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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.

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.

The steady-state and dynamic fluorescence spectral properties of 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) and several other naphthalene derivatives are summarized to illustrate their sensitivity to the polarity of the environment. Results obtained both in solvents of different polarity and in phospholipid vesicles in two phase states are presented. The emission red shift observed in polar solvents and in the phospholipid liquid-crystalline phase is explained on the basis of dipolar relaxation of solvent molecules surrounding the fluorescent naphthalene moiety of these probes. In phospholipid environments, experimental evidence is shown that excludes the intramolecular relative reorientation of the dimethylamino and carbonyl groups in the naphthalene and the reorientation of the entire fluorescent moiety. The solvent dipolar relaxation observed for LAURDAN and PRODAN in phospholipid bilayers has been attributed to a small number of water molecules present at the membrane interface. A comparison between LAURDAN emission in phospholipid vesicles prepared in D2O and in H2O is also presented. The definition and the derivation of the generalized polarization function are also discussed.

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.

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.

2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation. In bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). TheGP value can be used for the quantitation of each phase. The wavelength dependence of theGP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined.GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in natural membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 Å.

Membranes are complex biological systems that display heterogeneity at all spatial scales. At a molecular level, the heterogeneity arises from lipid and protein composition. At the cellular level, heterogeneity is due to membrane organization and large scale morphology. A quantitative evaluation of membrane heterogeneity at a microscopic level is very important for several fields of membrane studies. We have developed a method for the analysis of the decay of fluorescent membrane probes that can provide a quantity sensitive to membrane heterogeneity. This method is based on the analysis of the fluorescence decay using continuous lifetime distributions. The major challenge in the interpretation of the analysis results is in the identification, at a molecular level, of the mechanisms that influence the fluorescence decay. In this review we illustrate the principles of data analysis and we show examples of identification of the measured parameters with specific variables that affect membrane heterogeneity.

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%.

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.

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.

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.

Research reveals that 17-beta-estradiol slows down low-density lipoprotein (LDL) lipid peroxidation not by scavenging of free radicals but by altering the secondary structure and conformation of apoB-100. Data indicate that estradiol modifies the protein's interaction with the outer layer of LDL leading to an increase in oxidation resistance.

The fluorescence lifetimes of parinaric acid (PnA) isomers have been measured in pure solvents and in synthetic phospholipid membranes over a wide temperature range. In pure solvents, dodecane, ethanol, and cyclohexanol, the emission is well described by three exponential components of approximately 40, 12, and 2 ns. The preexponential factor of the long-lifetime component is quite small, and this component is neglected in the present study. The preexponential factors of the shorter lifetime components are strongly temperature dependent. Both cis- and trans-PnA show an inversion of the fractional contribution of these two components in a naroow temperature range. The inversion temperature is lower for trans-PnA than for cis-PnA, both in ethanol and in dodecane. The different temperature behavior of the decay of the two PnA isomers can influence their behavior in phospholipids. In L-α-dimyristoylphosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) membranes, the emission is more complex, and more than three components are necessary to describe the fluorescence decay accurately. On the basis of their different behavior in pure solvents, we anticipate that cis-PnA will detect the phospholipid transition in DMPC and DPPC at lower temperature. In fact, this effect has been observed, but it has been attributed to different partitioning of the two isomers in the phospholipid phases rather than to their different photophysics as we suggest here. © 1984, American Chemical Society. All rights reserved.

Abstract Background Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation 1. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. Methods In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. Results Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. Conclusion NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action.

Electronegative LDL(-) is spontaneously produced in vitro by plasma incubation through phospholipid hydrolysis catalyzed by the activity of endogeneous phospholipases and hence apoprotein misfolding is proposed as a possible antherogenic modification. The studies have shown that electronegative charge and misfolding are independent and distinct features of LDL(-) and apoprotein and misfolding rather than the increase in the negative charge has emerged both as a valid biomarker and as an appealing pharmacological and nutritional target.

Objective: To evaluate whether aorta balloon occlusion decreases the rate of hysterectomies and maternal morbidity during extirpative surgery of placenta previa accreta or increta. Methods: We prospectively assessed 33 consecutive patients with placenta praevia and MRI diagnosis of multifocal accreta or increta. Manual removal of the placenta was performed during a scheduled caesarean delivery. In 15 patients, surgery was preceded by balloon catheterization of the abdominal aorta (Intra Abdominal Balloon Occlusion: IABO); 18 patients refusing IABO were considered as controls. We used Fisher's exact test for categorical variables and Mann-Whitney test for continuous variables. Results: In the IABO group we observed significant decreases in incidence of hysterectomy, estimated blood loss, number of transfused units of red blood cells, postoperative stay and admission to Intensive Care Unit. No IABO-related complications were reported. Conclusions: During scheduled caesarean section for placenta previa multifocally accreta or increta, IABO can prevent hysterectomy in many cases and improves perioperative outcome as it gives the operator time to achieve the haemostasis via curettage and oversewing of the implantation site with acceptable blood loss. [ABSTRACT FROM AUTHOR]

Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo. [ABSTRACT FROM AUTHOR]

Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy. [ABSTRACT FROM AUTHOR]

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP. [ABSTRACT FROM AUTHOR]

In early atherogenesis, subendothelial retention of lipidic droplets is associated with an inflammatory response-to-injury, culminating in the formation of foam cells and plaque. Low density lipoprotein (LDL) is the main constituent of subendothelial lipidic droplets. The process is believed to occur following LDL modification. Searching for a modified LDL in plasma, electronegative LDL [LDL(-)] was identified and found to be associated with major risk biomarkers. The apoprotein in LDL(-) is misfolded, and we show here that this modification primes the aggregation of native LDL, conforming to the typical pattern of protein amyloidogenesis. After a lag phase, whose length depends on LDL(-) concentration, light scattering and atomic force microscopy reveal early exponential growth of intermediate globules, which evolve into fibrils. These globules are remarkably similar to subendothelial droplets in atheromatous lesions and different from those produced by oxidation or biochemical manipulation. During aggregation, ellipticity and tryptophan fluorescence measurements reveal a domino-style spread of apoprotein misfolding from LDL(-) to all of the LDL. Computational analysis of the apoprotein primary sequence predicts an unstable, aggregation-prone domain in the regulatory α2 region. Apoprotein misfolding well represents an LDL modification able to transform this cholesterol carrier into a trigger for a response-to-injury in the artery wall.--Parasassi, T., De Spirito, M., Mei, G., Brunelli, R., Greco, G., Lenzi, L., Maulucci, G., Nicolai, E., Papi, M., Arcovito, G., Tosatto, S. C. E., Ursini, F. Low density lipoprotein misfolding and amyloidogenesis. [ABSTRACT FROM AUTHOR]

In this study we report that apoptotic death of primary cultures of cerebellar granule neurons is accompanied by release of thioflavin-binding proteins – indicative of the presence of β-sheet structures – and fibril formation in the culture medium. When the same neurons are subjected to an excytotoxic death caused by 100 μM glutamate exposure, the amount of thioflavin binding is markedly reduced. Western blot analysis shows that fibrils contain monomers, dimers and trimers of amyloid-β (Aβ) which, when observed at the electron microscope, have morphologies reminiscent of fibrils of senile plaques. These findings demonstrate that triggering an apoptotic pathway leads to β-sheet transition and fibril formation of a protein primarily involved in Alzheimer's disease and may be of direct relevance to the possible link between apoptosis and this neuropathology. [ABSTRACT FROM AUTHOR]

Human cervical mucus is a heterogeneous mixture of mucin glycoproteins whose relative concentration changes during the ovulatory phases, thereby producing different mucus aggregation structures that can periodically permit the transit of spermatozoa for fertilization. In preovulatory phase, mucus is arranged in compact fiber-like structures where sperm transit is hindered. Previously, through observations made of fixed and dehydrated samples, a permissive structure in the ovulatory phase was attributed to the larger diameters of pores in the mucus network. Instead, by means of atomic force microscopy, we can show, for the first time, that unfixed ovulatory mucus is composed by floating globules of mucin aggregates. This finding sheds new light on the mechanism that governs spermatozoa transit toward the uterine cavity. In addition, we demonstrate that the switch from globular ovulatory to fibrous preovulatory mucus largely depends on a pH-driven mechanism. Analysis of mucin 5B primary sequence, the main mucin in ovulatory mucus, highlights pH-sensitive domains that are associated to flexible regions prone to drive aggregation. We suggest an involvement of these domains in the fiber-to-globule switch in cervical mucus. [ABSTRACT FROM AUTHOR]

Background: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. Methods: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. Results: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. Conclusion: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action. [ABSTRACT FROM AUTHOR]

We have investigated the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, using atomic force spectroscopy. The response of the membrane to mechanical stress has been reconstructed using a modified Hertz model. The transition from elastic behavior, which occurs when low stress forces are applied (characterized by a Young’s modulus E=(22±5) kPa), toward plastic behavior is observed. The critical indentation necessary to induce plastic deformations occurs at δyield=(550±50) nm. This high critical value, corresponding to two ZP layers, well supports the noncovalent long lifetimes of interactions that take place between constituent glycoproteins. [ABSTRACT FROM AUTHOR]

Based on our previous report on structural and conformational modifications of apoB-100 in the presence of 17β-estradiol (E2), we characterized the interaction between this hormone and the apoβ-100 and explored the induced alterations in terms of the structural arrangement of the whole LDL particle. We report evidence for the existence on the apoB-100 of a single specific and saturable binding site for E2, whose occupancy modifies the overall structure of the protein, inducing an increase in the α-helix fraction. [ABSTRACT FROM AUTHOR]

The zona pellucida (ZP) is a multilayer glycoprotein spherical shell surrounding mammalian eggs. The ZP's mechanical response plays a crucial role in mammalian fertilization and is a parameter commonly adopted in 'in vitro fertilization' to characterize the oocytes quality. While it is assumed that ZP mechanical response is purely elastic, here we prove that dissipative forces cannot be neglected. Physiologically, this evidence implies that an increase in the spermatozoa motility can induce dramatic changes on the ZP reaction force turning ZP shell in an impenetrable barrier leading to fertility impairments. [ABSTRACT FROM AUTHOR]

Seeking for a modified lipoprotein present in plasma that could account for the atherogenic effect of high cholesterol, several years ago electronegative LDL(-) was identified. The peculiar feature of LDL(-) is an apoprotein misfolding that triggers the formation of aggregates, perfectly fitting in size the subendothelial droplets observed in early phases of atherogenesis. Apoprotein misfolding was therefore proposed as a possible atherogenic modification. LDL(-) can be spontaneously produced in euro by plasma incubation through phospholipid hydrolysis catalyzed by the activity of endogenous phospholipases. As a consequence, apoprotein is misfolded. 17β-Estradiol (E2), a specific ligand to apoB-100, was used to unravel the relationship between negative charge of the lipoprotein and apoprotein structural/conformational shift. Although E2 addition to plasma does not prevent LDL(-) generation nor phospholipase activity, it deeply stabilizes apoB-lOO structure, thus preventing its structural and conformational shift. Apoprotein stabilization extends to lipids. Indeed, while a loosening of lipid packing is observed together with apoprotein misfolding, conversely, when E2 stabilizes apoprotein, lipid structure is preserved. Finally, even in the presence of LDL(-), the E2-stabilized LDL is resistant to aggregation, unambiguously demonstrating that misfolding, but not negative charge, primes aggregation. In conclusion, electronegative charge and misfolding are independent and distinct features of LDL(-), and apoprotein misfolding rather than the increase in the negative charge emerges both as a valid biomarker and as an appealing pharmacological and nutritional target. [ABSTRACT FROM AUTHOR]

Urged by the unmet medical needs in endometriosis treatment, often with undesirable side effects, and encouraged by N-acetylcysteine (NAC) efficacy in an animal model of endometriosis and by the virtual absence of toxicity of this natural compound, we performed an observational cohort study on ovarian endometriosis. NAC treatment or no treatment was offered to 92 consecutive Italian women referred to our university hospital with ultrasound confirmed diagnosis of ovarian endometriosis and scheduled to undergo laparoscopy 3 months later. According to patients acceptance or refusal, NAC-treated and untreated groups finally comprised 73 and 72 endometriomas, respectively. After 3 months, within NAC-treated patients cyst mean diameter was slightly reduced (-1.5 mm) versus a significant increase (+6.6 mm) in untreated patients (P = 0.001). Particularly, during NAC treatment, more cysts reduced and fewer cysts increased their size. Our results are better than those reported after hormonal treatments. Twenty-four NAC-treated patients-versus 1 within controls-cancelled scheduled laparoscopy due to cysts decrease/disappearance and/or relevant pain reduction (21 cases) or pregnancy (1 case). Eight pregnancies occurred in NAC-treated patients and 6 in untreated patients. We can conclude that NAC actually represents a simple effective treatment for endometriosis, without side effects, and a suitable approach for women desiring a pregnancy.

The zona pellucida (ZP) is an extracellular membrane surrounding mammalian oocytes. The so-called zona hardening plays a key role in fertilization process, as it blocks polyspermy, which may also be caused by an increase in the mechanical stiffness of the ZP membrane. However, structural reorganization mechanisms leading to ZP's biomechanical hardening are not fully understood yet. Furthermore, a correct estimate of the elastic properties of the ZP is still lacking. Therefore, the aim of the present study was to investigate the biomechanical behaviour of ZP membranes extracted from mature and fertilized bovine oocytes to better understand the mechanisms involved in the structural reorganization of the ZP that may lead to the biomechanical hardening of the ZP. For that purpose, a hybrid procedure is developed by combining atomic force microscopy nanoindentation measurements, nonlinear finite element analysis and nonlinear optimization. The proposed approach allows us to determine the biomechanical properties of the ZP more realistically than the classical analysis based on Hertz's contact theory, as it accounts for the nonlinearity of finite indentation process, hyperelastic behaviour and material heterogeneity. Experimental results show the presence of significant biomechanical hardening induced by the fertilization process. By comparing various hyperelastic constitutive models, it is found that the Arruda-Boyce eight-chain model best describes the biomechanical response of the ZP. Fertilization leads to an increase in the degree of heterogeneity of membrane elastic properties. The Young modulus changes sharply within a superficial layer whose thickness is related to the characteristic distance between cross-links in the ZP filamentous network. These findings support the hypothesis that biomechanical hardening of bovine ZP is caused by an increase in the number of inter-filaments cross-links whose density should be higher in the ZP inner side.

The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.