Impact of electronegative low-density lipoprotein on angiographic coronary atherosclerotic burden.

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.

The thermal structural transition of alpha-crystallin modulates subunit interactions and increases protein solubility.

BACKGROUND: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract), is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K). METHODS/RESULTS: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δµ) and the interfacial tension (γ) of the aggregating phase, that characterize subunit interactions. CONCLUSIONS/GENERAL SIGNIFICANCE: The decrease of both Δµ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.

The thermal structural transition of α-crystallin inhibits the heat induced self-aggregation.

α-crystallin, the major constituent of human lens, is a member of the heat-shock proteins family and it is known to have a quaternary structural transition at . The presence of calcium ions and/or temperature changes induce supramolecular self-aggregation, a process of relevance in the cataractogenesis. Here we investigate the potential effect of the bovine α-crystallin's structural transition on the self-aggregation process. Along all the temperatures investigated, aggregation proceeds by forming intermediate molecular assemblies that successively aggregate in clusters. The final morphology of the aggregates, above and below Tc, is similar, but the aggregation kinetics are completely different. The size of the intermediate molecular assemblies, and their repulsive energy barrier show a marked increase while crossing . Our results highlight the key role of heat modified form of α-crystallin in protecting from aggregation and preserving the transparency of the lens under hyperthermic conditions.

Ristocetin-induced self-aggregation of von Willebrand factor.

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.

Mechanical properties of zona pellucida hardening.

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.

High-resolution imaging of redox signaling in live cells through an oxidation-sensitive yellow fluorescent protein.

We present the application of a redox-sensitive mutant of the yellow fluorescent protein (rxYFP) to image, with elevated sensitivity and high temporal and spatial resolution, oxidative responses of eukaryotic cells to pathophysiological stimuli. The method presented, based on the ratiometric quantitation of the distribution of fluorescence by confocal microscopy, allows us to draw real-time "redox maps" of adherent cells and to score subtle changes in the intracellular redox state, such as those induced by overexpression of redox-active proteins. This strategy for in vivo imaging of redox signaling circumvents many of the technical limitations currently encountered in the study of complex redox-based phenomena and promises to contribute substantially to this expanding area of signal transduction.

Modifications in solvent clusters embedded along the fibers of a cellulose polymer network cause paper degradation.

Plants, algae, and their derivatives (paper, textiles, etc.) are complex systems that are chiefly composed of a web of cellulose fibers. The arrangement of solvents within the polymeric structure is of great importance since cellulose degradation is strongly influenced by water accessibility and external agents. In this paper we develop a model that is able to deconvolve the scattering contributions of both polymeric structures and solvent clusters trapped along the polymeric fibers. The surface morphology of cellulose fibers and the spatial distribution of water-filled pores and their dimensions have been recovered from small angle neutron scattering and atomic force microscopy data in function with paper degradation. In addition to providing a boost to the effort to preserve cellulose-supported material (included cultural heritage), the relevance of our model resides in the exploitation of a large number of biopolymer networks that are known to share structures similar to that of cellulose.

Low density lipoprotein misfolding and amyloidogenesis.

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 alpha2 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.

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes.

Sphingosine 1-phosphate (S1P) has recently been described to induce antimycobacterial activity. The present study analyses the role played by S1P in antigen presentation of monocytes and in the next activation of Mycobacterium tuberculosis (MTB)-specific CD4+ T cell response. Results reported herein show that S1P stimulation of MTB-infected monocytes (i) inhibits intracellular mycobacterial growth, (ii) enhances phagolysosome maturation and the transit of mycobacteria in MHC class II compartments, (iii) increases the frequency of MTB-specific CD4+CD69+ T cells, expressing the inflammatory homing receptor CCR5, derived from tuberculosis patients and PPD+, BCG naïve, healthy subjects, and (iv) induces IFN-gamma production in CD4+CD69+CCR5+ T cells derived from PPD+ healthy individuals, only. Altogether, these results show that S1P promotes antigen processing and presentation in monocytes, increases the frequency of MTB-specific CD4+ T cells and can regulate IFN-gamma production by antigen specific CD4+ T cells in the course of active disease.

Globular structure of human ovulatory cervical mucus.

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.

Low density lipoprotein aged in plasma forms clusters resembling subendothelial droplets: aggregation via surface sites.

In early phases of atherogenesis, droplets and vesicles accumulate in the subendothelial extracellular space of arterial intima. There is much evidence to suggest that these droplets, ranging between 100 and 400 nm, derive from modified low-density lipoprotein (LDL). In investigations of the formation mechanism of these droplets, LDL fusion was previously induced in vitro by proteolysis, lipolysis, oxidation, and vigorous shaking, but all treatments failed to reproduce the size distribution range of in vivo droplets, mostly resulting, instead, in particles with a diameter intermediate between that of one and two LDL. Our approach was meant to mimic LDL aging in plasma. LDL isolated from plasma that was incubated overnight at 37 degrees C is slightly modified in the secondary structure of its protein component and is primed to form very large aggregates according to a reaction-limited mechanism. This mechanism requires interactions between selected surface sites, whereas massive fusion is ruled out. In the frame of the general theory for colloids, the aggregation of LDL aged in plasma fulfills all the requirements of the reaction-limited mechanism, encompassing 1), exponential growth; 2), fractal structure, with the dimension of elementary constituent still consistent with a single LDL; and 3), extreme polydispersity of aggregates, with shape and dimension very close to that of droplets observed in vivo.

Modifications of the mesoscopic structure of cellulose in paper degradation.

Paper is the main component of a huge quantity of cultural heritage. It is primarily composed of cellulose that undergoes significant degradation with the passage of time. By using small angle neutron scattering (SANS), we investigated cellulose's supramolecular structure, which allows access to degradation agents, in ancient and modern samples. For the first time, SANS data were interpreted in terms of water-filled pores, with their sizes increasing from 1.61 nm up to 1.97 nm in natural and artificially aged papers. The protective effect of gelatine sizing was also observed.

Particle size distribution in DMPC vesicles solutions undergoing different sonication times.

Size distribution of dimyristoylphosphatidylcholine liposome suspensions was investigated by dynamic-light scattering (DLS) as a function of the sonication time (t(s)). Cumulant expansion (second- and third-order) and regularized Laplace inversion (CONTIN) of dynamic single-angle laser light-scattering data were performed. With both methods, the intensity-weighted mean hydrodynamic radius r(I) depended on the investigated lengthscale. The number-weighted mean hydrodynamic radius (r(N)), obtained from CONTIN by modeling dimyristoylphosphatidylcholine vesicles as thin-walled hollow spheres, resulted as independent on the lengthscale. However, the r(N) value obtained from cumulant expansions remained lengthscale-dependent. Therefore, the number-weighted radius distribution function is highly asymmetric. The number-weighted mean radius, the standard deviation, and the number-weighted radius at the peak (r(N)(peak)) all decreased to a plateau when increasing sonication time. At t(s) longer than 1 h, the r(N)(peak) compares well with the radius of unilamellar vesicles in equilibrium with monomers predicted on a thermodynamic basis. The reliability of our analysis is proved by the comparison of experimental Rayleigh ratios with simulated ones, using the normalized number-weighted radius distribution function p(N)(r) determined by DLS data. A perfect agreement was obtained at longer sonication times, and the average aggregation number was determined. At lower t(s) values, simulations did not match experimental data, and this discrepancy was ascribed to the presence of large and floppy unilamellar vesicles with ellipsoidal shapes. Our investigation shows that, from single-angle DLS data, the radius distribution function of the vesicles can only be obtained if p(N)(r) is known.

Protofibrils within fibrin fibres are packed together in a regular array.

The inner structure of fibrin fibres grown from fibrinogen solution activated by human alpha-thrombin was investigated by means of an Energy Dispersive X-ray Diffraction technique. The experiments show evidence for the well-characterized 22.5 nm repeat distance, which indicates the high order of protofibril arrangement in the longitudinal direction of fibres. The diffraction pattern also manifested a further pronounced peak at 18.1 nm (and its second order reflection at 18.1/ radical 2) demonstrating the existence in fibrin of a high degree of lateral order. The reported results directly confirm, on unperturbed wet samples, that protofibrils closely associate giving rise to a crystalline axial and equatorial packing according to the conclusions of the multibundle model.

Structure of fibrin gels studied by elastic light scattering techniques: dependence of fractal dimension, gel crossover length, fiber diameter, and fiber density on monomer concentration.

The concentration dependence of the structure of fibrin gels, formed following fibrinogen activation by thrombin at a constant molar ratio, was investigated by means of elastic light scattering techniques. The scattered intensity distributions were measured in absolute units over a wave-vector range q of about three decades ( approximately 3x10(2)-3x10(5) cm(-1)). A set of gel-characterizing parameters were recovered by accurately fitting the data with a single function recently developed by us [F. Ferri et al., Phys. Rev. E 63, 031401 (2001)], based on a simple structural model. Accordingly, the gels can be described as random networks of fibers of average diameter d and density rho, entangled together to form densely packed and spatially correlated blobs of mass fractal dimension D(m) and average size (or crossover length) xi. As previously done for d, we show here that the recovered xi is also a good approximation of a weight average, namely, d approximately sqrt[(w)] and xi approximately (w). By varying the fibrinogen concentration c(F) between 0.034-0.81 mg/ml, gels with 100> or =xi> or =10 microm, 100< or =d< or =200 nm, 1.2< or =D(m)< or =1.4, and constant rho approximately 0.4 mg/ml were obtained. The power-law c(F) dependencies that we found for both xi and d are consistent with the model, provided that the blobs are allowed to partially overlap by a factor eta likewise scaling with c(F) (2> or =eta> or =1). Recasting the whole dataset on a single master curve provided further evidence of the similarity between the structures of all the gels, and confirmed the self-consistency of the model.