Low-Temperature Calcium Phosphate Ceramics Can Modulate Monocytes and Macrophages Inflammatory Response In Vitro.

Creating bioactive materials for bone tissue regeneration and augmentation remains a pertinent challenge. One of the most promising and rapidly advancing approaches involves the use of low-temperature ceramics that closely mimic the natural composition of the extracellular matrix of native bone tissue, such as Hydroxyapatite (HAp) and its phase precursors (Dicalcium Phosphate Dihydrate-DCPD, Octacalcium Phosphate-OCP, etc.). However, despite significant scientific interest, the current knowledge and understanding remain limited regarding the impact of these ceramics not only on reparative histogenesis processes but also on the immunostimulation and initiation of local aseptic inflammation leading to material rejection. Using the stable cell models of monocyte-like (THP-1ATRA) and macrophage-like (THP-1PMA) cells under the conditions of LPS-induced model inflammation in vitro, the influence of DCPD, OCP, and HAp on cell viability, ROS and intracellular NO production, phagocytosis, and the secretion of pro-inflammatory cytokines was assessed. The results demonstrate that all investigated ceramic particles exhibit biological activity toward human macrophage and monocyte cells in vitro, potentially providing conditions necessary for bone tissue restoration/regeneration in the peri-implant environment in vivo. Among the studied ceramics, DCPD appears to be the most preferable for implantation in patients with latent inflammation or unpredictable immune status, as this ceramic had the most favorable overall impact on the investigated cellular models.

Molecular and Ionic Diffusion in Ion Exchange Membranes and Biological Systems (Cells and Proteins) Studied by NMR.

The results of NMR, and especially pulsed field gradient NMR (PFG NMR) investigations, are summarized. Pulsed field gradient NMR technique makes it possible to investigate directly the partial self-diffusion processes in spatial scales from tenth micron to millimeters. Modern NMR spectrometer diffusive units enable to measure self-diffusion coefficients from 10-13 m2/s to 10-8 m2/s in different materials on 1 H, 2 H, 7 Li, 13 C, 19 F, 23 Na, 31 P, 133 Cs nuclei. PFG NMR became the method of choice for reveals of transport mechanism in polymeric electrolytes for lithium batteries and fuel cells. Second wide field of application this technique is the exchange processes and lateral diffusion in biological cells as well as molecular association of proteins. In this case a permeability, cell size, and associate lifetime could be estimated. The authors have presented the review of their research carried out in Karpov Institute of Physical Chemistry, Moscow, Russia; Institute of Problems of Chemical Physics RAS, Chernogolovka, Russia; Kazan Federal University, Kazan, Russia; Korea University, Seoul, South Korea; Yokohama National University, Yokohama, Japan. The results of water molecule and Li+, Na+, Cs+ cation self-diffusion in Nafion membranes and membranes based on sulfonated polystyrene, water (and water soluble) fullerene derivative permeability in RBC, casein molecule association have being discussed.

On Complex Formation between 5-Fluorouracil and ß-Cyclodextrin in Solution and in the Solid State: IR Markers and Detection of Short-Lived Complexes by Diffusion NMR.

In this work, the nuclear magnetic resonance (NMR) and IR spectroscopic markers of the complexation between 5-fluorouracil (5-FU) and ß-cyclodextrin (ß-CD) in solid state and in aqueous solution are investigated. In the attenuated total reflectance(ATR) spectra of 5-FU/ß-CD products obtained by physical mixing, kneading and co-precipitation, we have identified the two most promising marker bands that could be used to detect complex formations: the C=O and C-F stretching bands of 5-FU that experience a blue shift by ca. 8 and 2 cm-1 upon complexation. The aqueous solutions were studied by NMR spectroscopy. As routine NMR spectra did not show any signs of complexation, we have analyzed the diffusion attenuation of spin-echo signals and the dependence of the population factor of slowly diffusing components on the diffusion time (diffusion NMR of pulsed-field gradient (PFG) NMR). The analysis has revealed that, at each moment, ~60% of 5-FU molecules form a complex with ß-CD and its lifetime is ca. 13.5 ms. It is likely to be an inclusion complex, judging from the independence of the diffusion coefficient of ß-CD on complexation. The obtained results could be important for future attempts of finding better methods of targeted anticancer drug delivery.

Translational diffusion of unfolded and intrinsically disordered proteins.

Translational (or self-diffusion) coefficient in dilute solution is inversely proportional to the size of a diffusing molecule, and hence self-diffusion coefficient measurements have been applied to determine the effective hydrodynamic radii for a range of native and nonnative protein conformations. In particular, translational diffusion coefficient measurements are useful to estimate the hydrodynamic radius of natively (or intrinsically) disordered proteins in solution, and, thereby, probe the compactness of a protein as well as its change when environmental parameters such as temperature, solution pH, or protein concentration are varied. The situation becomes more complicated in concentrated solutions. In this review, we discuss the translational diffusion of disordered proteins in dilute and crowded solutions, focusing primarily on the information provided by pulsed-field gradient NMR technique, and draw analogies to well-structured globular proteins and synthetic polymers.

Effect of Reducing Agent TCEP on Translational Diffusion and Supramolecular Assembly in Aqueous Solutions of α-Casein.

The translational diffusion coefficient is highly sensitive to the size change of diffusing species and is ideally suited for the study of molecular association. Here, we used translational diffusion measurements by a pulsed-field gradient nuclear magnetic resonance (PFG NMR) technique to investigate the role of disulfide bonds in the formation of a supramolecular gel-like structure in the concentrated solution of α-casein. To reduce disulfide bonds, we added a commonly used reducing reagent tris(2-carboxyethyl)phosphine (TCEP) to α-casein solution. We found that the disruption of a disulfide bond Cys36-Cys40 in αs2-casein does not alter the translational diffusion or secondary structure of α-casein in dilute, 1 and 3% (wt %) solution. On the contrary, in concentrated, 15% (wt %) α-casein solution, in addition to the disruption of disulfide bonds, TCEP induced significant changes in gel properties. New long-lived intermolecular interactions formed, leading to the irreversible gel formation. While a few side reactions of TCEP (as well as other reducing agents, e.g., dithiothreitol) have been reported, this area is still understudied. Here, we provide new data on the side reaction of the reducing agent TCEP in concentrated protein solution, suggesting that at high protein concentrations TCEP should be used with caution.

Effect of Intrinsic Disorder and Self-Association on the Translational Diffusion of Proteins: The Case of α-Casein.

Translational diffusion is the major mode of macromolecular transport in leaving organisms, and therefore it is vital to many biological and biotechnological processes. Although translational diffusion of proteins has received considerable theoretical and experimental scrutiny, much of that attention has been directed toward the description of globular proteins. The translational diffusion of intrinsically disordered proteins (IDPs), however, is much less studied. Here, we use a pulsed-gradient nuclear magnetic resonance technique (PFG NMR) to investigate the translational diffusion of a disordered protein in a wide range of concentrations using α-casein that belongs to the class of natively disordered proteins as an example.

Spatial structure of peptides determined by residual dipolar couplings analysis.

The gated decoupled (13)C NMR spectra of a dipeptide (Glu-Trp) and a tetrapeptide (NAc-Ser-Phe-Val-Gly-OMe) were recorded in D(2)O and in a lyotropic alignment medium (pentaethylene glycol monododecyl ether/n-hexanol). The residual dipolar couplings were extracted as the differences between the observed couplings for the magnetic nuclei dissolved in the latter and former media. Using a computational optimization, the spatial structures of the compounds were calculated starting from their respective low energy conformations obtained on a semiempirical basis. The uniformity of each conformation was confirmed by the solid-state (13)C NMR spectra of powder samples. Differences between the starting structures and final ones, optimized when employing residual dipolar couplings, are discussed.

Porous media characterization by PFG and IMFG NMR.

Fully and partially filled with tridecane quartz sand was studied by different NMR techniques. The set of NMR experiments was carried out to obtain information about porous media geometry and fluid localization in it in case of partially filled porous space. The study was done using three NMR approaches: pulse field gradient NMR (PFG NMR), DDif experiment and tau-scanning experiment. The possibility to use all three approaches to study porous media properties even at the high resonance frequency is shown together with complementarity of the given by them information. Thus, first two approaches give information about porous sizes and geometry, at the same time tau-scanning experiment allows us to obtain information about distribution of internal magnetic field gradients in the porous space and draw conclusions about fluid localization in it.

Water self-diffusion behavior in yeast cells studied by pulsed field gradient NMR.

The water self-diffusion behavior in yeast cell water suspension was investigated by pulsed field gradient NMR techniques. Three types of water were detected, which differ according to the self-diffusion coefficients: bulk water, extracellular and intracellular water. Intracellular and extracellular water self-diffusion was restricted; the sizes of restriction regions were approximately 3 and 15-20 microm, respectively. The smallest restriction size was determined as inner cell size. This size and also cell permeability varied with the growth phase of yeast cell. Cell size increased, but permeability decreased with increasing growth time. The values of cell permeabilities P(1)(d) obtained from time dependence of water self-diffusion coefficient were in good agreement with the permeabilities obtained from the exchange rate constants P(1)(eff). The values of P(1)(eff) were 7 x 10(-6), 1.2 x 10(-6) and 1.6 x 10(-6) m/s, and P(1)(d) were 6.3 x 10(-6), 8.4 x 10(-7), 1.5 x 10(-6) m/s for yeast cells incubated for 9 h (exponential growth phase), 24 h (end of exponential growth phase), and 48 h (stationary growth phase), respectively.

Generalized concentration dependence of globular protein self-diffusion coefficients in aqueous solutions.

The self-diffusion coefficients of globular proteins (myoglobin, bovine serum albumin, barstar, lysozyme) in aqueous solutions at different temperatures and pH values are obtained by pulsed-gradient spin-echo NMR, and their concentration dependence is analyzed. The generalized concentration dependence of globular protein self-diffusion coefficients is empirically established, and compared to the concentration dependence of diffusion coefficients of flexible polymers and rigid Brownian particles.