Biomedical subjects

Claudio Nicolini

Publications and source records attributed to Claudio Nicolini.

At least 19 recordsLinked to original sources

PubMed2006

Gene expression of human T lymphocytes cell cycle: experimental and bioinformatic analysis.

Human lymphocytes gene expression is monitored before and after PHA stimulation over 72 h, using DNA microarray technology. Results are then compared with our previous bioinformatics predictions, which identified six leader genes of highest importance in human T lymphocytes cell cycle. Experimental data are strikingly compatible with bioinformatic predictions of the specific role and interaction of PCNA, CDC2, and CCNA2 at all phases of the cell cycle and of CHEK1 in regulating DNA repair and preservation. It does not escape our notice that the conception and use of ad hoc arrays, based on a bioinformatics prediction which identifies the most important genes involved in a particular biological process, can really be an added value in cell biology and cancer research alternative to massive frequently misleading molecular genomics.

CDC2 Protein Kinase↗

PubMed2006

Gene expression in the cell cycle of human T-lymphocytes: II. Experimental determination by DNASER technology.

Human lymphocytes gene expression before and after PHA stimulation is monitored by DNASER technology, a novel bioinstrumentation entirely constructed in our laboratories as previously reported. The validity of the DNASER measurements is confirmed by standard fluorescence microscopy equipped with CCD. The human lymphocytes gene expression here experimentally probed using commercially available DNA microarrays such as Human Starter, appears compatible both with independent bioinformatic prediction and with existing experimental data, pointing to MYC as the key gene in the G0-G1 transition induced by PHA in resting lymphocytes. It does not escape our notice that in cell biology and cancer research DNASER technology based on microarray constructed with few leader genes identified from bioinformatics represents a meaningful cost-effective route alternative to massive frequently misleading molecular genomics.

Cell Cycle↗

PubMed2006

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: I. microGISAXS and microdiffraction of P450scc.

Ultrasmall P450scc cytochrome microcrystals are grown by classical hanging vapor diffusion and by its modification using homologous protein thin-film template displaying a long-range order. The nucleation and growth mechanisms of P450scc microcrystals are studied at the thin cytochrome film surface by a new microbeam grazing incidence small angle X-ray scattering (microGISAXS) technique developed at the microfocus beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. P450scc cytochrome crystals of about 5 microm are also investigated by synchrotron radiation diffraction in order to attempt a preliminary analysis of the atomic structure of this unique protein system yet unsolved.

Cholesterol Side-Chain Cleavage Enzyme↗

PubMed2006

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: II. microGISAX and microscopy of lysozyme.

The early steps of growth and nucleation of the lysozyme microcrystals by classical and nanotemplate-based hanging vapor diffusion methods are studied using microGISAXS at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Out-of-plane cuts in the Yoneda regions of the 2D scattering profiles point to the detection of ultrasmall lysozyme crystals by microGISAXS quite before than by light microscopy. Furthermore lysozyme crystal formation occurs quite earlier with the nanotemplate than with the classical method. Our data are compatible with two distinct modes of crystal nucleation and growth for P450sc and lysozyme.

Crystallization↗

PubMed2006

Mapping electrostatic potential of a protein on its hydrophobic surface: implications for crystallization of Cytochrome P450scc.

Calculation and combined visualization of electrostatic and hydrophobic properties of Cytochrome P450scc based on two very different homology models allowed to identify extensive hydrophobic patches with neutral electrostatic potential and mutations removing such patches and thus expecting to facilitate crystallization of Cytochrome P450scc, especially for the nanotemplate crystallization method. Implications are discussed for optimizing crystallization and other aspects of protein surface properties and protein recognition.

Animals↗

PubMed2006

Nanostructured biofilms and biocrystals.

The design, the physical properties and the implementation of nanostructured protein biofilms and nanostructured biocrystals are here summarized from ab initio considerations. Particular emphasis is placed on those being developed in our Institute having unique properties useful in a wide range of applications.

Animals↗

PubMed2005

Theoretical framework for octopus rhodopsin crystallization.

Bacteriorhodopsin (bR) is presently a classical example of membrane protein crystallization. We are comparing the structure of bR with the homology model of octopus rhodopsin (octR), which is similar in topology to bR and as highly ordered in its native membranes as bR in purple membranes. Such comparison provides insights for optimization of present octR experimentation both for crystallization and for application in nanobiotechnology in a manner similar to bR, and possibly even superior in optical computation.

Animals↗

PubMed2005

microGISAXS and protein nanotemplate crystallization: methods and instrumentation.

Microbeam grazing-incidence small-angle X-ray scattering (microGISAXS) has been used and the technique has been improved in order to investigate protein nucleation and crystal growth, assisted by a protein nanotemplate. The aim is to understand the protein nanotemplate method in detail, as this method has been proved capable of accelerating and increasing crystal size and quality as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing lysozyme crystals at three different stages of growth.

Crystallography, X-Ray↗

PubMed2005

Ultrathin films of tetrasulfonated copper phthalocyanine-capped titanium dioxide nanoparticles: fabrication, characterization, and photovoltaic effect.

Tetrasulfonated copper phthalocyanine (CuTsPc)-capped TiO2 nanoparticle ultrathin films were fabricated by a layer-by-layer (LBL) self-assembly technique. Alternating bilayer structures were formed by consecutive adsorption of CuTsPc-capped TiO2 nanoparticles with poly(diallyldimethylammonium chloride). Optical and electrical measurements were carried out to characterize the CuTsPc-capped TiO2 nanoparticle ultrathin films. Optical measurements revealed uniform deposition. The surface morphology of such bilayer films showed a granular morphology similar to other classes of LBL films. Electrical measurements revealed an abnormal phenomenon under white light illumination. Electrochemical and photoelectrochemical measurements on such a bilayer system were carried out to elucidate the electron transport processes and the photovoltaic effect.

Indoles↗

PubMed2005

A heterostructure composed of conjugated polymer and copper sulfide nanoparticles.

A heterostructure formed by a conjugated polymer and semiconducting nanoparticles was produced. The conjugated polymer was synthesized by oxidative copolymerization of 3-thiopheneacetic acid and 3-hexylthiophene, thus obtaining an amphiphilic polythiophene that allows the formation of a stable polymer layer at the air-water interface. Different numbers of monolayers were deposited on solid substrates. CuS nanoparticles were grown directly in the polymeric matrix using the carboxylic groups as nucleation centers. The reactions were monitored by quartz crystal microbalance, Brewster angle, and fluorescence microscopy. The heterostructure showed increased conductivity as compared to the pristine polymer.

Copper↗

PubMed2005

Site-directed mutations (Asp405Ile and Glu124Ile) in cytochrome P450scc: effect on adrenodoxin binding.

Cytochrome P450scc, mitochondrial adrenodoxin (Adx), and adrenodoxin reductase (AdR) are an essential components in a steroid hydroxylation system. In particular, mytochondrial cytochrome P450scc enzyme catalyses the first step in steroid hormones biosynthesis, represented by the conversion of cholesterol to pregnenolone. In order to study the effect of single mutations on the Adx binding a model of bovine cytochrome P450scc, previously optimized by molecular modeling, was utilized. It was hypothesized by molecular docking that two residues (Asp405 and Glu124) are involved in Adx binding. By site-directed mutagenesis, two mutants of cytochrome P450scc (Asp405Ile and Glu124Ile) expressed in Escherichia coli, were realized by replacing with isoleucines. The site-directed mutations effect on Adx binding was evaluated by differential spectral titration. The apparent dissociation constant values for Asp405Ile and Glu124Ile cytochrome P450scc show that the mutated residues seem to be at the interaction domain with Adx or at least close to it, as predicted by molecular modeling study. Finally, the engineered enzymes were characterized by biochemical and biophysical techniques such as circular dichroism (CD), UV/Vis spectroscopy, and electrochemical analysis.

Adrenodoxin↗

PubMed2005

Homology modeling of cytochrome P450scc and the mutations for optimal amperometric sensor.

A new homology model of bovine cytochrome P450scc is obtained starting from the recently determined crystal structure of mammalian cytochrome P450 2B4. The new emerging structure appears compatible with recent diffraction patterns of bovine P450scc microcrystals as obtained at the Microfocus Beamline of the European Synchrotron Radiation in Grenoble and here reported for the first time. The same atomic structure is utilized thereby to predict the mutations needed for modifying redox potential. A comprehensive comparison is finally carried out with the previous model present in the RCSB Protein DataBank also in terms of the alternative mutations being predicted for the same functional modification. The implication of these studies for optimal sensor construction is discussed.

Animals↗

PubMed2005

Antiviral activity of ovotransferrin derived peptides.

Ovotransferrin and lactoferrin are iron-binding proteins with antiviral and antibacterial activities related to natural immunity, showing marked sequence and structural homologies. The antiviral activity of two hen ovotransferrin fragments DQKDEYELL (hOtrf(219-227)) and KDLLFK (hOtrf(269-301) and hOtrf(633-638)) towards Marek's disease virus infection of chicken embryo fibroblasts is reported here. These fragments have sequence homology with two bovine lactoferrin fragments with antiviral activity towards herpes simplex virus, suggesting that these fragments could have a role for the exploitation of the antiviral activity of the intact proteins towards herpes viruses. NMR analysis showed that these peptides, chemically synthetized, did not possess any favourite conformation in solution, indicating that both the aminoacid sequence and the conformation they display in the intact protein are essential for the antiviral activity.

Animals↗

PubMed2005

Investigating crystal-growth mechanisms with and without LB template: protein transfer from LB to crystal.

Protein nanocrystallography has recently been introduced as a unique nanotechnology-based approach to forming stable protein crystals and to characterize them down to atomic resolution. In particular, a protein nanostuctured template appears to be capable of stimulating nucleation and crystal growth of so far unsolved proteins. In the present communication, aimed at investigating the lysozyme crystal-growth mechanisms with and without nanotemplate, the lysozymes appear to transfer directly from the nanostructured film into the drop to trigger the formation of the crystal, therefore highlighting the physical interpretation of the mechanism for nanotemplate-induced protein crystallization.

Animals↗

PubMed2005

Comparison of lysozyme structures derived from thin-film-based and classical crystals.

The present report is dedicated to a systematic comparison of crystal structures produced by the nanobiofilm template method and by the classical hanging-drop vapour-diffusion method. Crystals grown by the innovative nanostructured template method appear indeed radiation-resistant even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The implications of this finding for protein crystallography are discussed here in terms of water redistribution and of the detailed atomic resolution comparative studies of the two crystal structures with or without nanobiofilm template, as emerging also from circular-dichroism and thermal denaturation studies.

Animals↗

PubMed2005

Langmuir--Schaefer films of Nafion with incorporated TiO(2) nanoparticles.

An easy method of incorporating TiO(2) nanoparticles into Nafion perfluorinated ionomer is proposed. Ultrathin films of Nafion were prepared by employing the Langmuir-Schaefer (LS) technique. The pressure-area isotherm study of a Langmuir monolayer of Nafion at the air-water interface on different concentrations of NaCl as the subphase allowed us to find the best experimental conditions for the deposition of stable Langmuir-Schaefer films. Incorporation of TiO(2) nanoparticles was performed by dipping Nafion LS films in a solution of TiO(2) nanoparticles. The uniformity of the TiO(2) incorporation was detected by UV-visible spectroscopy. The morphology of the Nafion, Nafion/TiO(2) nanoparticles thin films, and the changes due to the annealing procedure were investigated by atomic force microscopy. Interestingly, the AFM investigation showed that Nafion and Nafion/TiO(2) LS films have thermal stability up to 600 degrees C.

Journal Article↗

PubMed2005

An in-vitro study of the sterilization of titanium dental implants using low intensity UV-radiation.

OBJECTIVES: Commercial titanium dental implants are coated with nanostructured TiO2. The aim of the research reported in this paper was to assess whether the TiO2 at the surface of a dental implant is sufficiently photoactive to eradicate bacteria when illuminated with low intensity light. METHODS: The photoactivity of dental implants was established by studies of the photoenhanced decomposition of Rhodamine B. In vitro studies to establish the influence of irradiating with UV light an implant that is immersed in a solution containing Escherichia Coli were performed. RESULTS: It was demonstrated that under low UV intensity irradiation, 49 microW cm(-2), bacteria are killed at a rate of approximately 650 million per cm2 of implant per minute. SIGNIFICANCE: The results indicate that illumination of dental implants with UV light may be a suitable treatment for periimplantitis.

Dental Implants↗

PubMed2004

Recombinant cytochrome p450 immobilization for biosensor applications.

As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.

Biosensing Techniques↗