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Biomedical subjects

Claudio Nicolini

Publications and source records attributed to Claudio Nicolini.

At least 37 records · Page 2Linked to original sources
PubMed2004

Direct electron transfer between cytochrome P450scc and gold nanoparticles on screen-printed rhodium-graphite electrodes.

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) and gold nanoparticles immobilised on rhodium-graphite electrodes. Thin films of gold nanoparticles were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto both gold nanoparticle modified and bare rhodium-graphite electrodes. Cyclic voltammetry indicated enhanced activity of the enzyme at the gold nanoparticle modified surface. The role of the nanoparticles in mediating electron transfer to the cytochrome P450scc was verified using ac impedance spectroscopy. Equivalent circuit analysis of the impedance spectra was performed and the values of the individual components estimated. On addition of aliquots of cholesterol to the electrolyte bioelectrocatalytic reduction currents were obtained. The sensitivity of the nanoparticle modified biosensor to cholesterol was 0.13 microA microM-1 in a detection range between 10 and 70 microM of cholesterol. This confirms that gold nanoparticles enhance electron transfer to the P450scc when present on the rhodium-graphite electrodes.

Biosensing Techniques↗
PubMed2004

C-terminal region of protein kinase CK2 alpha: How the structure can affect function and stability of the catalytic subunit.

A novel mutant of the catalytic alpha subunit of human protein kinase CK2 (CK2 alpha) was designed in an attempt to clarify the role of the carboxylic-terminal segment characteristic of vertebrates, excluding fish. Starting from the sequence alignments, we constructed a phylogenetic tree of the primary structure of CK2 alpha. On this basis, we substituted two distal prolines with alanines (PA 382-384). Theoretical calculations and spectropolarimetry measurements, performed both on native and mutant subunits, indicate an increased content of alpha-helix after this double amino acidic substitution. In order to clarify the structure/function relationship of the C-terminal region, we verified if the structural change affects the catalytic activity of CK2 alpha. The mutant exhibits slightly increased phosphorylation efficiency, but reduced ability to transfer phosphate in comparison with the native subunit. At last, we compared the thermal stability of the mutant with respect to the native subunit and we tested the proteolytic degradability. The observation that the PA 382-384 mutant exhibits an increased thermal and proteolytic stability suggests that this mutant could be employed to solve the three-dimensional (3D) structure of human CK2 alpha and to overcome difficulties in crystallizing the native form.

Amino Acid Sequence↗
PubMed2004

Cholesterol amperometric biosensor based on cytochrome P450scc.

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min).

Biosensing Techniques↗
PubMed2004

Atomic structure of a CK2alpha human kinase by microfocus diffraction of extra-small microcrystals grown with nanobiofilm template.

Extra-small microcrystals of a human kinase CK2alpha were obtained for the first time by the optimization of a recent protein crystallization method based on highly packed protein nanofilm template. Protein crystal induction and growth appear indeed optimal at high surface pressure of the film template yielding high protein orientation and packing. The resulting extra-small CK2alpha microcrystals (of about 20 microm in diameter) was subsequently used for synchrotron radiation diffraction data collection, which proves possible by means of the Microfocus Beamline at the ESRF Synchrotron in Grenoble. The quality of the resulting crystal diffraction patterns and of its resulting atomic structure at 2.4 A resolution proves the unique validity of the above two combined frontier technologies in defining a new approach to structural proteomics capable to solve the atomic structure of proteins so far never been crystallized and of pharmaceutical relevance. Physical explanation in terms of template dipole moments and possibility of generalization of this method to the wide class of proteins not yet crystallized are finally discussed. The structure of our CK2alpha mutant is in the Protein Data Bank (PDB ID Code 1NA7, deposited on 27 November 2002).

Air Pressure↗
PubMed2004

Optical and electrochemical properties of poly(o-toluidine) multiwalled carbon nanotubes composite Langmuir-Schaefer films.

Conducting poly(o-toluidine) (POT) with multiwalled carbon nanotubes (MWNTs) nanocomposite (POT-MWNTs) was synthesized by oxidative polymerization. Chloroform solutions of the material were used for the optical characterizations by means of UV-visible spectroscopy and for the fabrication of Langmuir-Schaefer (LS) films. LS films were fabricated at the air-liquid interface by using 0.1 M HCl aqueous solution as the subphase to study the electrochemical properties of the nanocomposite by means of cyclic voltammetry and photoelectrochemical techniques. The optical characterizations gave proof that the presence of MWNTs inside the polymeric matrix produced no change in the (pi-pi*) transition of POT structure, indicating that the polymeric chains were simply wrapped around and not doped by MWNTs. The electrochemical investigations highlighted significant changes in the redox properties of POT-MWNTs LS films with respect to pure POT. The cyclic voltammetric study also revealed high electrochemical stability, confirmed by the estimation of the diffusion coefficient and the photoelectrochemical response of the nanocomposite LS films. This characteristic turned out to be more evident than that obtained in our earlier studied poly(o-anisidine)-MWNTs (POAS-MWNTs) system.

Journal Article↗
PubMed2004

Modifications of chromatin structure and gene expression following induced alterations of cellular shape.

In higher eukaryotes cellular shape is a dynamic element which can be altered by external and internal factors (i.e. surface interactions, temperature, ionic strength). Our question was: might modifications of cell shape reflect on nuclear morphology and architecture and hence on chromatin function, in order to represent a mechanism of cell regulation? We altered the shape of cultured fibroblasts by coating the growth substratum with synthetic polymers, which alternatively increased and decreased the adhesiveness. By means of Fluorescence microscopy we analysed the modifications of cell and nucleus architecture induced by the different substrata. Then we used differential scanning calorimetry to investigate if a remodelling of chromatin structure was associated with the induced morphological changes. Finally, we evaluated if the observed modifications of chromatin condensation affect the transcriptional profile. At this stage of the work we focused on just four genes (c-myc, c-fos, c-jun and collagen) and we analysed their expression by dot blot hybridization and RT-PCR. The results confirm that mechanical factors external to the cell, such as the physico-chemical features of the substratum, are able to modulate gene transcription through a remodelling of chromatin structure. Therefore the work supports our starting hypothesis of a regulatory pathway connecting in sequence cellular morphomety/nuclear architecture/chromatin structure/gene expression.

Animals↗
PubMed2004

Protein nanocrystallography: a new approach to structural proteomics.

This article describes a new approach to structural proteomics that can produce and characterize diffracting, stable and radiation-resistant crystals of miniscule dimensions using nanotechnology. We believe that the protein microcrystals obtained by nanotechnology-based protein thin-film template crystallization, as well as groundbreaking technology, such as atomic force microscopy, nanogravimetry and synchrotron microfocus, have enabled protein nanocrystallography to be defined as a unique technology capable of forming and characterizing stable protein microcrystals down to atomic resolution. A new route from art to science and technology has, therefore, been opened in protein crystallography, and it could be used to unravel the mysteries of many systems that remain unsolved.

Casein Kinase II↗
PubMed2004

P450scc mutant nanostructuring for optimal assembly.

Molecular modeling and protein engineering were synergically employed to improve the fabrication of cytochrome P450scc mutant nanostructures for biodevice assembly. The optimization of protein three-dimensional structure by molecular modeling was performed using two models: in vacuum and simulating the presence of a polar solvent. Calculations were performed on a model to predict a P450scc mutant which could improve the process of molecules' immobilization onto solid supports. Engineerized cytochrome P450scc thin films were prepared and characterized by various biophysical techniques such as pi-A isotherms, surface potential measurements, Brewster angle microscopy, UV-vis spectroscopy, circular dichroism, nanogravimetry, and electrochemical analysis. This paper takes into consideration biomolecules modified by protein engineering that represent a new and powerful approach for obtaining synthetic simpler artificial structures with new or improved properties (i.e., specificity, stability, sensitivity, etc.) useful for biosensors development.

Amino Acid Sequence↗
PubMed2003

Three-dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2.

The three-dimensional crystal structure of the triple-point mutant of the catalytic subunit of human protein kinase CK2alpha has been determined at 2.4 A resolution. Microcrystals of mutant CK2 catalytic subunit were obtained by a protein-crystallization method based on thin-film nanotechnology. These microcrystals (of about 20 micro m in diameter) were used for diffraction data collection by means of the microfocus beamline at the ESRF synchrotron. A comparison between the human protein kinase CK2alpha and the corresponding enzyme from a lower organism (Zea mays) is made.

Amino Acid Sequence↗
PubMed2003

Complex catalytic colloids on the basis of firefly luciferase as optical nanosensor platform.

In the present work the layer-by-layer nano-assembly technique was used for the development of complex catalytic microparticles on the basis of firefly luciferase (FL). FL films containing 1, 2, or 3 monolayers were assembled on silver electrode QCM-resonators and on 520-nm diameter sulfonated polystyrene latex by alternate adsorption of FL and polycations using electrostatic interactions for the interlayer interaction. The assembly process was studied with quartz crystal microbalance, UV-vis spectroscopy, and microelectrophoresis (surface potential). Structural studies of the resulting multilayers confirmed stepwise deposition of FL and cationic poly(dimethyldiallyl ammonium chloride) with a bilayer thickness of 14 nm; a systematic shift of the surface potential from +28 mV for poly(dimethyldiallyl ammonium chloride) to -14 mV for luciferase outermost layer was established. The functionality and stability of the biocolloids were demonstrated by monitoring the intensity of the light emission. Factors influencing the light emitted upon catalytic activity of FL such as the number of luciferase layers in the film and polyion layer at the outermost layer were studied.

Adenosine Triphosphate↗
PubMed2003

A structural characterization of in situ chromatin on cell lines isolated from patients affected by ataxia telangiectasia.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by numerous clinical and cellular features. The pleiotropic nature of the AT syndrome attests to the multiple roles of ATM, the protein codified by the gene altered in AT patients. We investigated if different mutations of ATM could reflect on different alterations of nuclear architecture and chromatin organization. We selected three lymphoblastoid cell lines isolated from AT patients affected by different mutations of ATM gene and one healthy control. We characterized the in situ chromatin structure of each cell line by a biophysical approach: (1) we evaluated the rearrangements of the chromatin domains at the level of single cell by quantitative fluorescence microscopy; (2) we analysed the changes of the average chromatin condensation by differential scanning calorimetry. The results show that the three different ATM mutations produce significant modifications of both nuclear architecture and chromatin condensation.

Ataxia Telangiectasia↗
PubMed2003

Expression, purification and characterisation of a novel mutant of the human protein kinase CK2.

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant a subunit. According to our theoretical predictions, we observed that the alpha-helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.

Amino Acid Sequence↗
PubMed2003

Bacteriorhodopsin-based Langmuir-Schaefer films for solar energy capture.

The photovoltaic (PV) solar cell, converting incident solar radiation directly into electrical energy, today represents the most common power source for the earth-orbiting spacecraft, and the utilization of organic materials in this context is here explored in comparison with the present state of the art placing emphasis in organic nanotechnology. Poly[3-3'(vinylcarbazole)] (PVK) was synthesized by oxidative polymerization with ferric chloride of N-vinylcarbazole. The resulting polymer was then deposited on solid support by using the Langmuir-Schaefer (LS) technique. The pressure-area isotherm of PVK revealed the possibility of compact monolayer formation at the air-water interface. Different layers of PVK were doped with iodine vapors. The cyclic voltammetry investigation of PVK-doped I2 showed a distinctive electrochemical behavior. The photoinduced charge transfer across a donor/acceptor (D/A) hybrid interface provided an effective method to study the PV properties of the composite LS films. The results are compared with other approaches within the biological framework, such as bacteriorhodopsin (BR), and organic nanostructured materials.

Bacteriorhodopsins↗
PubMed2003

Structural investigation of proapoptotic peptide by CD and NMR spectroscopy.

We have performed a systematic investigation of the structural features of the peptides Int (a sequence able to cross cell membranes) and Int-H1(S6A,F8A) (which shows interesting antitumoral properties). After screening in aqueous solution at different ionic strength and pH values, we analyzed the structures of the peptides in different water/trifluoroethanol mixtures by Circular Dichroism and Nuclear Magnetic Resonance techniques.

Amino Acid Sequence↗
PubMed2002

Protein nucleation and crystallization by homologous protein thin film template.

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.

Animals↗
PubMed2002

Chromatin of Trypanosoma cruzi: in situ analysis revealed its unusual structure and nuclear organization.

Chromatin of Trypanosoma cruzi is known to be organized in classical nucleosomal filaments, but surprisingly, these filaments do not fold in visible chromosomes and the nuclear envelope is preserved during cell division. Our hypothesis about the role of chromatin structure in regulating gene expression and, more generally, cell functioning, pressed us to verify if chromatin organization is modulated during the parasite life-cycle. To this end, we analyzed in situ the fine structural organization of T. cruzi chromatin by means of an integrated biophysical approach, using differential scanning calorimetry and fluorescence microscopy. We observed that logarithmic forms exhibit a less condensed chromatin with respect to the stationary ones. Thermal analysis revealed that parasite chromatin is organized in three main levels of condensation, barring from the polynucleosomal filament till to superstructured fibers. Besides, the fluorescence images of nuclei showed a characteristic chromatin distribution, with defined domains localized near to the nuclear envelope. While in stationary parasites, these regions are highly condensed, in logarithmic forms they unfold by extending themselves toward the center of nucleus. These observations suggest that, in comparison with higher eukaryotes, in T. cruzi the nuclear envelope plays an unusual and pivotal role in interphase and in mitosis.

Animals↗
PubMed2002

Expression, purification, and structural characterization of human histone H4.

Recombinant human histone H4 (hH4) was produced in milligrams quantities in Escherichia coli, without altering the codons of the original cDNA sequence. The hH4 cDNA was subcloned into the pQE30 expression vector, in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to electrophoretic homogeneity was obtained by nickel-chelating chromatography, followed by gel filtration. The final yield of the entire expression and purification process was about 1 mg of pure histone H4 per liter of bacterial culture. SDS-PAGE analysis showed for the recombinant H4 a molecular weight corresponding to the expected one (12,535 Da). Circular dichroism spectroscopy was used to estimate the secondary structural composition of recombinant histone, when it is isolated from the physiological core particle. It was observed that under these conditions histone H4 exhibits an altered secondary conformation. In order to induce the recombinant histone to assume a conformation more similar to the one measured when it is organized inside the nucleosome, we resuspended it in buffers at increasing ionic strengths and in the presence of different concentrations of trifluoroethanol. We tried also to mimic the physiological situation of histone H4 by adding an equimolar amount of a commercial DNA to the protein solution. Finally, an estimation of protein thermal stability was evaluated by spectropolarimetry.

Circular Dichroism↗