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The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

Polycrystalline samples of lysozyme were prepared with and without a Langmuir-Blodgett (LB) thin film template via both the hanging drop method and batch crystallisation. Powder diffraction methods are used to compare these samples and to measure their resistance to radiation damage at room temperature. The X-ray induced amorphisation of the samples was followed as a function of time and it was shown that diffraction does not entirely disappear even at very long exposure times. Two distinct kinetic timescales are evident suggesting that early and late stage processes are quite different. Radiation damage was also shown to be localized in the sample in the region where the beam impinges.

Biological Faculty, Lomonosov Moscow State University (MSU), Leninskie gory 1, Moscow 119234, Russia Department of Physics, University of Osnabruck, Barbarastr 7, Osnabruck 49076, Germany Department of Health Sciences (DISSAL), University of Genoa, Via Antonio Pastore 1, Genoa 16132, Italy Laboratories of Biophysics and Nanotechnologies (LBN), Department of Experimental Medicine (DIMES), University of Genoa, Via Antonio Pastore 3, Genoa 16132, Italy Nanoworld Institute Fondazione EL.B.A.Nicolini (NWI-FEN), Largo Redaelli 7, Pradalunga, Bergamo 24100, Italy

Conductometric monitoring of drug-gene and drug-protein interactions is of fundamental importance in the field of molecular pharmacology. Here, we present our main findings and characterizations of an important antiblastic used in neuro-oncology (Temozolomide), interacting with selected proteins that represent predictive biomarkers of the rate survival of the patients, of the outcome of chemotherapy and resistance to drug itself (namely, BRIP1 and MLH1). We use our previously introduced two genes along with previously described Nucleic Acid Programmable Protein Arrays (NAPPA)-based nanoconductometric sensor. We performed a positive control (Temozolomide plus MLH1 protein), a negative control (Temozolomide plus BRIP1 protein) and a multi-gene experiment (Temozolomide plus BRIP1&MLH1 being co-expressed), showing that we are able to properly perform pharmacoproteomics tasks, discriminating each protein and drug unique conductance curve as well as their interactions, even in the presence of multi-proteins being immobilized. Moreover, in the last part of our paper, we used a multiple regression model in order to predict the behavior of Temozolomide when exposed to BRIP1&MLH1 co-expressed and we showed that we are able to predict the drug-protein interaction profile with a good regression coefficient.

Laser-induced microfragmentation of LB nanotemplate-induced protein crystals in glycerol solution resu lts in distinct, coherently diffracting domains. Only c rystals produced according to the Langmuir-Blodgett (LB) nanotemplate technique reveal in all four prot eins being tested (lysozyme, insulin, thaumatin and ribonuclease) domains highly radiation resistant, w hile the crystals produced by the standard hanging drop crystallization method do not. Actually the very sa me laser exposure causes the disappearance of these “classical” protein crystals during the same time f rame of 40 min needed for the laser cutting in all four proteins being tested. The microdiffraction of micr ocrystals prepered by the combination of LangmuirBlodgett and Laser technologies proves that not onl y the Lysozyme survives the process, as shown recently by nanodifraction, but also all three other model p roteins appear to behave similarly well, namely ins ulin, thaumatin and ribonuclease. The result confirms the emerging of a new biophysical technique uniquely usefull for synchrotron radiation studies based on small protein microcrystals uniquely radiation resi stant when prepered by LB nanotemplate and subsequently fragmented by Laser.

Homogeneous matrices of calcium oxide (CaO) were prepared by mixing this material with polyethylene glycol (PEG) acting as malleable inert support in order to obtain processable composites. Preliminary tests were carried out to assess the best concentration of CaO in the composite, individuated in the CaO/PEG weight ratio of 1/4. Experimental data highlighted that the composite was able to selectively detect carbon dioxide (CO2) via a nanogravimetric method by performing the experiments inside an atmosphere-controlled chamber filled with CO2. Furthermore, the composite material showed a linear absorption of CO2 as a function of the gas concentration inside the atmosphere-controlled chamber, thus paving the way for the possible use of these matrices for applications in the field of sensor devices for long-term evaluation of accumulated environmental CO2.

Proteins represent versatile building blocks for realization of nanostructured materials to be applied in nanobiotechnology. In the present work, the Langmuir–Blodgett technique was utilized to develop nanobiodevices based on protein molecules. Particularly, lipase thin films were fabricated and characterized, with characterization performed in order to optimize the working parameters. As the first step the protein films were studied at the air–water interface and then transferred onto a solid support for further characterization. The films were characterized by different techniques such as UV–Vis spectroscopy, nanogravimetry, atomic force microscopy, and biochemical assays. Catalytic activity of lipase characterized by the maximal reaction rate found to increase over 10 times as a result of inclusion into LB films, while the substrate binding characterized by the Michaelis constant remain unchanged. Catalytic activity per mole of enzyme was found to increase with the increased number of LB layers up to five, and then decrease at 10, while the surface coverage ranged from 70% to 100% from 1 to 10 layers of lipase. This study exploits the possibility to employ LB based protein structures to use in biocatalysis, exemplified by lipase, which is known as an interfacially-activated enzyme, with olive oil as substrate, when lipase should already be in the maximally active state even without a film. We show, however, that it was possible to form even more active lipase nanostructures by the Langmuir–Blodgett technique at the air–water interface, proving that Langmuir-film provides a better catalytic effect in lipase than a mere oil–water boundary.

Chinese Hamster Ovary fibroblasts (CHO-K1) have shown different protein contents when undergoing differentiation by 3′,5′-cyclic adenosine monophosphate (cAMP), which is known to induce reverse transformation (RT) from malignancy to fibroblast-like characteristics. The mass spectrometry (MS) investigation here reported about the behavior of CHO-K1 cells before and after exposure to cAMP reveals a change in the composition of nuclear proteins associated to an inhibition of the protein expression. Possible implications of this finding on the control of cell reverse transformation are discussed. J. Cell. Biochem. 102: 473–482, 2007. © 2007 Wiley-Liss, Inc.

A correlated investigation of cell metabolism and protein expression of Chinese Hamster Ovary cells (CHO-K1) under different growth conditions was performed by 1H nuclear magnetic resonance (1H-NMR), mass spectrometry (MS) and biochemical assays. CHO fibroblasts have shown different metabolic products when grown in different media and when differentiation is stimulated by adenosine 3’, 5’ cyclic monophosphate (cAMP). In particular, while addition of Fetal Calf Serum causes an increase in the glucose metabolism correlated to changes in lipid composition of the membrane observed by 1 H-NMR, differentiation induced by cAMP causes biochemical differences in glucose and lipidic metabolism uniquely correlated both to the specific changes in the composition of nuclear proteins, revealed by mass spectrometry, and to the differences in metabolism, determined by NMR.

The unique photochromic retinal protein from rhabdomeric octopus membranes – octopus rhodopsin (OctR) has emerged as promising material for biomolecular photonic applications due to its unique properties and advantages. Here we report isolation of the novel full length octR gene from retina cDNA of Octopus vulgaris eyes and its sequence comparison with rhodopsins of other cephalopods and vertebrates. The isolated gene can be used to develop various expression systems for production of recombinant OctR for structural studies and novel optobioelectronic applications. The alignment of amino acid (a.a.) sequence with different opsins revealed similarity to cephalopoda rhodopsins (Rho) and to human melanopsin from intrinsically photosensitive retinal ganglion cells. The alingment of OctR a.a. sequence with mammalian and cephalopoda Rho with known 3D structures revealed promising substitutions V2C and W292C for developing stable and functionally active recombinant OctR after heterologous expression.

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.

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.

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.

(2004). Nanocrystallography: an emerging technology for structural proteomics. Expert Review of Proteomics: Vol. 1, No. 3, pp. 253-256.

The lipase-catalyzed degradation of poly(e-caprolactone) (PCL) is described in the present work. The enzymatic degradation of PCL, having number-averaged molecular weight (Mn) of about 87,000 g/mol and molecular weight dispersion (Mw/Mn) of 1.51, was carried out in organic solvent (toluene) and the effects of medium composition, temperature and source of lipase were evaluated and quantified. Three fungal lipases of Candida rugosa, Mucor miehei and Rhizopus delemar were tested to select the best biocatalyst for PCL degradation. The retention of catalytic activity of M. miehei lipase was determined in organic solvent at different temperatures by pH-stat titration, using olive oil as substrate. The experimental data of the activity coefficient versus time were then used to calculate, for each temperature, the corresponding value of the first-order denaturation constant (kd) and to estimate the main thermodynamic parameters of the enzyme thermal denaturation. The results of the degradation reaction, followed by size exclusion chromatography (SEC), showed that M. miehei lipase was able to catalyze the degradation of PCL with a maximum conversion degree of about 70% only after 1 h, within the temperature range of 40–60 °C. The catalytic activity of this biocatalyst demonstrated to be nearly stable at 40 °C, whereas the enzyme half-life was less than 2 h at 60 °C.

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.

Extra-small microcrystals of a human kinase CK2α 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 CK2α microcrystals (of about 20 μm 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 CK2α mutant is in the Protein Data Bank (PDB ID Code 1NA7, deposited on 27 November 2002). © 2004 Wiley-Liss, Inc.

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 α subunit. According to our theoretical predictions, we observed that the α-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.

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.

A review of the emerging trends and perspectives of Protein Nanocrystallography is here presented at the intersection of advances in nanotechnology (Langmuir-Blodgett and Anodic Porous Allumina), proteomics (microarray, cell free expression and SNAP) and synchrotron radiation (third generation sources trillion times more brilliant requiring quite smaller crystals and Montecarlo simulation). It should be noted that nanocrystallography here does not refer to crystals of nanometer size or to nanodrop crystallization technology, but to the significant applications for medicine emerging in our labs at the interface of Langmuir-Blodgett engineering, organic chemistry, molecular dynamics and label-free Protein Arrays, utilizing bacterial hell's gate globin, octapus rodhopsin, bovine cytochrome, human kinase, laccase and many other proteins.

International audience; Micro-beam Grazing-Incidence Small-Angle X-ray scattering (μ-GISAXS), exploiting both the advantages of elastic X-ray scattering and the highly focused third-generation synchrotron radiation micro-beams, is an advanced scattering technique that enables scientists to unravel the details of crystal growth processes and to investigate large-scale structures in thin films, including nanobiofilms or other different kinds of surfaces, such as surface gradients or confined surfaces. In this study, we analyze semi-quantitatively and we simulate our previously acquired μ-GISAXS experiments of Thaumatin and Lysozyme Langmuir-Blodgett (LB)-film, shedding light on nucleation and crystal growth processes. Here, we show that, during LB-thin film facilitated nucleation, the particle radius of Thaumatin and of Lysozyme crystal increases while the film thickness reduces. Structural re-organization inside and within the LB-thin film are likely to lead to the crystal nucleation and growth. These semi-quantitative findings are in agreement with the model previously hypothesized. New insights and implications for protein nanocrystallography are also discussed.

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 (hOtrf219–227) and KDLLFK (hOtrf269–301 and hOtrf633–638) 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.

The effect of histone acetylation was monitored on CHO chromatin structure, following the addition of 7 mM Na-butyrate to the cell culture medium. The properties of both control and hyperacetylated chromatins and nuclei were investigated by circular dichroism, ethidium bromide intercalation, differential scanning calorimetry, and affinity chromatography. Our results are compatible with modest but significant alterations in the various levels of chromatin organization, as a result of the charge neutralization of some lysine residues within the N-terminal region of the histonic octamer. Namely, large statistically significant differences do exist in the heat capacity thermograms of native nuclei, where unfolding into single nucleofilament of the highly packed native chromatin superfiber appears associated with acetylation; at the same time CD, EB, and affinity chromatography point to modest but consistent differences in the compactness of isolated nucleosomes and polynucleosomes.

New quantitative insights on the native high order chromatin-DNA structure existing within interphase nuclei are obtained by monitoring the effects of two common well-characterized fixatives, glutaraldehyde and ethanol/acetic acid mixture, at the level of the intranuclear DNA distribution and structures. Reproducible distinct levels of DNA fluorescence intensity and their intranuclear distribution are apparent in unfixed and fixed thymocytes by using DAPI and quantitative optical microscopy based on a charge coupled device. The fluorescent histograms correlated with the calorimetric thermograms on the very same thymocytes fixed and unfixed, establish an unequivocal baseline for the different levels of structural organization of the chromatin within the intact nucleus; namely their number, DNA packing ratio and fiber diameter. A systematic comparison among all the numerous models, being so far proposed for the quinternary and quaternary levels of DNA folding, to identifies the rope or ribbon-like and the chromonema as the ones that best fit with the in situ distribution.

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-β-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II.

Using Raman spectroscopy and lysozyme, this latter as model protein, we investigate the differences in protein conformation before and after LB nanotemplate-induced crystal nucleation and growth. It was found that the main difference in lysozyme conformation is associated to the higher amount of S-S bonds in lysozyme of LB crystals, probably in C-end of protein, resulting in the higher stiffness of the lysozyme molecules and LB crystal in a whole. Growth in size of LB crystal over time is also accompanied by the formation of S-S bonds. Atomic structure, determined by X-ray diffraction, correlates Raman spectroscopy results confirm the main differences between LB and classical crystals are in terms of water molecules environment previously associated to the increased radiation stability of LB crystals.

A silicon based potentiometric transducer (LAPS, Light Addressable Potentiometric Sensor) has been previously characterized both as a pH (Si 3 N 4 sensitive surface) and as a redox potential sensor (metal sensitive surface). The transducer, in the redox configuration, has been used to monitor the enzymatic activity of HRP (Horse Radish Peroxidase) which can be used as a label in an immunoenzymatic application. A competitive immunoenzymatic assay for the detection of the pesticide 2,4-D (2,4-dichlorophenoxyacetic acid) has been set up. A monoclonal antibody has been immobilized both on a preactivated membrane and glass. The values of enzymatic activity measured by the sensor in these samples versus the analyte concentration show the typical trend of competitive immunoassays and the limit of detection is comparable to the one obtained with the conventional colorimetric methods (Enzyme Linked Immunosorbent Assay (ELISA)). These standard assays have been used as a check for our results and also as guidelines for the setting up of the experiments with the sensor.

This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications.

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.

X-ray atomic structure of recombinant Hell's gate globin I (HGbI) from Methylacidophilum infernorum was calculated from the X-ray diffraction data of two different types of crystals: obtained by classical hanging drop and by LB nanotemplate method under the same crystallization conditions. After the accurate comparison of crystallographic parameters and electron density maps of two structures they appears to be quite similar, while the quality of the crystals grown by LB nanotemplate method was higher then of those grown by classical method. Indeed, the resolution of the LB crystal structure was 1.65 A, while classical crystals showed only 3.2 A resolution. Moreover, the reproducibility of this result in the case of LB crystals was much better-nine crystals from 10 gave the same structural results, while only two of 10 classical crystals were appropriate for the X-ray structure resolution.

Native chromatin has been characterized at different ionic strengths, according to different methods of preparation, by means of circular dichroism and ethidium bromide intercalation. For a more precise interpretation of these results, a series of analogous experiments on phagic DNA, as a function of its circularization, and on mononucleosomes have been performed. Results are discussed here in terms of high order chromatin structure, DNA supercoiling, and topological constraints.

Recent findings indicated that the SMILE gene may be involved in kidney graft operational tolerance in human. This gene was found to be up-regulated in blood from patients with a well functioning kidney transplant in the absence of immunosuppression compared to other transplanted recipients with clinically different status. A microarray study of SMILE knock-down and phorbol 12-myristate 13-acetate (PMA) activation in HeLa cells was herein compared to our earlier analysis based on microarray data of kidney allograft tolerance and rejection in humans and in a rat model of allograft transplantation to determine possible new genes and gene networks involved in kidney transplantation. The nearest neighbors at the intersection of the SMILE knock-down network with the human tolerance/rejection networks are shown to be NPHS1 and ARRB2, the former (Nephrin) being involved in kidney podocyte function, and the decrease of the latter (Arrestin β2) being recently shown to be involved in monocyte activation during acute kidney allograft rejection in rat. Moreover, another one of the neighbors at the intersection of SMILE network and tolerance/rejection networks is XBP-1, that we report previously to be increased, at a transcript level, after ER stress in SMILE silenced cells. Finally, in this study, we also show that topological properties (both local and global) of joint SMILE knock-down network—tolerance/rejection networks and joint PMA activation network—tolerance/rejection networks in rat and human are essentially different, likely due to the inherent nature of the gene SMILE and the mitogen PMA, that do not act the same way on genes and do not interfere the same way on networks. We also show that interestingly SMILE networks contain more feed-forward loop (FFL) motifs and thus SMILE calls for a more fine-tuned genetic regulation. J. Cell. Biochem. 113: 1820–1832, 2012. © 2011 Wiley Periodicals, Inc.

DNA microarrays are one of the most promising methods for molecular genomics, but this technique is often associated with experimental complications and difficulties in the analysis. Moreover, the greatest part of genes displayed on an array is often not directly involved in the cellular process being studied. Recently, we proposed a data mining algorithm, based on the identification of genes involved in a given process, the calculation of interactions among them and their ranking according to number of interactions. Genes in the highest cluster are defined as "leader genes". These findings may lead to an ad hoc and therefore more significant experimentation. However, at present this complex process is performed manually. In this work, we present the general architecture of LeaderGene, an automated tool for ab-initio molecular genomics. Three different and independent parts: (1) Identification of gene list; (2) Calculation of weighted number of links; (3) Genes clustering. Initial inputs are provided by user; then, output of part 1 and part 2, respectively, become inputs of parts 2 and 3. The development of an user-friendly software capable to automatically compute leader genes in a given cellular system will allow further progresses in this field of molecular genomics.

To the Editor, Much attention has been focused on the attempt to undertake global analysis of protein expression and in particular to analyze the changes in protein expression associated with several processes such as differentiation and cell cycle progression. In general, cell differentiation— or proliferation—requires de novo protein synthesis and/or post-translational modification, in particular phosphorylation of proteins such as transcription factors. Consequently, there is a need to identify the proteins that are being modified, as well as to know when and where in the cell modifications occur. We focused our studies on the Chinese hamster ovary (CHO) mammalian tumor cell line, a good model to study the cell differentiation. It is known that cyclic 30,50monophosphate adenosine (cAMP) transforms CHO cells, in vitro: they lose characteristics associated with malignancy and adopt characteristics of normal fibroblasts (‘‘reverse transformation’’) [3, 7, 9]. Puck et al. [8–10] found that cAMP regulates the structure of the cell cytoskeleton, which in turn strongly influences many differentiation properties including the assumption of normal or cancerous behavior in mammalian cells. In a previous study, we analyzed how CHO-K1 cells display different nuclear protein contents when undergoing differentiation by cAMP [14]; our Mass Spectrometry (MS) investigation revealed a change in the composition of nuclear proteins associated to an inhibition of the protein expression with possible implications for the control of cell reverse transformation [14]. The mechanism of the transformation, however, is as yet obscure and need further exploration in all compartments of cell proteome which we have carried out with this study. Fundamental changes in properties of the cell membrane have indeed been reported but the molecular mechanisms generating these structural changes are largely unknown. The changes in the concentration of the different metabolites and the different protein composition associated with morphological changes observed are also largely unknown [8]. This study wants to explore more deeply the effect of cAMP differentiation on the proteome of CHO cells, considering that the changes in the levels of nuclear envelopeassociated proteins and chromatin-condensing proteins [14] constitutes only the first step toward developing an understanding of cellular mechanisms for controlling nuclear shape. Due to the complexity of eukaryotic proteomes, and the divergence of protein properties, it is beneficial to prepare standardized partial proteomes in order to maximize the coverage of the proteome and to increase the chance to visualize low abundance proteins and make them accessible for subsequent analysis [1]. The strategies used to prepare such a complex proteomic samples for MS analysis involve many steps. For protein extraction we chose to perform a subcellular fractionation to separate the whole cellular proteome in four fractions. In literature, there are several works in which this approach to proteome analysis is adopted [1, 6, 12, 13], but always to analyze the behavior of a restrict number of proteins, for example to investigate changes in subcellular localization of regulatory proteins impacted by experimental or disease parameters. R. Spera C. Nicolini Nanoworld Institute, Fondazione El.B.A. Nicolini, Largo Redaelli 7, Pradalunga, Bergamo, Italy e-mail: rspera@ibf.unige.it

Tolerance is the so-called "Holy Grail" of transplantation but achieving this state is proving a major challenge, particularly in the clinical settings. This tolerance state can be induced in rodent models using a variety of maneuvers. This phenomenon is classically characterized by donor specificity (recipients accept a secondary donor-specific allograft but reject third-party allograft) as well as by the absence of chronic rejection lesion. We previously showed that administration and anti-donor anti-class II serum on the day of transplantation induce tolerance to a kidney allograft in the LEW-1W to LEW-1A strain combination. In this study, we used DNA microarrays to compare gene patterns involved in anti-donor anti-class II tolerated or untreated syngeneic kidney transplants in this strain combination. Statistical and non-statistical analyses were combined with ab initio analysis, using the recently developed leader gene approach, to shed new light on this phenomenon. Theoretical and experimental results suggest that tolerance and rejection outcome may be in large part determined by low expression variations of some genes, which can form a core gene network around specific genes such as Rac1, NFKB1, RelA, AKT1, IKBKB, BCL2, BCLX, and CHUK. Through this model, we showed that AKT1 gene, WNT pathway and NO synthesis are strictly connected to each other and may play an important role in kidney tolerance and rejection processes, with AKT1 gene being the center of this complex network of interactions.

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-β tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1–2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells. © 1992 Wiley-Liss, Inc.

Three different approaches (propensity curve shifting, hydropathy index evaluation, and iterative attribution/ cancellation of secondary structure) to the use of secondary structure percentages derived from circular dichroism measurements to improve the success rate of a protein secondary structure prediction method, without using decision constants, are described and compared. Propensity-curve shifting appears to be the best-performing approach, bearing an increase of 5.3% in the success rate of single-residue structural prediction when exact information on the secondary structure, obtained by X-ray crystallography, is employed; with information of an accuracy comparable to that obtainable by circular dichroism, the improvement stays between 3.5 and 4.9%, for a three-state prediction. Although developed with circular dichroism in mind, the method can use percentages of secondary structure obtained by any other experimental methodology from which they can be inferred, for instance Raman spectroscopy and infrared spectroscopy.

Single crystals of ribosomal proteins obtained for the first time by Langmuir-Blodgett (LB) nanotemplate confirm earlier findings (Pechkova et al., 2008), pointing to a new generation of bionanomaterials of unique structure-function relationship. The ribosomal protein phage GroEL was overexpressed in E. coli. Since these protein's samples have some difficulties by classical vapour diffusion method to yield optimal diffraction quality and order (GroEL), the LB nanotemplate method has been applied and compared to the classical method. With the thin film nanotemplate method large phage GroEL crystals appeared in few days and were subsequently characterized by MALDI-TOF Mass Spectroscopy and by a very preliminary X-ray diffraction.

Survival of solid organ grafts depends on life-long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous "operational tolerance" can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross-validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis.

A comprehensive bioinformatic analysis was performed on all protein homologous pairs from mesophilic and thermophilic microorganisms present in the RCSB Protein Data Bank in order to yield a clue on the role of protein structure and aqueous environment. Subsequently self-assembly and LB studies were carried out at increasing temperature by nanogravimetry with thermostable thioredoxin (Trx) from Alicyclobacillus acidocaldarius (BacTrx) versus the mesophilic Escherichia coli counterpart (EcTrx). The comparison with earlier 3D atomic structure determined on the same proteins by X-ray crystallographic diffraction and nuclear magnetic resonance confirm the role inner bound water in determining protein thermostability, as suggested by the bioinformatic and nanogravimetric analysis. The above comparative characterizations in protein solution, thin film and crystal allow to draw a possible coherent explanation for the origin and the molecular mechanisms of both heat stability and radiation resistance in proteins.

Cytochromes P450 are a large superfamily of heme-thiolate enzymes involved in the metabolism of many different organic substrates such as drugs, fatty acids and toxic compounds. The aim of this work is to analyse the binding between the cytochrome P4501A2, in solution and in gel-matrix, and its substrate (clozapine), utilising voltammetric tests. The interaction measurements were carried out using two different screen printed electrodes (rhodium-graphite and graphite-riboflavin), and the results were compared. It was demonstrated that it is possible to realise a biosensor prototype to detect the presence of clozapine indirectly by chronoamperometry.

The early steps of growth and nucleation of the lysozyme microcrystals by classical and nanotemplate-based hanging vapor diffusion methods are studied using µGISAXS 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 µGISAXS 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. J. Cell. Biochem. © 2005 Wiley-Liss, Inc.

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. J. Cell. Biochem. 97: 1151–1159, 2006. © 2005 Wiley-Liss, Inc.

The key genes involved in the cell cycle of human T lymphocytes were identified by iterative searches of gene-related databases, as derived also from DNA microarray experimentation, revealing and predicting interactions between those genes, assigning scores to each of the genes according to numbers of interaction for each gene weighted by significance of each interaction, and finally applying several types of clustering algorithms to genes basing on the assigned scores. All clustering algorithms applied, both hierarchical and K-means, invariably selected the same six "leader" genes involved in controlling the cell cycle of human T lymphocytes. Relations of the six genes to experimental data describing switching between stages of cell cycle of human T lymphocytes are discussed.