RESUMEN
The umbilical cord is a complex structure containing three vessels, one straight vein and two coiled arteries, encased by the Wharton Jelly (WJ) a spongy structure made of collagen and hydrated macromolecules. Fetal blood reaches the placenta through the arteries and flows back to the fetus through the vein. The role of the WJ in maintaining cord circulation proficiency and the ultimate reason for arterial coiling still lack of reasonable mechanistic interpretations. We performed biaxial tension tests and evidenced significant differences in the mechanical properties of the core and peripheral WJ. The core region, located between the arteries and the vein, resulted rather stiffer close to the fetus. Finite element modelling and optimization based inverse method were used to create 2D and 3D models of the cord and to simulate stress distribution in different hemodynamic conditions, compressive loads and arterial coiling. We recorded a facilitated stress transmission from the arteries to the vein through the soft core of periplacental WJ. This condition generates a pressure gradient that boosts the venous backflow circulation towards the fetus. Peripheral WJ allows arteries to act as pressure buffering chambers during the cardiac diastole and helps to dissipate compressive forces away from vessels. Altered WJ biomechanics may represent the structural basis of cord vulnerability in many high-risk clinical conditions.
Asunto(s)
Fenómenos Biomecánicos , Fuerza Compresiva , Cordón Umbilical/fisiología , Gelatina de Wharton/fisiología , Adulto , Algoritmos , Anisotropía , Colágeno/fisiología , Elasticidad , Femenino , Análisis de Elementos Finitos , Análisis de Fourier , Hemodinámica , Humanos , Imagenología Tridimensional , Sustancias Macromoleculares , Placenta/fisiología , Embarazo , Presión , Estrés Mecánico , Resistencia a la Tracción , Adulto JovenRESUMEN
The long quest for a missing mechanistic rationale accounting for the correlation between plasma cholesterol levels and cardiovascular disease (CVD) has been focused on various possible modifications of low density lipoprotein (LDL), turning this physiological cholesterol carrier into a damaging agent able to trigger atherogenesis and later the onset of the disease. In addition to the debated oxidized LDL (oxLDL), a modified LDL with a misfolded apoprotein B-100, called electronegative LDL(-) for its negative charge due to an increased amount of free fatty acids, is commonly present in plasma. LDL(-) is generated by the action of secretory calcium dependent phospholipase A2. LDL(-) primes LDL aggregation and amyloid formation according to mechanisms very similar to those observed in other misfolding diseases. The LDL particle aggregates recall the structure and size of the subendothelial lipid droplets described in early atherogenesis and elicit a powerful inflammatory response. The use of 17-ß-estradiol (E2) confirmed that the suggested atherogenicity of LDL (-) is mostly dependent on the misfolded character of its apoprotein. E2 binding to the apoprotein of native LDL, through a specific and saturable receptor, inhibits misfolding phenomenon despite an unaffected production of LDL (-) by phospholipase A2, ultimately preventing LDL aggregation. The apoprotein misfolding in LDL(-) emerges as a possible significant trigger mechanism of atherogenesis. Potential implications for the development of novel therapeutic approaches might be hypothesized in perspective. The existing evidence is discussed and reported in this review.
Asunto(s)
Apolipoproteínas B/metabolismo , Aterosclerosis/metabolismo , Estradiol/metabolismo , Lipoproteínas LDL/metabolismo , Pliegue de Proteína , Apolipoproteínas B/química , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/ultraestructura , Oxidación-ReducciónRESUMEN
Magnetic resonance imaging requested for a potentially serious indication, provided a unique opportunity to explore the intervillous circulation of placentas from pregnancies complicated by Intra Uterine Growth Restriction (IUGR) and to compare them to normal cases. This allowed an innovative characterization of in vivo utero-placental blood flow, correlating a compromised intervillous circulation in IUGR to the deterioration of fetal condition. MR imaging was requested to rule out suspected posterior placental adhesive disorders in 26 patients. Twelve patients had fetuses appropriate for gestational age, while in 14 patients fetuses were affected by severe IUGR. Multiphasic dynamic contrast-enhanced sagittal sequences were acquired and a quantitative analysis of signal intensity and enhancement kinetics was performed for both the entire placenta and for selected regions. Images disclosed a homogeneous perfusion overall the placenta in normal cases, while IUGR placentas displayed a slow intervillous blood flow, along with many patchy unperfused areas. Intermittent stops worsen the perfusion dynamics of the intervillous mostly in IUGR cases with an elevated ductus venosus pulsatility index. In conclusion, we proved that in IUGR placenta maternal placental blood flow is extremely compromised and that superimposed dynamic phenomena concur to worsen the intervillous circulation leading to an end-stage fetal decompensation.
Asunto(s)
Retardo del Crecimiento Fetal/fisiopatología , Circulación Placentaria , Adulto , Femenino , Humanos , Imagen por Resonancia Magnética , Placenta/fisiopatología , Embarazo , Estudios Retrospectivos , Adulto JovenRESUMEN
We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.
Asunto(s)
Diferenciación Celular/fisiología , Neoplasias del Colon/patología , Neoplasias Ováricas/patología , Compuestos de Sulfhidrilo/metabolismo , Acetilcisteína/farmacología , Proteína Tirosina Quinasa CSK , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Proteínas del Citoesqueleto/metabolismo , Femenino , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Neoplasias Ováricas/metabolismo , Fosfotransferasas/antagonistas & inhibidores , Fosfotransferasas/metabolismo , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timidina/metabolismo , Transactivadores , beta Catenina , Familia-src QuinasasRESUMEN
Photosensitization induced by the fluoroquinolone ofloxacin (OFLX) has been studied using two human cell lines, HL60 and K562, two UV wavelengths, 290 and 330 nm, and two different exposure protocols, acute and protracted. The examined endpoints are the cellular lethality and recovery and the membrane changes produced by the oxidative damage, studied using cloning and counting techniques and the measurement of the generalized polarization (GP) of the fluorescent membrane probe 2-dimethylamino-6-lauroyl-naphthalene (Laurdan). The results show that: (i) the photosensitizing effect is detectable at concentrations similar to those found in patients treated with OFLX only when the cells are irradiated with 330 nm; (ii) the amount of photodamage is a function of the drug concentration and of UV dose and persists also after the removal of the drug; (iii) during the first 24 h after OFLX treatment, a large decrease of the cell number can be observed due to cell lysis; (iv) the OFLX is inserted in the cell membranes at concentrations directly related with the drug concentration and incubation time; (v) the OFLX produces an increase in the GP values similar to that produced by membrane lipid oxidation which persists for hours after the removal of the drug. The overall results suggest the cell membrane as the main target of the OFLX adverse action, with a possible mechanism involving the formation of reactive oxygen species (ROS), which triggers, in turn, the lipid peroxidation chain reaction.
Asunto(s)
Antiinfecciosos/toxicidad , Peroxidación de Lípido , Ofloxacino/toxicidad , Rayos Ultravioleta/efectos adversos , Muerte Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/patología , Eritrocitos , Células HL-60 , Humanos , Estrés Oxidativo , Trastornos por FotosensibilidadRESUMEN
A subpopulation of low-density lipoproteins (LDL) is present in human plasma that contains lipid hydroperoxides and is more negatively charged (LDL(-)) than normal native LDL. By circular dichroism and tryptophan lifetime measurements we found that apoB-100 secondary structure is markedly decreased and its conformation is severely altered in LDL(-). The low tryptophan fluorescence intensity confirms the oxidative degradation of the lipoprotein, and the very long lifetime value of one of its decay components indicates a low polarity environment for the remaining unbleached residues. Either a peculiar folding or, most likely, a sinking of the apoB-100 into the lipid core can account for the observed long lifetime component. Oxidation in vitro produces a similar unfolding of the apolipoprotein but the lifetime of tryptophan fluorescence is shifted to lower values, indicating that the denatured apoprotein remains at the hydrophilic surface of the lipoprotein particle. A disordering and an increased polarity of the LDL(-) surface lipids was demonstrated by measuring the generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). The looser monolayer packing apparently favors the new conformation of apoB-100 and its sinking into a more hydrophobic environment, possibly accounting for it reduced receptor binding properties.
Asunto(s)
2-Naftilamina/análogos & derivados , Apolipoproteínas B/química , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , 2-Naftilamina/química , Adulto , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Dicroismo Circular , Colorantes Fluorescentes/química , Humanos , Peróxido de Hidrógeno/química , Lauratos/química , Conformación Proteica , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Triptófano/química , Venas/fisiologíaRESUMEN
The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase model for cholesterol-containing membranes and show that the presence of cholesterol, in addition to modification to the phase state of the hydrophobic portion of the bilayer, strongly affects the packing and the polarity of the membrane hydrophobic-hydrophilic interface.
Asunto(s)
2-Naftilamina/análogos & derivados , Colesterol/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , 1,2-Dipalmitoilfosfatidilcolina , Colorantes Fluorescentes , Lauratos , Microscopía Fluorescente , Propiedades de Superficie , TemperaturaRESUMEN
Among different proposed mechanisms to account for the protection exerted by estrogens against cardiovascular diseases, the antioxidant effect has attracted considerable attention. We confirmed that 17-beta-estradiol (E2), when added to human LDL at a 6:1 ratio to apoB-100, markedly delays the phase of massive LDL lipid peroxidation induced by Cu(2+). We also observed an increased oxidative resistance of E2-treated LDL by monitoring the early phase of oxidative degradation on the basis of increased LDL surface polarity by the generalized polarization of the lipophilic fluorescent probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan). A scavenging of free radicals by E2 is ruled out since, consistent with its structure, its rate constant for the reduction of peroxy radicals is extremely low, i.e., 0.02% of that of vitamin E. Tryptophan fluorescence lifetime and circular dichroism measurements revealed that (i) apoB-100 undergoes a conformational modification and a progressive loss of secondary structure during lipid peroxidation; (ii) E2 increases apoB-100 secondary structure and modifies its conformation; and (iii) the apoB-100 conformational change induced by E2 makes this protein resistant to modifications brought about by lipid peroxidation. We propose that E2, by affecting apoB-100 secondary structure and conformation, modifies the interaction of this protein with the outer layer of the LDL particle thus increasing its overall oxidative resistance.
Asunto(s)
2-Naftilamina/análogos & derivados , Apolipoproteínas B/química , Estradiol/química , Peroxidación de Lípido , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , 2-Naftilamina/química , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacología , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Dicroismo Circular , Estradiol/metabolismo , Estradiol/farmacología , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/metabolismo , Humanos , Lauratos/química , Peroxidación de Lípido/efectos de los fármacos , Conformación Proteica , Espectrometría de Fluorescencia , Triptófano/químicaRESUMEN
Oxidatively modified LDL mimics several aspects of atherogenesis. In this disease, degradation of the matrix proteins' network also occurs. By a new morphological ex vivo approach, not requiring sample processing, we explored the relationship between the degradation of matrix protein and oxidatively modified LDL. Two-photon excitation fluorescence microscopy images of fresh cross-section rings of rat aorta, acquired while the sample was maintained in a glucose- and oxygen-supplemented buffer, showed straight, parallel, thick, long extracellular matrix proteins. Traditional microscopic examination, requiring sample fixation and staining, shows smaller and curved fibers. Instead, we observed curved and broken fibers after a 30-min incubation of aorta with either LDL containing lipid hydroperoxides, or tert-butyl-hydroperoxide. The adhesion of LDL to the endothelium and its internalization was directly visualized by using a lipid fluorophore. The damage to aorta matrix proteins induced by LDL and tert-butyl-hydroperoxide was fully prevented by antioxidants, such as ascorbate or Trolox C, or inhibitors of proteases. The image spectroscopy of the fibers' autofluorescence (polarization and lifetime) revealed an increased mobility of the fluorescent cross-link in fibers. Damaged matrix proteins were also imaged in aorta samples from apolipoprotein E knock-out mice. Our ex vivo images directly visualized the activation of a fast redox-sensitive proteolytic process in the arterial wall triggered by lipid hydroperoxides in LDL.
Asunto(s)
Aorta/efectos de los fármacos , Aorta/patología , Lipoproteínas LDL/toxicidad , Animales , Aorta/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Endopeptidasas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Técnicas In Vitro , Peróxidos Lipídicos/toxicidad , Masculino , Ratones , Ratones Noqueados , Microscopía Fluorescente/métodos , Oxidación-Reducción , Ratas , terc-Butilhidroperóxido/toxicidadRESUMEN
Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics--in terms of lamellarity and morphology--of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicle's preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene (LAURDAN) and using the LAURDAN generalized polarization function we show that the lipid packing in these tethers or tubes is similar to those found in the phospholipid vesicles. The fact that both vesicles and tethers are found in the lipid preparations indicates similar stability between these structures.
Asunto(s)
Fosfolípidos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Microscopía Electrónica , Microscopía Fluorescente , Fosfatidilcolinas/química , Fotones , Solventes/química , Suspensiones , Temperatura , Agua/metabolismoRESUMEN
Although 6-lauroyl-2-(N,N-dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase-transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well-resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN dimethylamino group and the ester linkage of ester phospholipids is responsible for the strong stabilization of LAURDAN's red excitation band in the gel phase of ester phospholipid vesicles. We discuss the consequence of these proposed ground-state interactions on LAURDAN's emission generalized polarization function. In the context of variable excitation wavelengths, information concerning solvent dipolar relaxation through excitation generalized polarization function is also discussed.
Asunto(s)
2-Naftilamina/análogos & derivados , Colorantes Fluorescentes/química , Lauratos/química , 2-Naftilamina/química , Técnicas In Vitro , Lípidos/química , Liposomas , Modelos Químicos , Fotoquímica , Espectrometría de FluorescenciaAsunto(s)
Colorantes Fluorescentes/análisis , Procesamiento de Imagen Asistido por Computador , Lípidos de la Membrana/química , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Animales , Células Cultivadas , Corteza Renal/ultraestructura , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Microvellosidades/ultraestructura , Modelos Biológicos , Zarigüeyas , Fosfolípidos/químicaRESUMEN
PURPOSE: To investigate the production and repair of lipid oxidative damage in two human cell lines exposed to acute and fractionated dose of ionizing radiation. Radiation dose was in the range from 0.1 to 44 Gy. MATERIALS AND METHODS: K562 and HL60 human cell lines have been used, 24 and 96 h after seeding. Membrane lipid oxidative damage has been detected by the measurement of the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), its polarization value and the conjugated dienes concentration. The modification of DPH decay has been previously reported to be directly related to the lipid hydroperoxide concentration. RESULTS: A modification of the DPH decay has been observed as a linear function of the logarithm of the radiation dose and only when the irradiation was performed in the presence of oxygen. The amount of the damage is related to the time after the cell medium change. By exposing the cells to fractionated radiation doses for several days (10 cGy day(-1)), the oxidative damage has been found to be cumulative. After a single acute dose, evidence of repair of the lipid oxidative damage was not obtained. CONCLUSIONS: Following a previously developed method, the membrane damage was attributed to the production of hydroperoxide residues in the lipid acyl chains with the consequence of water penetration into the external portion of the bilayer, from the aqueous environment to the position of hydroperoxides. This damage is not repaired. The results obtained by measuring the DPH fluorescence decay have been compared with those obtained using other current optical and biochemical methods. None of these techniques could detect membrane oxidative damage at doses < 10 Gy. Finally, the different sensitivity of 'young' and 'old' cells to the oxidative damage can be related to different cholesterol concentrations.
Asunto(s)
Peroxidación de Lípido , Lípidos de la Membrana/efectos de la radiación , Membrana Celular/efectos de la radiación , Difenilhexatrieno/metabolismo , Relación Dosis-Respuesta en la Radiación , Colorantes Fluorescentes/metabolismo , Células HL-60/efectos de la radiación , Humanos , Peróxido de Hidrógeno/metabolismo , Células K562/efectos de la radiación , Lípidos de la Membrana/metabolismo , Oxidación-ReducciónRESUMEN
Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.
Asunto(s)
2-Naftilamina/análogos & derivados , Colorantes Fluorescentes , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Fenómenos Biofísicos , Biofisica , Cristalización , Lauratos , Espectrometría de Fluorescencia , Propiedades de Superficie , Temperatura , Agua/químicaRESUMEN
The generalized polarization function of the fluorescent probe 2-dimethylamino-6-lauroylnaphthalene has been used to evaluate the lipid dynamics in Friend erythroleukemia cell membrane. The values of this function varied during the culture growth cycle, showing decreased lipid dynamics 24-48 h from the cell seeding. When the cycle occurred in a solenoid producing a magnetic field of 70 microT at 50 Hz in addition to the 45 microT DC of the earth (short-term 4-day exposure), the membrane lipid dynamics during this same time-period decreased by about 10% (P < .04). After long-term (184 days) or extremely long-term (395 days) exposure of the cells to the magnetic field, little additional variation in the membrane lipid dynamics was observed, suggesting an adaptation phenomenon. A variation of membrane lipid dynamics was also observed due to in vitro cell differentiation (P < .02). Nevertheless, the exposure of both undifferentiating and differentiating cells to a highly attenuated magnetic field in a magnetically shielded room (20 nT DC plus 2.5 pT AC) did not induce any modification of membrane lipid dynamics.
Asunto(s)
Campos Electromagnéticos , Ambiente Controlado , Lípidos de la Membrana/metabolismo , Animales , Ciclo Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , División Celular/efectos de la radiación , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Conductividad Eléctrica , Campos Electromagnéticos/efectos adversos , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Lípidos de la Membrana/efectos de la radiación , Ratones , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
In a cell culture model of aging we have previously shown that there is an age-related decrease in the lipid dynamics of the proerythropoetic K562 cell membranes, as determined by the generalized polarization (GP) of the phase-sensitive lipid probe 2-dimethylamino-6-lauroylnaphthalene (Laurdan) (T. Parasassi, M. Di Stefano, G. Ravagnan, O. Sapora and E. Gratton. Exp. Cell Res., 202 (1992) 432-439). In the present study we also extended our observations to the lymphoblastoid HL60 cell line. In both K562 and HL60 cells during the four days after the last cell culture medium renewal the GP Laurdan value increased in a linear fashion indicating a time-dependent decrease in lipid dynamics. The initial membrane physical properties were almost completely restored upon renewal of the cell culture medium. We measured lipid composition, including individual and total phospholipids, free and esterified cholesterol at the first ('young') and at the fourth ('aged') day after culture medium renewal. We found that the decreased membrane lipid 'fluidity' at the fourth day of cell growth was associated with a 40% increase in cholesterol concentration in both cell lines. This increase in cholesterol concentration was reversible 24 h following the culture medium change. We conclude that in K562 and HL60 cells the 'age-related' decrease in membrane lipid dynamics is mediated by an 'age-related' increase in cell cholesterol content.
Asunto(s)
Envejecimiento/metabolismo , Colesterol/metabolismo , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , División Celular , Membrana Celular/metabolismo , Senescencia Celular , Ésteres del Colesterol/metabolismo , Colorantes Fluorescentes/química , Células HL-60 , Humanos , Lauratos/química , Modelos Biológicos , Fosfolípidos/metabolismo , Células Tumorales CultivadasRESUMEN
Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.
Asunto(s)
Membrana Celular/química , Membranas Artificiales , Microscopía Fluorescente/métodos , 2-Naftilamina/análogos & derivados , Animales , Fenómenos Biofísicos , Biofisica , Línea Celular , Colesterol/química , Cristalización , Membrana Eritrocítica/química , Polarización de Fluorescencia , Colorantes Fluorescentes , Geles , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Riñón/química , Lauratos , Liposomas/química , Lípidos de la Membrana/química , Zarigüeyas , Ratas , TermodinámicaRESUMEN
The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.
Asunto(s)
Colesterol/farmacología , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/efectos de la radiación , Estrés Oxidativo , Fosfolípidos/metabolismo , Difenilhexatrieno/química , Fluorescencia , Radicales Libres , Peróxido de Hidrógeno/metabolismo , Mediciones Luminiscentes , Oxidación-Reducción , FosfatidilcolinasRESUMEN
The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.