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The interaction of the cytotoxic metals cadmium, zinc, and lead with pancreatic cells was studied by atomic force/lateral Force microscopy (AFM/LFM), an approach that provides both topographic (with nanometer scale lateral resolution) and chemical information on the membrane. Different morphological modifications of the overall cell shape and roughness took place as consequence of 100 μM metal-dependent treatment. Furthermore, after exposure to Cd(Cl2) and Zn(Cl2), but not Pb(Cl2), the LFM images revealed several areas of the cell's surface showing lateral friction contrasts that have been interpreted as marker of different alterations of the cell physiology induced by the metal loading. Thus, the coupling of LFM detection to topographic AFM characterization allows to distinguish, through a nondestructive and surface characterising approach, between different metal-induced cytotoxic effects on cells. In this framework, the role of the LFM as an important tool to discriminate between different alteration of a biological system has to be highlighted. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

Proteins of the transferrin (Tf) family have a role in metal transport in vertebrates and have beenbeen 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

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.

The effect of allosteric effectors, such as inositol hexakisphosphate and/or bezafibrate, has been investigated on the unliganded human adult hemoglobin both spectroscopically (employing electronic absorption, circular dichroism, resonance Raman, and x-ray absorption near-edge spectroscopies) and functionally (following the kinetics of the first CO binding step up to a final 4% ligand saturation degree). All data indicate that the unliganded T-state is not perturbed by the interaction with either one or both effectors, suggesting that their functional influence is only exerted when a ligand molecule is bound to the heme. This is confirmed by the observation that CO dissociation from partially liganded hemoglobin ( Y ¯ ≤ 0.04) is strongly altered by the presence of either effector, and the effect is enhanced whenever the two effectors are simultaneously present. Altogether, these data are a direct demonstration of the occurrence of a strain induced by the presence of a ligand molecule bound to the heme, and for the first time there is a clear indication that the expression of the functional heterotropic effect by these non-heme ligands requires this strain, which is not present in the unliganded molecule.

Fourier transform infrared spectroscopy in attenuated total reflection can be used to discriminate the necrotic from the apoptotic cell death in a tumoral T cell line irradiated by a UV source able to induce both apoptosis and necrosis. Using Jurkat cells as the model system, significant spectral differences in the irradiated cells vs. time were observed in the lipid-proteins ratio absorbance band at 1,397 cm(-1) and in lactic acid IR band at 1,122 cm(-1); these spectral features are inversely correlated with the percentage of apoptotic cells assessed by flow cytometry. From the analysis of second derivatives in the IR spectral region between 1,800 and 900 cm(-1), we have detected two significant spectral changes: the first centered at 1,621 cm(-1) by analyzing the components of the amide I band and the second centered at 1,069 cm(-1) due to C-O stretching vibration of the DNA backbone sensitive to the dehydrated state of DNA; these identified differences in the intracellular biomolecules have been allowed to monitor the necrotic process. The variations in the spectral data set have been identified by the Kruskal-Wallis test and confirmed by the hierarchical cluster analysis.

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.

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.

Pozzi, D., Grimaldi, P., Gaudenzi, S., Di Giambattista, L., Silvestri, I., Morrone, S. and Congiu Castellano, A. UVB-Radiation-Induced Apoptosis in Jurkat Cells: A Coordinated Fourier Transform Infrared Spectroscopy-Flow Cytometry Study. Radiat. Res. 168, 698–705 (2007). We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290–320 nm) at a dose of 310 mJ/ cm2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I β-sheet component and the area of the 1083 cm−1 band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area sh...

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.

We investigated for the first time the haem stereochemistry in the nitrosylated derivative of two amphibian haemoglobins, Xenopus laevis and Ambystoma mexicanum, by means of X-ray absorption spectroscopy technique with the aim to explain the relationships between the active site structure and physiological function of these proteins, compared to that from humans. Our results show that while the Fe site local structure of human HbNO is modulated by an allosteric effector such as IHP shifting the T-R equilibrium towards the T-state, the Fe site local structure of amphibians HbNO is stabilized in a particularly tensed T-state also without IHP.

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.

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.

The Fe K-edge X-ray absorption near-edge structure (XANES) spectra of two irreversible human hemichromes, spontaneously formed from HbA and HbMetSO (a hemoglobin derivative, where MetD6(55)beta has been previously oxidized to sulfoxide by chloramine T) were determined. The results show that the hemichrome from HbMetSO is characterized by the distal histidyl imidazole moved within the bonding distance of the heme iron. Such structure is different from that of the hemichrome spontaneously produced from native human hemoglobin, which probably has a hydroxide group as sixth heme ligand.

We report experimental evidence that the pH value of the micro-environment of the protein is the key for the conformational changes of transferrin, rather than the ligated or unligated state of the binding sites. The "open" and "closed" conformation of protein, suggested by X-ray crystallography in static condition, is triggered by the pH value and the effect is independent of the metal ion being transported. We propose a simple description of the structure-function relationship that allows this protein to deliver, in particular, iron or aluminum ions in the biological cycle.

X-ray small angle scattering experiments, using a pin hole SAXS camera with Synchrotron radiation source, have been performed to study the conformational changes of lyophilized samples of Apo-, Mono-, and Diferric- human transferrin. We report the experimental evidence that the analysis of the scattered intensity through the fractal theory may give information on the particle size and its variation upon iron binding.

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.

The X-ray absorption near edge structure (XANES) spectra of the human adult and foetal hemoglobin, of the isolated alpha and beta chains, in the oxygenated forms, and of the oxymyoglobin and carp oxyhemoglobin have been measured at the wiggler beam line of the Frascati Synchrotron radiation facility. The bonding angle of oxygen molecule at the iron site in these hemoproteins in solution, has been measured using the multiple scattering theory for data analysis.

Iron X-ray absorption near edge structure (XANES) spectra of human fetal (F) and adult (A) deoxyhemoglobin (deoxyHb) measured at the Frascati synchrotron radiation facility reveal the different geometrical structure of the Fe-porphyrin complexes in the two proteins. By this method, having determined for the first time the variation of atomic positions in fetal and adult hemoglobin in solution (close to the 'in vivo' situation), we give further insight into the structure-function relationship in hemoglobins.

The XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe-porphyrin geometry.

We use the recently developed multiple scattering theory to give a quantitative analysis of the X-ray absorption near edge structure (XANES) of haemoglobin and some of its substituents. We demonstrate that the XANES may contain information not provided by the extended X-ray absorption fine structure (EXAFS) part of the spectrum about the coordination geometry around the Fe atom, and in particular discuss the sensitivity of the XANES to the orientation of the CN group in HbCN. The anisotropy of the system leads to a strong dependence of the calculated spectrum on the polarisation of the X-rays. We show how this effect can be exploited in further XANES structural studies.

The Fe-site structure variation in the transition from the low-affinity tense (T) quaternary structure to the high-affinity relaxed (R) structure in carp deoxyhemoglobin was studied by analysis of multiple scattering resonances in the XANES (x-ray absorption near edge structure) spectra. High signal-to-noise XANES spectra were measured at the Frascati "wiggler" synchrotron radiation facility. We find that the forces on the Fe active site due to the change of quaternary protein conformation do not induce variations greater than 0.01 A in interatomic Fe-N distances, variations greater than 0.1 A in the Fe displacement toward the heme plane, or the "doming" of the heme. The relevance of these results to the mechanism of protein control of ligand binding is discussed.

When the magnetic susceptibility of frozen aqueous solutions of human oxyhemoglobin was measured in the range between 25 and 250 K, it showed a temperature-dependent behavior typical of a thermal equilibrium between a ground singlet state and an excited triplet state for two electrons per heme, the energy separation being [2J] = 146 cm-1. By contrast, within the same temperature range, carboxyhemoglobin was found to be diamagnetic, as already reported.

SummaryThe inner structure of fibrin fibres grown from fibrinogen solution activated by human α-thrombin was investigated by means of an Energy Dispersive X-ray Diffraction technique. The experiments show evidence for the well-characterized 22.5 nm repeat distance, which indicates the high order of protofibril arrangement in the longitudinal direction of fibres. The diffraction pattern also manifested a further pronounced peak at 18.1 nm (and its second order reflection at 18.1/√2) demonstrating the existence in fibrin of a high degree of lateral order. The reported results directly confirm, on unperturbed wet samples, that protofibrils closely associate giving rise to a crystalline axial and equatorial packing according to the conclusions of the multibundle model.Theme paper: Part of this work was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.

The x-ray absorption near edge structure (XANES) spectra of hemoglobin and myoglobin have been measured at the wiggler beam line of the Frascati Synchrotron Radiation Facility. The energy shifts of the iron absorption jump edge and the chemical shifts of the bound excited state at threshold of 1s core excitations, going from deoxygenated to oxygenated form, are interpreted as evidence of some increase of the positive effective charge on the iron atom upon oxygenation.