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T Parasassi

Publications and source records attributed to T Parasassi.

At least 37 records · Page 2Linked to original sources
PubMed1994

Evidence for an increase in water concentration in bilayers after oxidative damage of phospholipids induced by ionizing radiation.

The two membrane fluorescent probes 2-dimethyl-amino-6-lauroyl-naphthalene (Laurdan) and 2-dimethylamino-6-propionyl-naphthalene (Prodan) have been used to study the molecular basis of the damage induced in phospholipid membranes by ionizing radiation. Laurdan and Prodan display a spectral sensitivity to the polarity of their environment, showing a red shift of both excitation and emission spectra with increase of the polarity of their environment. Owing to their chemical differences, the two probes are anchored in the membrane with different strengths. In aqueous environments Laurdan is not fluorescent while Prodan shows appreciable fluorescence. Laurdan and Prodan show an opposite response to oxidative damage produced in phospholipid bilayers by ionizing radiation. The results support the model recently developed of water penetration in the bilayer as a consequence of oxidative damage.

Lipid Bilayers↗
PubMed1994

Hoechst 33258 staining for detecting mycoplasma contamination in cell cultures: a method for reducing fluorescence photobleaching.

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.

3T3 Cells↗
PubMed1993

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.

2-Naphthylamine↗
PubMed1993

Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence.

Steady-state and dynamic fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl- and dipalmitoyl-phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time-resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Membrane aging during cell growth ascertained by Laurdan generalized polarization.

The sensitivity of the fluorescent probe Laurdan to the phase state of lipids has been utilized to detect modifications in the composition and physical state of cell membranes during cell growth. In phospholipid vesicles, the Laurdan emission spectrum shows a 50-nm red shift by passing from the gel to the liquid-crystalline phase. The Generalized Polarization (GP) value has been used for the data treatment instead of the ratiometric method common in investigations utilizing other fluorescent probes that display spectral sensitivity to medium properties. The GP value can be measured easily and quickly and possesses all the properties of "classical" polarization, including the additivity rule. Once Laurdan limiting GP values have been established for the gel and the liquid-crystalline phase of lipids, the quantitative determination of coexisting phases in natural samples is possible. In the present work the observation of a relevant decrease in the fractional intensity of the liquid-crystalline phase in K562 cell membranes during 5 days of asynchronous growth is reported. A decrease in the "fluidity" of cell membranes in K562 cells kept in culture for several months is also reported. The procedure developed for labeling cell membranes with Laurdan is reported and the influence of cell metabolism on fluorescence parameters is discussed. Also discussed is the influence of cholesterol on Laurdan GP.

2-Naphthylamine↗
PubMed1992

Membrane oxidative damage induced by ionizing radiation detected by diphenylhexatriene fluorescence lifetime distributions.

The sensitivity of the fluorescence lifetime of 1,6-diphenyl- 1,3,5-hexatriene (DPH) to the dielectric constant of its environment has been used to detect oxidative damage to phospholipid membranes induced by ionizing radiation. The DPH fluorescence decay in phospholipid vesicles is described well by a continuous distribution of lifetime values, reflecting the various DPH depths in the bilayer and related to the gradient of the dielectric constant. Ionizing radiation oxidizes unsaturated acyl residues of phospholipids, altering the dielectric constant across the bilayer, sharpening the distribution of DPH lifetimes and increasing the centre of the distribution. Ionizing radiation doses between 22 and 110 Gy were used, and were effective only in the presence of oxygen. A model based on the formation of packing defects in the bilayer describes the phenomenon.

Diphenylhexatriene↗
PubMed1991

Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence.

The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.

2-Naphthylamine↗
PubMed1991

A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in pure solvents and in phospholipid vesicles has been measured using frequency domain fluorometry. Data analysis uses a model with two energetically close excited states. The model explains the high quantum yield and the double exponential decay of DPH observed in some pure solvents and in phospholipid vesicles. This model assumes that after excitation to a first excited state, there is a rapid interconversion to a lower excited state and that most of the emission occurs from this state. The interconversion rates between the two excited states determine the average lifetime. For DPH in solvents, we find that the interconversion rates are solvent and temperature dependent. For DPH in phospholipid vesicles, we find that the back reaction rate from excited state 2 to excited state 1 (R12) is what determines the fluorescence properties. The phospholipid phase transition affects only this back reaction rate. The model was analyzed globally for a range of solvents, temperatures and vesicle composition. Of the six parameters of the model, only two, the interconversion rates between the two excited states, varied in all different samples examined. For DPH in phospholipid vesicles, there is an additional feature of the model, which is related to the apparent distribution of the rate R12. Significantly better fits were obtained using a continuous lorentzian distribution of interconversion rates. The resulting lifetime distribution was asymmetric and showed a definite narrowing above the phase transition.

Diphenylhexatriene↗
PubMed1991

Alterations in erythrocyte membrane lipids induced by low doses of ionizing radiation as revealed by 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime.

Damage in membrane lipids induced by low doses of ionizing radiation in the presence of oxygen has been detected in rabbit erythrocyte ghosts labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH). Multifrequency phase and modulation fluorometry was used to measure DPH fluorescence lifetime. This technique is particularly suited for the observation of heterogeneous fluorescence decays. DPH decay in erythrocyte membranes is described by a two-component continuous distribution of lifetimes. The value of the distribution width of the long-lived component is found to be affected by radiation-induced membrane lipid damage at doses as low as 0.5 Gy, well within the dose range used to measure cell survival. The width of the DPH lifetime distribution decreases when the ghosts are irradiated in the presence of oxygen. Such a decrease is a linear function of the logarithm of the dose. After a dose of 110 Gy and above, the fractional intensity of the short-lived component of the DPH decay increases linearly, indicating severe membrane damage. Experiments performed in the absence of oxygen do not show any change in the fluorescence parameters up to a dose of 550 Gy. The molecular identification of the produced damage has not been accomplished, but the necessity of oxygen to observe the damage suggests that hydroperoxides and lipids crosslinks are produced.

Animals↗
PubMed1990

Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence.

The organization of lipids surrounding membrane proteins can influence their properties. We have used 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) to study phase coexistence and phase interconversion in membrane model systems. The fluorescence properties of Laurdan provide a unique possibility to study lipid domains because of the different excitation and emission spectra of this probe in the gel and in the liquid-crystalline phase. The difference in excitation spectra allows photoselection of Laurdan molecules in one of the two phases. Using the difference in emission spectra it is then possible to observe interconversion between the two phases. We have performed experiments in dipalmitoyl-phosphatidylcholine (DPPC) vesicles at different temperatures, in particular in the region of the phase transition, where phase coexistence and interconversion between phases is likely to be maximal. We have also studied vesicles of different lipids and mixtures dilauroyl-phosphatidylcholine (DLPC), DPPC, and 50% DLPC in DPPC. Both steady-state fluorescence intensity and polarization data have been collected. To quantitate phase coexistence and interconversion we have introduced the concept of "generalized polarization." We have also performed time-resolved experiments to directly prove the interconversion process. We have found that in DLPC-DPPC mixtures, at 20 degrees C, phase interconversion occurs in approximately 30-40 ns.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Abscisic acid-induced microheterogeneity in phospholipid vesicle: a fluorescence study.

Changes in the thermal behavior of DMPC (dimyristoyl-r-phosphatidylcholine) and an equimolar mixture of DMPC and DMPE (dimyristoyl-L-phosphatidylethanolamine) induced by the plant hormone abscisic acid (ABA) have been investigated using fluorescent probes. The fluorescence decay of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in these vesicles has been measured using frequency-domain fluorometry, and has been analyzed using both models of discrete exponential components and continuous lifetime distributions. In the DMPC vesicles, using the distributional approach, higher center and width values were observed in the presence of abscisic acid (ABA), indicating a decrease in the dielectric constant of the lipid phase that we attribute to a decrease in the water concentration within the bilayer. Moreover, the presence of ABA in the liposomes increased the phospholipid phase transition temperature. The addition of ABA to the DMPC/DMPE mixture strongly increased the microheterogeneity of the system as reported by the FWHM (full-width at half-maximum) of the distributional approach.

Journal Article↗
PubMed1990

Abscisic acid-induced microheterogeneity in phospholipid vesicles. A fluorescence study.

Changes in the thermal behavior of DMPC (dimyristoyl-L-phosphatidylcholine) and an equimolar mixture of DMPC and DMPE (dimyristoyl-L-phosphatidylethanolamine) induced by the plant hormone abscisic acid (ABA) have been investigated using fluorescent probes. The fluorescence decay of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in these vesicles has been measured using frequency-domain fluorometry, and has been analyzed using both models of discrete exponential components and continuous lifetime distributions. In the DMPC vesicles, using the distributional approach, higher center and width values were observed in the presence of abscisic acid (ABA), indicating a decrease in the dielectric constant of the lipid phase that we attribute to a decrease in the water concentration within the bilayer. Moreover, the presence of ABA in the liposomes increased the phospholipid phase transition temperature. The addition of ABA to the DMPC/DMPE mixture strongly increased the microheterogeneity of the system as reported by the FWHM (full-width at half-maximum) of the distributional approach.

Abscisic Acid↗
PubMed1987

Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry.

The fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) in K562 cell membranes has been studied using multifrequency phase and modulation fluorimetry. The DPH decay data collected at various modulation frequencies were analysed by assuming either a model of discrete exponential components or a model of continuous lifetime distribution. The fits showed smaller values of the reduced chi square using the model of continuous lifetime distribution. The K562 cell membranes dynamics were investigated during the cell differentiation along the erythroid pathway. By using the continuous lifetime distribution method for the analysis of the DPH decay, marked variations were observed during the four initial days of the erythroid differentiation. Namely, the width of the DPH lifetime distribution increased by a factor of about two, while the center value of the distribution remained constant. By using the discrete exponential components model for the analysis of the DPH decay no variations were observed during the K562 differentiation.

Cell Differentiation↗
PubMed1984

Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence.

Using multifrequency phase and modulation fluorometry and a nonlinear least-squares analysis of lifetime data, we were able to determine the complex decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in synthetic phospholipid bilayers. Our results showed a monoexponential decay of DPH in the pure isotropic solvents studied, over a wide temperature range, and a double-exponential decay of DPH in phospholipids, both above and below the transition. During the transition, and in mixed-phase phospholipids, a three-component analysis was successfully accomplished, and the pre-exponential factors of the two main components have been shown to be quantitatively representative of the gel and liquid-crystalline phases of the bilayer. The fractional intensity of the shorter lifetime component depends on the modalities of the sample preparation. The factors affecting this component are discussed. From the DPH fluorescence lifetime and from the anisotropy data in L-alpha-dimyristoyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidyl choline mixtures, a phase diagram was independently constructed. Conclusions about the sensitivity and the partition of the probe between gel and the liquid-crystalline phases of the bilayer are derived. Lifetime experiments on DPH in a L-alpha-dilauroyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidylch oline mixture suggested a general method for the determination and quantitation of the two different phases in the bilayer.

Dimyristoylphosphatidylcholine↗
PubMed1984

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids.

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.

Cell Aggregation↗