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S100B is an astrocytic cytokine that has been shown to be involved in several neurodegenerative diseases. We used an astrocytoma cell line (U373 MG) silenced for S100B, and stimulated it with amyloid beta-peptide (Aβ) as a known paradigm factor for astrocyte activation, and showed that the ability of the cell (including the gene machinery) to express S100B is a prerequisite for inducing reactive astrocytic features, such as ROS generation, NOS activation and cytotoxicity. Our results showed that control astrocytoma cell line exhibited overexpression of S100B after Aβ treatment, and subsequently cytotoxicity, increased ROS generation and NOS activation. In contrast, cells silenced with S100B were essentially protected, consistently reducing cell death, significantly decreasing oxygen radical generation and nitric oxide synthase activity. The conclusive aim of the present study was to show a causative linkage between the cell expression of S100B and induction of astrocyte activation processes, such as cytotoxicity, ROS and NOS activation.

Diabetes-induced oxidative stress induces the development of vascular complications, which are significant causes of morbidity and mortality in diabetic patients. Among these, diabetic retinopathy (DR) is often caused by functional changes in the blood-retinal barrier (BRB) due to harmful oxidative stress events in lipids, proteins, and DNA. Docosahexaenoic acid (DHA) has a potential therapeutic effect against hyperglycemia-induced oxidative damage and apoptotic pathways in the main constituents of BRB, retinal pigment epithelium cells (ARPE-19). Effective antioxidant response elicited by DHA is driven by the activation of the Nrf2/Nqo1 signaling cascade, which leads to the formation of NADH, a reductive agent found in the cytoplasm. Nrf2 also induces the expression of genes encoding enzymes involved in lipid metabolism. This study, therefore, aims at investigating the modulation of lipid metabolism induced by high-glucose (HG) on ARPE-19 cells through the integration of metabolic imaging and molecular biology to provide a comprehensive functional and molecular characterization of the mechanisms activated in the disease, as well the therapeutic role of DHA. This study shows that HG augments RPE metabolic processes by enhancing lipid metabolism, from fatty acid uptake and turnover to lipid biosynthesis and β-oxidation. DHA exerts its beneficial effect by ameliorating lipid metabolism and reducing the increased ROS production under HG conditions. This investigation may provide novel insight for formulating novel treatments for DR by targeting lipid metabolism pathways.

Diabetes-induced oxidative stress leads to the onset of vascular complications, which are major causes of disability and death in diabetic patients. Among these, diabetic retinopathy (DR) often arises from functional alterations of the blood-retinal barrier (BRB) due to damaging oxidative stress reactions in lipids, proteins, and DNA. This study aimed to investigate the impact of the ω3-polyunsaturated docosahexaenoic acid (DHA) on the regulation of redox homeostasis in the human retinal pigment epithelial (RPE) cell line (ARPE-19) under hyperglycemic-like conditions. The present results show that the treatment with DHA under high-glucose conditions activated erythroid 2-related factor Nrf2, which orchestrates the activation of cellular antioxidant pathways and ultimately inhibits apoptosis. This process was accompanied by a marked increase in the expression of NADH (Nicotinamide Adenine Dinucleotide plus Hydrogen) Quinone Oxidoreductase 1 (Nqo1), which is correlated with a contextual modulation and intracellular re-organization of the NAD+/NADH redox balance. This investigation of the mechanisms underlying the impairment induced by high levels of glucose on redox homeostasis of the BRB and the subsequent recovery provided by DHA provides both a powerful indicator for the detection of RPE cell impairment as well as a potential metabolic therapeutic target for the early intervention in its treatment.

Idebenone is a ubiquinone short-chain synthetic analog with antioxidant properties, which is believed to restore mitochondrial ATP synthesis. As such, idebenone is investigated in numerous clinical trials for diseases of mitochondrial aetiology and it is authorized as a drug for the treatment of Leber's hereditary optic neuropathy. Mitochondria of retinal pigment epithelium (RPE) are particularly vulnerable to oxidative damage associated with cellular senescence. Therefore, the aim of this study was to explore idebenone's cytoprotective effect and its underlying mechanism. We used a human-RPE cell line (ARPE-19) exposed to idebenone pre-treatment for 24 h followed by conditions inducing H 2 O 2 oxidative damage for a further 24 h. We found that idebenone: (a) ameliorated H 2 O 2 -lowered cell viability in the RPE culture; (b) activated Nrf2 signaling pathway by promoting Nrf2 nuclear translocation; (c) increased Bcl-2 protein levels, leaving unmodified those of Bax, thereby reducing the Bax/Bcl-2 ratio; (d) maintained the mitochondrial membrane potential (ΔΨ m ) at physiological levels, preserving the functionality of mitochondrial respiratory complexes and counteracting the excessive production of ROS; and (e) reduced mitochondrial cytochrome C-mediated caspase-3 activity. Taken together, our findings show that idebenone protects RPE from oxidative damage by modulating the intrinsic mitochondrial pathway of apoptosis, suggesting its possible role in retinal epitheliopathies associated with mitochondrial dysfunction.

S100B is an astrocytic protein behaving at high concentration as a damage-associated molecular pattern molecule. A direct correlation between the increased amount of S100B and inflammatory processes has been demonstrated, and in particular, the inhibitor of S100B activity pentamidine has been shown to ameliorate clinical scores and neuropathologic-biomolecular parameters in the relapsing-remitting experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. This study investigates the effect of arundic acid (AA), a known inhibitor of astrocytic S100B synthesis, in the chronic experimental autoimmune encephalomyelitis, which is another mouse model of multiple sclerosis usually studied. By the daily evaluation of clinical scores and neuropathologic-molecular analysis performed in the spinal cord, we observed that the AA-treated group showed lower severity compared to the vehicle-treated mice, particularly in the early phase of disease onset. We also observed a significant reduction of astrocytosis, demyelination, immune infiltrates, proinflammatory cytokines expression and enzymatic oxidative reactivity in the AA-treated group. Overall, our results reinforce the involvement of S100B in the development of animal models of multiple sclerosis and propose AA targeting the S100B protein as a focused potential drug to be considered for multiple sclerosis treatment.

In the current work, a series of novel 4-benzyloxy and 4-(2-phenylethoxy) chalcone fibrate hybrids (10a-o) and (11a-e) were synthesized and evaluated as new PPARα agonists in order to find new agents with higher activity and fewer side effects. The 2-propanoic acid derivative 10a and the 2-butanoic acid congener 10i showed the best overall PPARα agonistic activity showing E max % values of 50.80 and 90.55%, respectively, and EC 50 values of 8.9 and 25.0 μM, respectively, compared to fenofibric acid with E max  = 100% and EC 50  = 23.22 μM, respectively. These two compounds also stimulated carnitine palmitoyltransferase 1A gene transcription in HepG2 cells and PPARα protein expression. Molecular docking simulations were performed for the newly synthesized compounds to study their predicted binding pattern and energies in PPARα active site to rationalize their promising activity. In vivo, compounds 10a and 10i elicited a significant hypolipidemic activity improving the lipid profile in triton WR-1339-induced hyperlipidemic rats, including serum triglycerides, total cholesterol, LDL, HDL and VLDL levels. Compound 10i possessed better anti-hyperlipidemic activity than 10a. At a dose of 200 mg/kg, it demonstrated significantly lower TC, TG, LDL and VLDL levels than that of fenofibrate at the same dose with similar HDL levels. Compounds 10i and 10a possessed atherogenic indices (CRR, AC, AI, CRI-II) like that of fenofibrate. Additionally, a promising antioxidant activity indicated by the increased tissue reduced glutathione and plasma total antioxidant capacity with decreased plasma malondialdehyde levels was demonstrated by compounds 10a and 10i. No histopathological alterations were recorded in the hepatic tissue of compound 10i (200 mg/kg)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

S100B is a calcium-binding protein mainly expressed by astrocytes, but also localized in other definite neural and extra-neural cell types. While its presence in biological fluids is widely recognized as a reliable biomarker of active injury, growing evidence now indicates that high levels of S100B are suggestive of pathogenic processes in different neural, but also extra-neural, disorders. Indeed, modulation of S100B levels correlates with the occurrence of clinical and/or toxic parameters in experimental models of diseases such as Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, muscular dystrophy, multiple sclerosis, acute neural injury, inflammatory bowel disease, uveal and retinal disorders, obesity, diabetes and cancer, thus directly linking the levels of S100B to pathogenic mechanisms. In general, deletion/inactivation of the protein causes the improvement of the disease, whereas its over-expression/administration induces a worse clinical presentation. This scenario reasonably proposes S100B as a common therapeutic target for several different disorders, also offering new clues to individuate possible unexpected connections among these diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The retinal pigment epithelium (RPE) is a densely pigmented, monostratified epithelium that provides metabolic and functional support to the outer segments of photoreceptors. Endogenous or exogenous oxidative stimuli determine a switch from physiological to pathological conditions, characterized by an increase of intracellular levels of reactive oxygen species (ROS). Accumulating evidence has elucidated that punicalagin (PUN), the major ellagitannin in pomegranate, is a potent antioxidant in several cell types. The present study aimed to investigate the protective effect of PUN on mitochondrial dysfunction associated with hydrogen peroxide (H 2 O 2 )-induced oxidative stress. For this purpose, we used a human RPE cell line (ARPE-19) exposed to H 2 O 2 for 24 h. The effects of PUN pre-treatment (24 h) were examined on cell viability, mitochondrial ROS levels, mitochondrial membrane potential, and respiratory chain complexes, then finally on caspase-3 enzymatic activity. The results showed that supplementation with PUN: (a) significantly increased cell viability; (b) kept the mitochondrial membrane potential (ΔΨm) at healthy levels and limited ROS production; (c) preserved the activity of respiratory complexes; (d) reduced caspase-3 activity. In conclusion, due to its activity in helping mitochondrial functions, reducing oxidative stress, and subsequent induction of cellular apoptosis, PUN might be considered a useful nutraceutical agent in the treatment of oxidation-associated disorders of RPE.

The oxidative damage of the retinal pigment epithelium (RPE) is the early event that underlies the pathogenesis of maculopathies. Numerous studies have shown that punicalagin (PUN), a polyphenol present in pomegranate, can protect several cell types from oxidative stress. Our study aims to establish if PUN protects RPE from UV radiation-induced oxidative damage. We used an experimental model which involves the use of a human-RPE cell line (ARPE-19) exposed to UV-A radiation for 1, 3, and 5 hours. ARPE-19 cells were pre-treated with PUN (24 h) followed by UV-A irradiation; controls were treated identically, except for UV-A. Effects of pre-treatment with PUN on cell viability, intracellular reactive oxygen species ROS levels, modulation of Nrf2 and its antioxidant target genes, and finally apoptosis were examined. We found that pre‑treatment with PUN: (1) antagonized the decrease in cell viability and reduced high levels of ROS associated with UV-A-induced oxidative stress; (2) activated Nrf2 signaling pathway by promoting Nrf2 nuclear translocation and upregulating its downstream antioxidant target genes (HO-1 and NQO1); (3) induced an anti-apoptotic effect by decreasing Bax/Bcl-2 ratio. These findings provide the first evidence that PUN can prevent UV-A-induced oxidative damage in RPE, offering itself as a possible antioxidant agent capable of contrasting degenerative eye diseases., Competing Interests: The authors declare no conflict of interest.

S100B is an astrocytic protein acting either as an intracellular regulator or an extracellular signaling molecule. A direct correlation between increased amount of S100B and demyelination and inflammatory processes has been demonstrated. The aim of this study is to investigate the possible role of a small molecule able to bind and inhibit S100B, pentamidine, in the modulation of disease progression in the relapsing-remitting experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. By the daily evaluation of clinical scores and neuropathologic-molecular analysis performed in the central nervous system, we observed that pentamidine is able to delay the acute phase of the disease and to inhibit remission, resulting in an amelioration of clinical score when compared with untreated relapsing-remitting experimental autoimmune encephalomyelitis mice. Moreover, we observed a significant reduction of proinflammatory cytokines expression levels in the brains of treated versus untreated mice, in addition to a reduction of nitric oxide synthase activity. Immunohistochemistry confirmed that the inhibition of S100B was able to modify the neuropathology of the disease, reducing immune infiltrates and partially protecting the brain from the damage. Overall, our results indicate that pentamidine targeting the S100B protein is a novel potential drug to be considered for multiple sclerosis treatment., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Docosahexaenoic acid (DHA) is an omega‑3 polyunsaturated fatty acid, derived mainly from fish oil. It is well known that DHA is present in high concentrations in nervous tissue and plays an important role in brain development and neuroprotection. However, the molecular mechanisms underlying its role remain to be fully elucidated. In this study, to enhance our understanding of the pathophysiological role of DHA, we investigated the possible neuroprotective mechanisms of action of DHA against hydrogen peroxide (H2O2)‑induced oxidative damage in a rat pheochromocytoma cell line (PC12). Specifically, we evaluated the viability, oxidation potential, and the expression and production of antioxidant/cytoprotective enzymes, and eventual apoptosis. We found that pre‑treatment with DHA (24 h) protected the cells from H2O2‑induced oxidative damage. In particular, pre‑treatment with DHA: i) Antagonized the consistent decrease in viability observed following exposure to H2O2 for 24 h; ii) reduced the high levels of intracellular reactive oxygen species (ROS) associated with H2O2‑induced oxidative stress; iii) increased the intracellular levels of enzymatic antioxidants [superoxide dismutase (SOD) and glutathione peroxidase (GSH‑Px)] both under basal conditions and following H2O2 exposure; iv) augmented the intracellular levels of reduced glutathione (GSH) and ascorbic acid, while it reduced the malondialdehyde (MDA) levels under conditions of oxidative stress; v) upregulated the expression of nuclear factor (erythroid‑derived 2)‑like 2 (NFE2L2) and its downstream target protein, heme‑oxygenase‑1 (HO‑1); and vi) induced an anti‑apoptotic effect by decreasing Bax and increasing Bcl2 expression. These findings provide evidence suggesting that DHA is able to prevent H2O2‑induced oxidative damage to PC12 cells, which is attributed to its antioxidant and anti‑apoptotic effects via the regulation NFE2L2/HO‑1 signaling. Therefore, DHA may play protective role in neurodegenerative diseases associated with oxidative stress.

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1-42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects. Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites. As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Oxidative stress has long been linked to neuronal cell death in many neurodegenerative diseases. Antioxidant conventional supplements are poorly effective in preventing neuronal damage caused by oxidative stress due to their inability to cross the blood brain barrier. Hence the use of molecules extracted from plants and fruits such as phenolics, flavonoids, and terpenoids compounds constitute a new wave of antioxidant therapies to defend against free radicals., Objective: In this study we examined the effects of punicalagin, a ellagitannin isolated from the pomegranate juice, on a rat adrenal pheochromocytoma cell line, treated with hydrogen peroxide, evaluating the viability, oxidation potential, mitochondrial function, and eventual apoptosis., Methods: This study was performed on PC12 cells pretreated with punicalagin (0.5, 1, 5, 10 e 20 µM) 24 hours before of the damage by hydrogen peroxide (H 2 O 2 ). H 2 O 2 concentration (300 µM) used in our study was determined by preliminary experiments of time course. The cell viability and ROS production were evaluated by MTS assay and cytofluorometry assays, respectively. Subsequently, the number of apoptotic-positive cells and mitochondrial transmembrane potential, were measured by flow cytometry, in the same experimental paradigm. Finally, the expression of Bax and enzymatic activity of Caspase 3, some of the principle actors of programmed cell death, were investigated by semiquantitative PCR and utilizing a colorimetric assay kit, respectively., Results: We found that pretreatment with punicalagin protected the cells from H 2 O 2 -induced damage. In particular, the protective effect seemed to be correlated with a control both in radical oxygen species production and in mitochondrial functions. In fact the cells treated with H 2 O 2 showed an altered mitochondrial membrane integrity while the pretreatment with punicalagin retained both the cellular viability and the mitochondrial membrane potential similar to the control. Furthermore, the punicalagin, modulated the apoptotic cascade triggered reducing Bax gene expression and Caspase 3 activity., Discussion: Results of the present study demonstrated a neuroprotective effect of punicalagin on H 2 O 2 -induced PC12 cell death, including mitochondria damage and expression of apoptotic gene Bax; therefore we hypothesize a possible prevent role for this molecule in neurodegenerative diseases related to oxidative stress.

Long-term exposure to ultraviolet (UV) radiation is associated with pathological alterations of the retinal pigment epithelium (RPE). It has been indicated that Cortistatin (CST) and somatostatin (SST) are able to inhibit the neurodegeneration of the RPE associated with diabetic retinopathy and retinal ischemia via activation of SST receptors (SSTRs). To the best of our knowledge, the present study indicated for the first time that treatment with UV‑A (30 and 60 min) causes an increase of CST expression, rather than SST, which was linked with the upregulation of STTR3,4,5 subtype receptor gene expression levels. The study revealed that: i) SST and CST mRNA expression were both detected under basal conditions in a human retinal pigment epithelial cell line (Arpe‑19); ii) SST expression remained constant from baseline to 1 h of UV‑A treatment; iii) CST mRNA expression levels were 80 times increased compared with time 0 and after 30 min of exposition to ultraviolet irradiation; iv) SSTR1, SSTR2 mRNA and low levels of SSTR4 were expressed in basal conditions, whereas SSTR3 and SSTR5 mRNA were not detected under the same conditions; and v) only SSTR3, SSTR4 and SSTR5 were overexpressed after UV‑A treatment, although in a different way. In conclusion, the findings provide reasonable evidence to support the pathophysiological role of the CST/SST/SSTRs system in the adaptive response of the RPE exposed to UV‑A radiation.

Pathological alterations to the retinal pigment epithelium underlie several eye diseases, which lead to visual impairment and even blindness. Exposure to ultraviolet (UV) radiation is associated with some skin and ocular pathologies; UV radiation may induce DNA breakdown and cause cellular damage through the production of reactive oxygen species (ROS), thus leading to programmed cell death. The present study aimed to investigate the production of ROS and the gene expression levels of anti‑ and proapoptotic proteins [B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax) and caspase‑3] in human retinal pigment epithelial cells (ARPE‑19) treated with UV‑A for 5 h consecutively. The results demonstrated that prolonged exposure to UV‑A induced: i) Cell death, the decrease in cell viability was time‑dependent and reached statistical significance after 3 h; ii) a significant and substantial increase in ROS levels that remained constant for the duration of the experiment, the levels were significantly increased after 1 h of exposure; iii) an activation of apoptotic genes (Bax and caspase‑3) after 1 h of treatment, which was accompanied by a decrease in the anti‑apoptotic gene Bcl‑2; and iv) a loss of apoptotic signals and a rapid decrease in cellular viability after 3 h of consecutive treatment. These processes may trigger necrosis, which was observed in the cells following treatment with UV‑A for 5 consecutive hours. In conclusion, the present study is the first, to the best of our knowledge, to provide in vitro evidence regarding the sequence of events that underlie the cellular damage induced by prolonged UV‑A radiation, starting from the first 30 min of treatment. UV‑A radiation resulted in the activation of apoptotic events, and subsequently led to irreversible cell necrosis.

Background: In this study, human neuroblastoma cells (IMR32) treated with Amyloid Beta Peptide (APβ), were used as model to evaluate the molecular basis of protective role of S100b, a neurotrophic factor and neuronal survival protein, highly expressed by reactive astrocytes close to amyloid deposition in the cortex of Alzheimer's patients. The aim of this work is to value the effect of S100b on ROS production in cells treated with Amyloid Beta Peptide and the subsequent influence on globin gene expression., Method: In this study we investigated the effect of S100b on ROS production and on globin gene expression in human neuroblastoma cells (IMR32) treated with Amyloid Beta Peptide (APβ)., Results: Our results have shown that at nanomolar concentrations, S100b protects cells against AP. mediated cytotoxicity and the protective mechanism could be related, almost in part, to the control of ROS production through an over expression of Myoglobin gene., Conclusion: In light of our results, we speculate that over-expression of the Myoglobin gene could be read as a possible attempt of the cell to increase the scavengers of reactive oxygen species (ROS).

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimer's disease disease., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Lamellar ichthyosis (LI) is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. The pathology may be caused by mutations in transglutaminase 1 (TGM1) gene that encodes an enzyme critical for terminally differentiating keratinocytes. Because of evidences that transglutaminase enzymes are involved in programmed cell death, we investigated morphological and biochemical apoptotic parameters in cultured skin fibroblasts from a patient with a severe LI and homozygous for the TGM1 R142H mutation., Method: The principle apoptotic signals (mitochondrial membrane potential, analysis of oxygen consumption, DNA fragmentation and Bax/Bcl-2 gene expression) were analyzed in cultured fibroblasts from a LI patient, his mother (TGM1 mutation carrier) and a control subject., Results: LI fibroblasts showing a reduction of fibronectin expression evidenced a strong inhibition of oxygen consumption, a dramatic drop in the mitochondrial membrane potential (Delta psi(m)), and a higher apoptotic index., Conclusion: The present results suggest a possible connection between the alterations in the keratinization process leading to LI and the observed increased fibroblast apoptosis.

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.

The beta amyloid (Abeta), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals toxic to brain tissue and the redox state of Met-35 residue seems to play a particular and critical role in peptide's neurotoxic actions. In this study, we investigated, in human neuroblastoma cells (IMR-32), the relationship between the oxidative state of methionine, and both neurotoxic and pro-apoptotic actions induced by Abeta-peptide, comparing the effects of native peptide, in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 (Abeta(1-42)(35Met-ox)), as well as an Abeta-derivative with Met-35 substituted with norleucine (Abeta(1-42)(35Nle)). The obtained results show that Abeta induces a time-dependent decrease in cell viability; Abeta(1-42)(35Met-ox) was significantly less potent, though inducing a remarkable decrease in cell viability compared to control. On the contrary, no toxic effects were observed after treatment with Abeta(1-42)(35Nle). Abeta-peptide as well as the amyloid modified peptide with oxidized Met-35 induced the pro-apoptotic gene bax over-expression after 24 h, whereas Abeta(1-42)(35Nle) had no effect. Conversely, bcl-2, an anti-apoptotic gene, became highly down-regulated by Abeta peptide treatment, in contrast to that evidenced by the Abeta(1-42)(35Met-ox) peptide. Finally, Abeta caused an increase in caspase-3 activity to be higher with respect to that shown by Abeta(1-42)(35Met-ox) while Abeta(1-42)(35Nle) had no effect. These results support the hypothesis that Abeta-induced neurotoxicity occurs via bax over-expression, bcl-2 down-regulation, and caspase-3 activation, first indicating that methionine 35 redox state may alter this cell death pathway.

The sequence-dependent curvature is generally recognized as an important and biologically relevant property of DNA because it is involved in the formation and stability of association complexes with proteins. When a DNA tract, intrinsically curved for the periodical recurrence on the same strand of A-tracts phased with the B-DNA periodicity, is deposited on a flat surface, it exposes to that surface either a T- or an A-rich face. The surface of a freshly cleaved mica crystal recognizes those two faces and preferentially interacts with the former one. Statistical analysis of scanning force microscopy (SFM) images provides evidence of this recognition between an inorganic crystal surface and nanoscale structures of double-stranded DNA. This finding could open the way toward the use of the sequence-dependent adhesion to specific crystal faces for nanotechnological purposes.

The preferential assembly of specialized nucleosomes on budding yeast centromeres can be due either to the higher stability of specialized centromeric nucleosomes and/or to the lower stability of canonical centromeric nucleosomes with respect to bulk nucleosomes. We have evaluated the thermodynamic stability of canonical nucleosomes, assembled on Kluyveromyces lactis centromeric DNAs, with a competitive reconstitution assay and a theoretical method recently developed by us. The results, obtained by both methods, show that all five known centromeric DNAs from K. lactis are able to organize canonical nucleosomes, characterized by higher stability with respect those of bulk DNA. With 'footprinting' and theoretical prediction, based on sequence-dependent DNA elasticity, we have found that centromeric canonical nucleosomes are characterized by nucleosome dyad axis multiple positioning, rotationally phased. The isoenergetic nucleosome multiple positions are relevant in understanding the transition from canonical to specialized nucleosomes in interacting with centromere protein complexes. The satisfactory agreement between the results obtained from theoretical and experimental methods shows that sequence-dependent centromeric DNA elasticity has a main role in nucleosome thermodynamic stability and positioning.

Superstructural features of the Kluyveromyces lactis chromosome 1 (KlCEN1) and of the Saccharomyces cerevisiae chromosome 6 (SCEN6) centromeric DNAs were evaluated using a theoretical method, developed by our group, and experimentally measured by gel electrophoretic retardation. Both methods show that, in spite of the remarkable AT richness of the two centromeric sequences, their curvature is not very high. However the peculiar sequence features of the two centromeres allow to organize highly stable nucleosomes, with a free energy about that of the nucleosome formed on the 5S RNA gene. The good agreement between experimental and theoretical evaluation of nucleosome free energies as well as of their multiple positioning shows that in centromeres both DNA curvature and flexibility are relevant in determining nucleosomal features.

Recently, we have found that the assembly of nucleosomes reconstituted on negatively supercoiled DNA is cooperative. In the present paper the role of DNA topology and of histone tails in nucleosome assembly was explored. Reconstituted minichromosomes on relaxed DNA at different histone/DNA ratios (R) were assayed by topological analysis and electron microscopy visualization. Both methods show a linear relationship between average nucleosome number (N) and R. This suggests that in the case of relaxed DNA, cooperative internucleosomal interactions are small or absent. The influence of histone tails in nucleosome assembly was studied on minichromosomes reconstituted with trypsinized histone octamer on negatively supercoiled DNA by topological analysis. The topoisomers distribution, after trypsinization, dramatically changes, indicating that nucleosome-nucleosome interactions are remarkably decreased. These results show that, in chromatin folding, in addition to the well known role of histone H1, the interactions between histone octamer tails and DNA are also of importance.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.