Humanos , Membrana Dobles de Lípidos/metabolismo , Membranas Artificiales , Imitación Molecular , Fosfolípidos/clasificación , Fosfolípidos/metabolismo , Epitelio Pigmentado de la Retina/química , Epitelio Pigmentado de la Retina/metabolismo , Espectrometría de Masas en TándemRESUMEN
Fourier Transform Infrared (FTIR) microspectroscopy was used to highlight the interactions between two photosensitisers (PS) of different geometries, TPPmOH4 and a glycoconjugated analogous, TPPDegMan, and lipid bilayers modelling retinoblastoma cell membranes. Retinoblastoma is a rare disease occurring in young infants, for whom conservative treatments may present harmful side-effects. Photodynamic therapy (PDT) is expected to induce less side-effects, as the photosensitiser is only activated when the tumour is illuminated. Since efficiency of the treatment relies on photosensitiser penetration in cancer cells, bilayers with three lipid compositions - pure SOPC, SOPC/SOPE/SOPS/Chol (56:23:11:10) and SOPC/SOPE/SOPS/Chol/CL (42:32:9:8:6) - were used as plasma and mitochondria model membranes. FTIR spectra showed that the interaction of the PSs with the lipid bilayers impacted the lipid organization of the latter, causing significant spectral variations. Both studied photosensitisers inserted at the level of lipid hydrophobic chains, increasing chain fluidity and disorder. This was confirmed by surface pressure measurements. Photosensitisers - TPPmOH4 more than TPPDegMan - also interacted with the polar region of the bilayer, forming hydrogen bonds with phosphate groups that induced major shifts of phosphate absorption bands. This difference in PS interaction with moieties in the polar region was more pronounced with the models with complex lipid composition.
Asunto(s)
Membrana Dobles de Lípidos/química , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Neoplasias de la Retina/radioterapia , Retinoblastoma/radioterapia , Antineoplásicos/farmacología , Membrana Celular/química , Humanos , Estructura Molecular , Fosfolípidos/química , Fotoquimioterapia , Espectroscopía Infrarroja por Transformada de Fourier , Tensión SuperficialRESUMEN
Hearing loss is the most frequent sensory disorder. It affects 3 in 1000 newborns. It is genetically heterogeneous with 60 causally-related genes identified to date. Mutations in GJB2 gene account for half of all cases of non-syndromic deafness. The aim of this study was to determine the relative frequency of GJB2 allele variants in Tunisia. In this study, we screened 138 patients with congenital hearing loss belonging to 131 families originating from different parts of Tunisia for mutations in GJB2 gene. GJB2 mutations were found in 39% of families (51/131). The most common mutation was c.35delG accounting for 35% of all cases (46/131). The second most frequent mutation was p.E47X present in 3.8% of families. Four identified mutations in our cohort have not been reported in Tunisia; p.V37I, c.235delC, p.G130A and the splice site mutation IVS1+1G>A (0.76%). These previously described mutations were detected only in families originating from Northern and not from other geographical regions in Tunisia. In conclusion we have confirmed the high frequency of c.35delG in Tunisia which represents 85.4% of all GJB2 mutant alleles. We have also extended the mutational spectrum of GJB2 gene in Tunisia and revealed a more pronounced allelic heterogeneity in the North compared to the rest of the country.