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

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

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

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

G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent Kd's of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these Kd's limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structure

Recombinant Fibrinogen Vlissingen/Frankfurt IV: THE DELETION OF RESIDUES 319 AND 320 FROM THE γ CHAIN OF FIBRINOGEN ALTERS CALCIUM BINDING, FIBRIN POLYMERIZATION, CROSS-LINKING, AND PLATELET AGGREGATION

We synthesized a variant, recombinant fibrinogen modeled after the heterozygous dysfibrinogen Vlissingen/Frankfurt IV, a deletion of two residues, gammaAsn-319 and gammaAsp-320, located within the high affinity calcium-binding pocket. Turbidity studies showed no evidence of fibrin polymerization, although size exclusion chromatography, transmission electron microscopy, and dynamic light scattering studies showed small aggregates. These aggregates did not resemble normal protofibrils nor did they clot. Fibrinopeptide A release was normal, whereas fibrinopeptide B release was delayed approximately 3-fold. Plasmin cleavage of this fibrinogen was not changed by the presence of calcium or Gly-Pro-Arg-Pro, indicating that both the calcium-binding site and the "a" polymerization site were non-functional. We conclude that the loss of normal polymerization was due to the lack of "A-a" interactions. Moreover, functions associated with the C-terminal end of the gamma chain, such as platelet aggregation and factor XIII cross-linking, were also disrupted, suggesting that this deletion of two residues affected the overall structure of the C-terminal domain of the gamma chain

Haptoglobin improves shock, lung injury, and survival in canine pneumonia

During the last half-century, numerous antiinflammatory agents were tested in dozens of clinical trials and have proven ineffective for treating septic shock. The observation in multiple studies that cell-free hemoglobin (CFH) levels are elevated during clinical sepsis and that the degree of increase correlates with higher mortality suggests an alternative approach. Human haptoglobin binds CFH with high affinity and, therefore, can potentially reduce iron availability and oxidative activity. CFH levels are elevated over approximately 24-48 hours in our antibiotic-treated canine model of S. aureus pneumonia that simulates the cardiovascular abnormalities of human septic shock. In this 96-hour model, resuscitative treatments, mechanical ventilation, sedation, and continuous care are translatable to management in human intensive care units. We found, in this S. aureus pneumonia model inducing septic shock, that commercial human haptoglobin concentrate infusions over 48-hours bind canine CFH, increase CFH clearance, and lower circulating iron. Over the 96-hour study, this treatment was associated with an improved metabolic profile (pH, lactate), less lung injury, reversal of shock, and increased survival. Haptoglobin binding compartmentalized CFH to the intravascular space. This observation, in combination with increasing CFHs clearance, reduced available iron as a potential source of bacterial nutrition while decreasing the ability for CFH and iron to cause extravascular oxidative tissue injury. In contrast, haptoglobin therapy had no measurable antiinflammatory effect on elevations in proinflammatory C-reactive protein and cytokine levels. Haptoglobin therapy enhances normal host defense mechanisms in contrast to previously studied antiinflammatory sepsis therapies, making it a biologically plausible novel approach to treat septic shock