Biomedical subjects

Alessandra Gliozzi

Publications and source records attributed to Alessandra Gliozzi.

14 recordsLinked to original sources

PubMed2006

Natively folded HypF-N and its early amyloid aggregates interact with phospholipid monolayers and destabilize supported phospholipid bilayers.

Recent data depict membranes as the main sites where proteins/peptides are recruited and concentrated, misfold, and nucleate amyloids; at the same time, membranes are considered key triggers of amyloid toxicity. The N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) in 30% trifluoroethanol undergoes a complex path of fibrillation starting with initial 2-3-nm oligomers and culminating with the appearance of mature fibrils. Oligomers are highly cytotoxic and permeabilize lipid membranes, both biological and synthetic. In this article, we report an in-depth study aimed at providing information on the surface activity of HypF-N and its interaction with synthetic membranes of different lipid composition, either in the native conformation or as amyloid oligomers or fibrils. Like other amyloidogenic peptides, the natively folded HypF-N forms stable films at the air/water interface and inserts into synthetic phospholipid bilayers with efficiencies depending on the type of phospholipid. In addition, HypF-N prefibrillar aggregates interact with, insert into, and disassemble supported phospholipid bilayers similarly to other amyloidogenic peptides. These results support the idea that, at least in most cases, early amyloid aggregates of different peptides and proteins produce similar effects on the integrity of membrane assembly and hence on cell viability.

Amyloid↗

PubMed2006

Amyloid fibrils formation and amorphous aggregation in concanavalin A.

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.

Amyloid↗

PubMed2006

Collagen plays an active role in the aggregation of beta2-microglobulin under physiopathological conditions of dialysis-related amyloidosis.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of beta(2)-microglobulin (beta(2)-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, beta(2)-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of beta(2)-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in beta(2)-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role in beta(2)-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-L-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-L-lysine is dissolved in solution with beta(2)-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as beta(2)-m, that decay on moving away from the fiber surface.

Amyloid↗

PubMed2006

Mechanical properties of single living cells encapsulated in polyelectrolyte matrixes.

We have studied the mechanical properties of encapsulated Saccharomyces cerevisiae yeast cells by performing AFM force measurements. Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte layers and mechanically trapped into a porous membrane. Coated and uncoated cells in presence/absence of bud scars, i.e. scars resulting from previous budding events, have been investigated. No significant differences between encapsulated and bare cells could be inferred from AFM topographs. On the other hand, investigation on the system elasticity through the acquisition and analysis of force curves allowed us to put in evidence the differences in the mechanical properties between the hybrid cell/polyelectrolyte system and the uncoated cells. Analysis of the curves contact region indicates that the polyelectrolyte coating increases the system rigidity. Quantitative evaluation of the cell rigidity through the Hertz-Sneddon model showed that coated cells are characterized by a Young's modulus higher than the value obtained for uncoated cells and similar to the value observed on the bud scar region of uncoated cells.

Biomechanical Phenomena↗

PubMed2006

Multilayer nanoencapsulation. New approach for immune protection of human pancreatic islets.

Immune protection of artificial tissue by means of pancreatic islet microencapsulation is a very ambitious new approach to avoid life-long immune suppression. But the success in the utilization of the alginate-beads with incorporated islets is unfortunately limited. Some of the problems cannot be solved by a two-component system, so polymer encapsulation of the microbeads was tested to improve the properties. In the present paper a pure nanoencapsulation multilayer approach was tested in order to reduce the size of the capsule and possibly apply in the future a multilayer capsule with individual properties in each layer or region of the capsule. Different polycations were attached in a self-assembly process. The advantage in using the surface charge of islets as binding site for the polyions is the guarantee of complete coverage after the second layer. Release of insulin was determined to characterize the function of the islets after encapsulation as well as the permeability of the capsule. Fluorescence microscopy was used to visualize the polyelectrolyte layers. Finally by means of an immune assay, the protection capability of the capsule was proved. In these first measurements the encapsulation with a multilayer nanocapsule was shown to be a possible alternative to the more space-consuming and random islet-trapping microencapsulation.

Cell Culture Techniques↗

PubMed2005

beta-amyloid is different in normal aging and in Alzheimer disease.

The mechanism of neurodegeneration caused by beta-amyloid in Alzheimer disease is controversial. Neuronal toxicity is exerted mostly by various species of soluble beta-amyloid oligomers that differ in their N- and C-terminal domains. However, abundant accumulation of beta-amyloid also occurs in the brains of cognitively normal elderly people, in the absence of obvious neuronal dysfunction. We postulated that neuronal toxicity depends on the molecular composition, rather than the amount, of the soluble beta-amyloid oligomers. Here we show that soluble beta-amyloid aggregates that accumulate in Alzheimer disease are different from those of normal aging in regard to the composition as well as the aggregation and toxicity properties.

Aged↗

PubMed2005

Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes.

The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells.

Calcium↗

PubMed2005

Encapsulated living cells on microstructured surfaces.

The immobilization of cells in defined arrays (cell patterning) is a key step towards cell-based biosensors or other cell-based devices. While cell patterning is usually achieved by modifying the surface on which only the cells should adhere and leaving the cells unmodified, we present here a different approach in which cells are first coated with polyelectrolytes and subsequently immobilized on patterned surfaces. By coating, the cells are protected and their interactions with the substrate are modified such that patterning is simplified. We used microcontact printing of polyelectrolytes to structure surfaces such that regions of opposite charges and the same charge as the cell coating were present and found that we can thus achieve patterning of the coated yeast cells. In accordance with prior work, we find that coating does not kill the cells and coated GFP-expressing cells still function after immobilization, which we checked by fluorescence microscopy.

Cell Adhesion↗

PubMed2005

Interaction of polyelectrolytes and their composites with living cells.

Since the layer-wise polyelectrolyte deposition offers the opportunity to modify surfaces for biomedical applications, interactions and toxicity between polyelectrolytes and living cells become interesting. The aim of the present work is to determine the different factors such as contact area, charge, and transplantation site that influence the cell reaction to a specific polymer. We found that toxicity is influenced by all these factors and cannot be tested easily in a model.

Animals↗

PubMed2004

Ultrastructural organization of ex vivo amyloid fibrils formed by the apolipoprotein A-I Leu174Ser variant: an atomic force microscopy study.

Atomic force microscopy was employed to study ex vivo amyloid material isolated from the transplanted hearts of two patients affected by systemic amyloidosis caused by the Leu174Ser apolipoprotein A-I variant. The purified material consists of fibrils and globular aggregates. For both patients the same morphological patterns are observed; in addition, fibril diameters obtained for the two patients turn out to be compatible, both in air (2.00+/-0.02 and 2.04+/-0.04 nm) and under liquid (10.7+/-0.4 and 11.3+/-0.5 nm). Fibrils display heterogeneous morphologies, occasionally showing a left-handed twist. Inspection of fibril ends, the study of fibril contour shape and the analysis of partially unfolded fibrils yield independent evidences suggesting that most twisted fibrils are composed of three protofilaments. The size of globular aggregates is the same for both patients (4.4+/-0.4 and 5.1+/-0.5 nm, measured under liquid) and is compatible with the protofilament expected diameter, suggesting that globules may represent protofilament precursors.

Amyloid↗

PubMed2004

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils.

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.

Bacterial Proteins↗

PubMed2004

Nanocapsules with functionalized surfaces and walls.

With the possibility of creating and handling nanometer-sized objects, it became popular to dream of future miniaturized tools allowing completely new applications in medicine and technology. Mother nature created during evolution many such self-assembled highly sophisticated and robust objects, and it is very tempting to modify them for other purposes: artificial cells as intelligent submarines floating in the bloodstream attacking cancer cells, single-molecule detection and manipulation allowing the bottom-up construction of devices, and so on. Nanobiotechnology became a hot topic and significant budgets have been attributed to it. However, caution has to be paid that unrealistic dreams might have a fairly negative effect on the taxpayer seeing unkept promises. In this paper, we explore the possibilities of using biological substances in material science.

Biocompatible Materials↗

PubMed2004

Nanocapsules: coating for living cells.

One of the most promising tools for future applications in science and medicine is the use of nanotechnologies. Especially self-assembly systems, e.g., polyelectrolyte (PE) capsules prepared by means of the layer-by-layer technique with tailored properties, fulfill the requirements for nano-organized systems in a satisfactory manner. The nano-organized shells are suitable as coating for living cells or artificial tissue to prevent immune response. With these shells, material can be delivered to predefined organs. In this paper, some preliminary results are presented, giving a broad overview over the possibilities to use nano-organized capsules. Based on the observations that the cells while duplicating break the capsule a mutant yeast strain (Saccharomyces cerevisiae), which express GFP-tubulin under galactose promotion, was investigated by means of confocal laser scanning microscopy. The measurements reveal an increased surface charge in the region of buds developed prior encapsulation. In order to test the used PE pair for cytotoxicity, germinating conidia of the fungi Neurospora crassa were coated. The investigation with fluorescence microscopy shows a variation in the surface charge for the growing region and the conidium poles. The capsules exhibit interesting properties as valuable tool in science and a promising candidate for application in the field of medicine.

Animals↗

PubMed2002

Microscopical characterization of nanocapsules templated on ionic crystals and biological cells toward biomedical applications.

Nanocapsules, fuzzy assemblies of polyelectrolyte, represent a comparatively new class of colloids with controlled nanostructure and tunable properties. Due to the fact that the core as well as the dissolution influences the wall texture and the properties of the hollow capsules, the use of carbonate crystals as template is most convenient. Yeast cells constitute as a core candidate as well. They are a good system for testing the protective ability of shells and the permeability of the walls with respect to the needs of biological systems, namely, feeding and stability against attacks. The main features of the nanocapsules have been studied by two-photon, confocal, and atomic force microscopy. Nanocapsules are of biomedical interest because they can be used, for example, for the controlled release and targeting of drugs as well as for the protection of enzymes, proteins, and foreign cells.

Biocompatible Materials↗