Исследование механизмов синтеза керамических материалов нового поколения на основе тетрарных оксикарбонитридных фаз циркония и титана

Работа посвящена исследованию механизмов синтеза керамических материалов нового поколения на основе тетрарных оксикарбонитридных фаз циркония и титана. Были определены плотность спечённых образцов, измерены модуль упругости, нанотвердость и предел прочности с использованием методики наноиндентрования и скрэч-тестинга, исследованы фазовый состав и микроструктура спеченных образцов. В результате выполнения работ методом горячего прессования получена высокомодульная прочная оксинитридная керамика.The work is devoted to the study of mechanisms of synthesis of new generation ceramic materials based on tetrad oxycarbonitride phases of zirconium and titanium. The density of sintered samples was determined, the modulus of elasticity, nanohardness and ultimate strength were measured using the technique of nanoindentation and scratch testing, and the phase composition and microstructure of sintered samples were studied. As a result of hot pressing, high-modulus, strong oxynitride ceramics were obtained

C(60)-Fullerenes: detection of intracellular photoluminescence and lack of cytotoxic effects

We have developed a new method of application of C(60 )to cultured cells that does not require water-solubilization techniques. Normal and malignant cells take-up C(60 )and the inherent photoluminescence of C(60 )is detected within multiple cell lines. Treatment of cells with up to 200 μg/ml (200 ppm) of C(60 )does not alter morphology, cytoskeletal organization, cell cycle dynamics nor does it inhibit cell proliferation. Our work shows that pristine C(60 )is non-toxic to the cells, and suggests that fullerene-based nanocarriers may be used for biomedical applications

Structure of the fibrin protofibril.

We identified the two-stranded fibrin protofibril and studied its structure in electron micrographs of negatively stained specimens. Based on these images and on considerations of symmetry, we constructed a model of the protofibril in which the two strands of trinodular fibrin molecules are related by a two-fold screw axis between the strands and two-fold axes perpendicular to them. The two strands are held together by staggered lateral contacts between the central nodules of one strand and outer nodules of the other. The molecules within a strand are joined by longitudinal contacts between outer nodules. This interpretation of the structure of protofibrils is supported by images of trimer complexes whose preparation and structure are described here, in which the central nodule of a fibrin monomer is attached to the crosslinked outer nodules of two other molecules. We conclude that the association of protofibrils to form thicker fibers must involve a second type of lateral contact, probably between outer nodules of adjacent, in-register strands. In total, we identify three intermolecular contacts involved in the polymerization of fibrin

The Mechanical Properties of Individual, Electrospun Fibrinogen Fibers

We used a combined atomic force microscope (AFM)/fluorescence microscope technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 μm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22·106 Pa. We collected incremental stress-strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 16·106 Pa and the relaxed, elastic modulus was 6.7·106 Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 56 seconds. Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and resuspended in aqueous buffer. Circular dichroism spectra indicate that alpha-helical content of fibrinogen is ~70% higher in 90-HFP than in aqueous solution. These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other, nanoscopic fibers

Dynamic Regulation of Fibrinogen: Integrin αIIbβ3 Binding

This study demonstrates that two orthogonal events regulate integrin αIIbβ3’s interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the αIIb–β3 interface and (2) flexibility in the carboxy terminus of fibrinogen’s γ-module. The first postulate was tested by capturing αIIbβ3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed αIIbβ3 was characterized by a kon of ~2 × 104 L mol−1 s−1 and a koff of ~8 × 10−5 s−1 at 37 °C. In contrast, even at 150 nM fibrinogen, no binding was detected with resting αIIbβ3. Eptifibatide competitively inhibited fibrinogen’s interactions with primed αIIbβ3 (Ki ~ 0.4 nM), while a synthetic γ-module peptide (HHLGGAKQAGDV) was only weakly inhibitory (Ki > 10 µM). The second postulate was tested by measuring αIIbβ3’s interactions with recombinant fibrinogen, both normal (rFgn) and a deletion mutant lacking the γ-chain AGDV sites (rFgn γΔ408–411). Normal rFgn bound rapidly, tightly, and specifically to primed αIIbβ3; no interaction was detected with rFgn γΔ408–411. Equilibrium and transition-state thermodynamic data indicated that binding of fibrinogen to primed αIIbβ3, while enthalpy-favorable, must overcome an entropy-dominated activation energy barrier. The hypothesis that fibrinogen binding is enthalpy-driven fits with structural data showing that its γ-C peptide and eptifibatide exhibit comparable electrostatic contacts with αIIbβ3’s ectodomain. The concept that fibrinogen’s αIIbβ3 targeting sequence is intrinsically disordered may explain the entropy penalty that limits its binding rate. In the hemostatic milieu, platelet–platelet interactions may be localized to vascular injury sites because integrins must be activated before they can bind their most abundant ligand

Structural and Biochemical Studies of Human 4-hydroxy-2-oxoglutarate Aldolase: Implications for Hydroxyproline Metabolism in Primary Hyperoxaluria

4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined.In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding.The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored

Fibrin Fibers Have Extraordinary Extensibility and Elasticity

Blood clots perform an essential mechanical task, yet the mechanical behavior of fibrin fibers, which form the structural framework of a clot, is largely unknown. By using combined atomic force-fluorescence microscopy, we determined the elastic limit and extensibility of individual fibers. Fibrin fibers can be strained 180% (2.8-fold extension) without sustaining permanent lengthening, and they can be strained up to 525% (average 330%) before rupturing. This is the largest extensibility observed for protein fibers. The data imply that fibrin monomers must be able to undergo sizeable, reversible structural changes and that deformations in clots can be accommodated by individual fiber stretching

A Comparison of the Mechanical and Structural Properties of Fibrin Fibers with Other Protein Fibers

In the past few years a great deal of progress has been made in studying the mechanical and structural properties of biological protein fibers. Here, we compare and review the stiffness (Young's modulus, E) and breaking strain (also called rupture strain or extensibility, εmax) of numerous biological protein fibers in light of the recently reported mechanical properties of fibrin fibers. Emphasis is also placed on the structural features and molecular mechanisms that endow biological protein fibers with their respective mechanical properties. Generally, stiff biological protein fibers have a Young's modulus on the order of a few Gigapascal and are not very extensible (εmax 100%). These soft, extensible fibers employ a variety of molecular mechanisms, such as extending amorphous regions or unfolding protein domains, to accommodate large strains. We conclude our review by proposing a novel model of how fibrin fibers might achieve their extremely large extensibility, despite the regular arrangement of the monomeric fibrin units within a fiber. We propose that fibrin fibers accommodate large strains by two major mechanisms: (1) an α-helix to β-strand conversion of the coiled coils; (2) a partial unfolding of the globular C-terminal domain of the γ-chain