Search

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]

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]

In Kluyveromyces lactis yeast, OCH1 encodes for the α-1,6-mannosyltrasferase that adds the initial α-1,6-mannose to the outer-chains of N-glycoproteins. Kloch1-1 mutant cells showed altered calcium homeostasis and endoplasmic reticulum (ER) stress. Since ER plays a major role in lipid biosynthesis and lipid droplet (LD) formation, herein the impact of Och1p depletion on lipid homeostasis was investigated. Transcriptional profiles of genes involved in biosynthesis of fatty acids, their amount and composition changed in mutant cells. An increased amount of ergosterol was determined in these cells. Enhanced transcription of genes involved in both synthesis and mobilization of LDs was also found in Kloch1-1 cells, accompanied by a reduced amount of LDs. We provide evidence that ER alterations, determined by protein misfolding as a result of reduced N-glycosylation, induced altered lipid homeostasis in Kloch1-1 cells. Chemical chaperone 4-phenyl butyrate (4-PBA) slightly alleviated the LD phenotype in cells depleted of Och1p. Remarkably, complete suppression of ER stress, via increased expression of plasma membrane calcium channel subunit Mid1, fully restored lipid homeostasis in mutant cells. To further reinforce this finding, low numbers of LDs were observed in wild type cells when ER stress was triggered by DTT treatment. [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. [ABSTRACT FROM AUTHOR]

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]

Abstract: Objective: Low density lipoproteins (LDL) with an electronegative charge [LDL(−)] may cause endothelial injury. We assessed the association between serum LDL(−) levels and coronary artery disease (CAD) severity. Methods: We prospectively enrolled patients with CAD angiographic evidence [stable angina (SA) or non-ST-elevation-acute coronary syndrome (NSTE-ACS)], or with normal coronary arteries (NCA). Baseline LDL(−) serum levels were measured in all patients. Angiographic CAD extent was assessed by using the Bogaty extent index, while CAD severity by evaluating the presence of multi-vessel disease. Results: Forty-seven patients (age 61 ± 9 years, male sex 60%) were enrolled (17 SA, 15 NSTE-ACS and 15 NCA patients). LDL(−) levels were significantly higher in SA [21% (18–34) p = 0.0001] and NSTE-ACS [22% (18–28), p = 0.0001] as compared to NCA [6% (5–8)], without significant differences between SA and NSTE-ACS (p = 0.92). Multi-vessel disease patients had higher LDL(−) levels as compared to single-vessel disease patients (p = 0.002) but similar total LDL levels (p = 0.66). LDL(−) significantly correlated with extent index (r = 0.38, p = 0.03), while total LDL did not (p = 0.24). Conclusion: LDL(−) serum levels are associated with CAD angiographic severity and extent. This exploratory analysis should prime further larger studies in order to assess LDL(−) proatherogenic role. [Copyright &y& Elsevier]

Objectives: To compare the accuracy of magnetic resonance (MRI) and colour Doppler-ultrasound (US) in the diagnosis of late pregnancy bleeding and to assess the accuracy of the different MR sequences in visualizing the origin of haemorrhage.Methods: 42 patients in the third trimester of pregnancy underwent to US and MRI for the evaluation of painless vaginal bleeding. Multiplanar HASTE, True Fisp, 3D T1 GRE and sagittal DWI sequences were acquired. Two radiologists, blinded to the results of US, reviewed each case, resolving by consensus any discrepancy. Reference standards were surgical and pathological findings.Results: The reference standards identified 22 placenta previa, 11 placental abruptions (1 coincident with a placental chorioangioma), 1 thrombohaematoma and 1 fibroma with haemorrhagic degeneration. MRI identified correctly all these condition with an interobserver agreement of 0.955. DWI and T1 weighted sequences were statistically superior to Haste and True Fisp sequences in detecting the cause of bleeding (p < .001). US had 6 false negatives and 2 false positive results, its diagnostic accuracy resulting lower than MRI (p = .001).Conclusions: MRI accurately evaluates pregnancy bleeding with an excellent interobserver agreement and can grant new and additional data when US is negative. [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]

N-acetyl-L-cysteine exerts a complex action on endometrial cells, involving regulation of gene expression and protein activity and location, all converging into a decreased proliferation and a switch toward a differentiating, less invasive, and less inflammatory phenotype. Also considering the lack of undesired side effects, including unaffected fertility potential, this suggests a beneficial use of NAC in endometriosis clinical treatment. [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]

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]

Abstract: The non-receptor-protein tyrosine kinase c-Src is overexpressed and activated in a large number of human cancers, in which it is associated with tumor development and progression. Canonical regulation takes place by means of an alternative phosphorylation of tyrosine residues—Tyr419 for activation and Tyr530 for inactivation. An independent redox regulation mechanism, involving cysteine residues, has also been proposed, in which oxidation activates the enzyme. Here we present a kinetic analysis of the effect of N-acetyl-l-cysteine (NAC) on c-Src, demonstrating that reduction reverts the oxidation-driven activation. In cancer cells, we show that NAC treatment produces an increase in specifically labeled reduced thiols of c-Src cysteines, thus confirming a redox transition. In addition to a decrease in Tyr419 phosphorylation, this leads to a massive shift of c-Src from plasma membranes—where its active form is located—to endolysosomal compartments. With the objective of deciphering the complex issue of c-Src regulation and of devising new strategies to revert its activation in cancers, redox regulation thus emerges as a promising area for study. [Copyright &y& Elsevier]

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]

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]

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 beta-sheet structures - and fibril formation in the culture medium. When the same neurons are subjected to an excytotoxic death caused by 100 microM glutamate exposure, the amount of thioflavin binding is markedly reduced. Western blot analysis shows that fibrils contain monomers, dimers and trimers of amyloid-beta (Abeta) 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 beta-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]

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]

The secondary structure and conformation of apo-B 100 in low-density lipoproteins (LDL) are imposed by lipid–protein interactions and dynamics, and affected by the introduction or removal of lipids during the course of lipoprotein metabolism. Following an alteration of the water–lipid interface as a result of, for example, oxidation of lipids, the supramolecular structure becomes destabilized and apoB can misfold. These events have been observed in LDL−, a fraction of oxidatively modified LDL isolated in vivo. This modified lipoprotein possesses several atherogenic properties and represents an in vivo counterpart of in vitro modified LDL that is implicated in atherosclerosis. The misfolding of apoB, its aggregation, resistance to proteolysis, and cytotoxicity are common motifs shared by LDL− and amyloidogenic proteins. Based on these analogies, we propose that atherogenesis could be considered as a disease produced by the accumulation of cytotoxic and pro-inflammatory misfolded lipoproteins. [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]