Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line.

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

Deprotonation of glutamic acid induced by weak magnetic field: an FTIR-ATR study.

It has been claimed that weak extremely low frequency electromagnetic fields (ELF-EMFs) can affect biochemical reactions and a wide-ranging body of literature is available on this topic. Nevertheless, the physical nature of these effects remains largely unknown. We investigated the influence of ELF-EMF on glutamic acid solutions using Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectra. Samples were exposed for 10, 20, or 30 min to a weak EMF generated by Helmoltz coils, and then placed in a spectrometer. After exposure, those solutions that had a pH lower than the isoelectric point tended to show a shift toward the deprotonation of the carboxylic group, while solutions having a pH greater than the isoelectric point showed the deprotonation of the residual amine group. Moreover, at low pH values, we also detected a shift of the δ(antisym) band of the amine. The effects lasted a few minutes after exposure before the native configuration was restored. The spectral modifications were observed after each independent exposure to EMFs, and the same effects were seen by varying the frequencies in the range of 0-7 kHz. Therefore, the hypothesis of the existence of a resonant frequency that has been proposed elsewhere cannot be supported by the results of this study. The most surprising characteristic of this effect is the long-lasting nature of the perturbation, which is hard to be explained in terms of short-living excitations in biological matter.

X-ray absorption spectroscopy studies of the adducts formed between cytotoxic gold compounds and two major serum proteins.

Gold metallodrugs form a class of promising antiproliferative agents showing a high propensity to react with proteins. We exploit here X-ray absorption spectroscopy (XAS) methods [both X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS)] to gain insight into the nature of the adducts formed between three representative gold(I, III) metallodrugs (i.e., auranofin, [Au(2,2'-bipyridine)(OH)(2)](PF(6)), Aubipy, and dinuclear [Au(2)(6,6'-dimethyl-2,2'-bipyridine)(2)(µ-O)(2)](PF(6))(2), Auoxo6) and two major plasma proteins, namely, bovine serum albumin (BSA) and human serum apotransferrin (apoTf). The following metallodrug-protein systems were investigated in depth: auranofin/apoTf, Aubipy/BSA, and Auoxo6/apoTf. XANES spectra revealed that auranofin, upon protein binding, conserves its gold(I) oxidation state. Protein binding most probably takes place through release of the thiosugar ligand and its subsequent replacement by a thiol (or a thioether) from the protein. This hypothesis is independently supported by EXAFS results. In contrast, the reactions of Aubipy with serum albumin and of Auoxo6 with serum apoTf invariantly result in gold(III) to gold(I) reduction. Gold(III) reduction, clearly documented by XANES, is accompanied, in both cases, by release of the bipyridyl ligands; for Auoxo6 cleavage of the gold-gold dioxo bridge is also observed. Gold(III) reduction leads to formation of protein-bound gold(I) species, with deeply modified metal coordination environments, as evidenced by EXAFS. In these adducts, the gold(I) centers are probably anchored to the protein through nitrogen donors. In general, these two XAS methods, i.e., XANES and EXAFS, used here jointly, allowed us to gain independent structural information on metallodrug/protein systems; detailed insight into the gold oxidation state and the local environment of protein-bound metal atoms was achieved in the various cases.

UVB radiation induced effects on cells studied by FTIR spectroscopy.

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.

Structural changes induced in proteins by therapeutic ultrasounds.

The structural effect induced by therapeutic ultrasound on proteins in aqueous solution has been investigated with FTIR spectroscopy, UV-VIS spectroscopy, circular dichroism and light scattering. Six proteins (cytochrome, lysozyme, myoglobin, bovine serum albumin, trypsinogen, and alpha-chymotrypsinogen A) with different molecular weight and secondary structure have been studied. The experiment has been performed using an ultrasound source at resonant frequency of 1 MHz and sonication times of 10, 20, 30, 40, 50, and 60 min. A different behaviour of proteins under sonication depends on the dominant secondary structure type (alpha-helix or beta-sheets) and on the grade of the ordered structure. The results suggest that the free radicals, produced by water sonolysis, have an important role in the changes of structural order.

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: a quantitative atomic force microscopy investigation.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study.

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.

Dynamic properties of an oriented lipid/DNA complex studied by neutron scattering.

The formation of lipid-DNA (CL-DNA) complexes called lipoplexes, proposed as DNA vectors in gene therapy, is obtained by adding DNA to a solution containing liposomes composed of cationic and neutral lipids. The structural and dynamic properties of such lipoplexes are determined by a coupling between the electrostatic interactions and the elastic parameters of the lipid mixture. An attempt to achieve a better understanding of the structure-dynamics relationship is reported herein. In particular, an elastic neutron scattering investigation of DOTAP-DOPC (dioleoyl trimethylammonium propane-dioleoyl phosphatidylcoline) complexed with DNA is described. Proton dynamics in this oriented CL-DNA lipoplex is found to be strongly dependent upon DNA concentration. Our results show that a substantial modification of the membrane dynamics is accompanied by the balancing of the total net charge inside the complex, together with the consequent displacement of interlayer water molecules.

X-ray absorption near-edge spectroscopy of transferrins: a theoretical and experimental probe of the metal site local structure.

Proteins of the transferrin (Tf) family have a role in metal transport in vertebrates and have been extensively studied. The results here reported provide, for the first time, a detailed systematic comparison of metal sites in Tf complexes involving several atoms in the whole protein and in two different types of Tfs. The high interest in the structural variations induced in a metalloprotein upon the uptake of different metals is related to the hypothesis of the metals' involvement in some neuropathologies. We propose a comparative study of the X-ray absorption spectra at the K-edge of iron, copper, zinc and nickel in serotransferrin and ovotransferrin. The experimental data are simulated using an algorithm of the full multiple scattering method. Our results show that: (1) the local structure of each site (N-terminal and C-terminal) is correlated to the ligation state of the other site; (2) the difference between the two proteins is related to site local structure and depends on the metal ion nature being greater in the case of copper and zinc with respect to iron and nickel ions; (3) X-ray spectroscopy is confirmed as a suitable technique able to discriminate between coordination models proposed by X-ray diffraction.

Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy.

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.

MRS study of the interaction of dihydropyridines with lipid molecules in phosphatidylcholine vesicles.

Dihydropyridines (DHPs), synthetic molecules used as antihypertensive agents, bind to plasma membrane receptors following diffusion through the hydrophobic phase. In this study, MRS technique has been used to clarify the interactions of the dihydrophyridines Nifedipine and Lacidipine within the lipid bilayer. 1D and 2D 1H MRS at high field have been employed to examine the behavior of unilamellar dimyristoyl-phosphatidylcholine liposomes when the two drugs have been inserted in the bilayer. In particular, the study represents an innovative application of 2D 1H NOESY technique to clarify different mechanisms of interactions of small molecules inside model membranes. On the other hand, 31P measurements have been performed in multilamellar dimyristoyl-phosphatidylcholine lipsomes to detect alterations of lipid polymorphic phases. The experiments show that the two dihydropyridines interact with the lipids by different modalities. Lacidipine undergoes a very strong interaction with lipids, possibly inducing a phase segregation of lipid molecules into the bilayer, while self-association seems to be the prevalent interaction of Nifedipine inside the bilayer.

Structural characterization of a new lipid/DNA complex showing a selective transfection efficiency in ovarian cancer cells.

We investigated, for the first time, by using Energy Dispersive X-ray Diffraction, the structure of a new ternary cationic liposome formulated with dioleoyl trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) and cholesterol (Chol) (DDC) which has been recently found to have a selective high gene transfer ability in ovarian cancer cells. Our structural results provide a further experimental support to the widely accepted statement that there is not a simple and direct correlation between structure and transfection efficiency and that the factors controlling cationic lipid/DNA (CL-DNA) complexes-mediated gene transfer depend not only on the formulations of the cationic liposomes and their thermodynamic phase, but also significantly on the cell properties.

Artificially induced unusual shape of erythrocytes: an atomic force microscopy study.

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration. After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation.

Conformational study of proteins by SAXS and EDXD: the case of trypsin and trypsinogen.

The radius of gyration (Rg) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique (EDXD) and by small-angle X-ray scattering (SAXS), under different solvent conditions. Both techniques gave superimposable results. The experimental evidence demonstrated that: (1) no structural modifications and/or damage occurred during the data acquisition by EDXD; (2) at pH 4 the active enzyme has one class of chloride binding sites in common with the zymogen, whereas the latter protease shows an additional class able to reverse the effects on Rg induced by chloride at low concentration; and (3) the pH profile of the Rg of both proteases does not resemble at all the pH effect on beta-trypsin activity, a result in line with the finding that the electrical potentials induced by surface charge are small in absolute magnitude and produce no gradient across the active site.

Iron and copper K-edge XAS study of serotransferrin and ovotransferrin.

The active metal site structure of transferrin with iron and copper atoms is investigated using metal K-XANES. Theoretical analysis of experimental data has been performed on the basis of full multiple-scattering theory. This approach made it possible to study the origin of XANES fine details and to investigate the local structure around active metal sites. A deep insight into the local structure and electronic subsystem of Fe, Cu transferrins is obtained. For example, in the case of Cu substitution of Fe in the active centre, the best fit of theoretical spectra to experiment has been obtained for distances 3% smaller between the Cu atom and the nearest neighbours.

MXAN: a new software procedure to perform geometrical fitting of experimental XANES spectra.

A new software procedure, MXAN, to fit experimental XANES spectra is presented here. The method is based on the comparison between the experimental spectrum and several theoretical calculations generated by changing the relevant geometrical parameter of the site around the absorbing atom. The x-ray photoabsorption cross section is calculated using the general multiple-scattering scheme, utilizing a complex Hedin-Lunqvist energy-dependent potential to describe the exchange correlation interaction. Our method has been applied to the study of geometrical environment of the tetrahedral zinc site of the protein superoxide dismutase (SOD). The experimental Zn K-edge XANES spectrum has been fitted in the space of the first shell coordination parameters (ligand distances and angles) following the behavior of the chi-square as a function of the local distortion from the starting crystallographic structure. The recovered structure is found to be independent on the starting conditions, showing the theoretical uniqueness of the structural solution. Strengths and limitations of the application to real systems are also discussed.

pH-dependent local structure of ferricytochrome c studied by x-ray absorption spectroscopy.

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.

Comparative XANES study of serotransferrin and ovotransferrin at Cu K-edge: evidence of interactions among the metal sites.

The Cu site structure of human serotransferrin and hen ovotransferrin using XANES spectroscopy has been investigated. Although the transferrin family proteins have been extensively studied, the results reported herein are the first concerning the structure of the metal site in C-terminal and N-terminal in the whole protein. Our structural data show that these proteins differ with regard to the independence of the two binding sites and the geometry of copper coordination, ranging from a poorly to a significantly distorted octahedron.

Three dimensional (3D) analysis of the morphological changes induced by 50 Hz magnetic field exposure on human lymphoblastoid cells (Raji).

Human Raji B lymphoid cells after exposure for 64 h to a 1 mT (rms) 50 Hz sinusoidal magnetic field showed a reorganization of membrane and cytoskeletal components. Atomic force microscopy in air revealed several modifications in 80% of the exposed cells, such as loss of microvilli-like structures followed by progressive appearance of membrane introflections. This change in plasma membrane morphology was also accompanied by a different actin distribution, as detected by phalloidin fluorescence. These observations support our previous hypothesis that electric and magnetic fields may modify the plasma membrane structure.