Adsorption and stability of streptavidin on cluster-assembled nanostructured TiOx films

The study of the adsorption of proteins on nanostructured surfaces is of fundamental importance to understand and control cell-surface interactions and, notably, cell adhesion and proliferation; it can also play a strategic role in the design and fabrication of nanostructured devices for postgenomic and proteomic applications. We have recently demonstrated that cluster-assembled nanostructured TiO x films produced by supersonic cluster beam deposition possess excellent biocompatibility and that these films can be functionalized with streptavidin, allowing the immobilization of biotinylated retroviral particles and the realization of living-cell microarrays for phenotype screening. Here we present a multitechnique investigation of the adsorption mechanisms of streptavidin on cluster-assembled TiO x films. We show that this nanostructured surface provides an optimal balance between adsorption efficacy and protein functionality. By using low-resolution protein arrays, we demonstrate that a layer of adsorbed streptavidin can be stably maintained on a cluster-assembled TiO x surface under cell culture conditions and that streptavidin retains its biological activity in the adsorbed layer. The adsorption mechanisms are investigated by atomic force microscopy in force spectroscopy mode and by valence-band photoemission spectroscopy, highlighting the potential role of the interaction of the exposed carboxyl groups on streptavidin with the titanium atoms of the nanostructured surface.     

Site selectivity in the protonation of a phosphinito bridged Pt(I)-Pt(I) complex: a combined NMR and density-functional theory mechanistic study

The protonation of the dinuclear phosphinito bridged complex [(PHCy2)Pt(mu-PCy2){kappa(2)P,O-mu-P(O)Cy2}Pt(PHCy2)] (Pt-Pt) (1) by Br?nsted acids affords hydrido bridged Pt-Pt species the structure of which depends on the nature and on the amount of the acid used. The addition of 1 equiv of HX (X = Cl, Br, I) gives products of formal protonation of the Pt-Pt bond of formula syn-[(PHCy2)(X)Pt(mu-PCy2)(mu-H)Pt(PHCy2){kappaP-P(O)Cy2}] (Pt-Pt) (5, X = Cl; 6, X = Br; 8, X = I), containing a Pt-X bond and a dangling kappa P-P(O)Cy2 ligand. Uptake of a second equivalent of HX results in the protonation of the P(O)Cy2 ligand with formation of the complexes [(PHCy2)(X)Pt(mu-PCy2)(mu-H)Pt(PHCy2){kappaP-P(OH)Cy2}]X (Pt-Pt) (3, X = Cl; 4, X = Br; 9, X = I). Each step of protonation is reversible, thus reactions of 3, 4, with NaOH give, first, the corresponding neutral complexes 5, 6, and then the parent compound 1. While the complexes 3 and 4 are indefinitely stable, the iodine analogue 9 transforms into anti-[(PHCy2)(I)Pt(mu-PCy2)(mu-H)Pt(PHCy2)(I)] (Pt-Pt) (7) deriving from substitution of an iodo group for the P(OH)Cy2 ligand. Complexes 3 and 4 are isomorphous crystallizing in the triclinic space group P1 and show an intramolecular hydrogen bond and an interaction between the halide counteranion and the POH hydrogen. The occurrence of such an interaction also in solution was ascertained for 3 by (35)Cl NMR. Multinuclear NMR spectroscopy (including (31)P-(1)H HOESY) and density-functional theory calculations indicate that the mechanism of the reaction starts with a prior protonation of the oxygen with formation of an intermediate (12) endowed with a six membered Pt(1)-X...H-O-P-Pt(2) ring that evolves into thermodynamically stable products featuring the hydride ligand bridging the Pt atoms. Energy profiles calculated for the various steps of the reaction between 1 and HCl showed very low barriers for the proton transfer and the subsequent rearrangement to 12, while a barrier of 29 kcal mol(-1) was found for the transformation of 12 into 5.     

HPLC with diode-array detection for the simultaneous determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in seminal plasma

A high-performance liquid chromatographic method for the simultaneous determination of di(2-ethylhexyl)phthalate (DEHP) and its major metabolite mono(2-ethylhexyl)phthalate (MEHP) in seminal plasma was developed and validated. The method involves liquid-liquid extraction followed by isocratic reversed-phase chromatography with diode-array detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from the analysis of spiked seminal plasma samples. The effect of mobile-phase composition and pH on the retention of the target analytes was investigated. The limits of detection were 0.010 and 0.015 microg/mL, for DEHP and MEHP, respectively. This method was used to analyze real samples in support of clinical studies on these potential endocrine disruptors.     

Mass spectrometry analysis of gliadins in celiac disease

In recent years, scientific research on wheat gluten proteins has followed three main directions aimed at (1) finding relationships between individual genetic alleles coding for gliadins, high or low molecular weight glutenin subunits, and the viscoelastic dough properties of flour-derived products such as pasta and bread; (2) identifying prolamins and derived peptides involved in celiac disease, a pathological condition in which the small intestine of genetically predisposed individuals is reversibly damaged; and (3) developing and validating sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for celiac disease patients. In this review, the main aspects of current and perspective applications of mass spectrometry and proteomic technologies to the structural characterization of gliadins are presented, with focus on issues related to detection, identification, and quantification of intact gliadins, as well as gliadin-derived peptides relevant to the biochemical, immunological, and toxicological aspects of celiac disease.     

DNA and RNA noncovalent interaction of platinum(II) polypyridine complexes

A comparative investigation of the noncovalent interaction of the platinum(II) polypyridine complexes [Pt(dipy)(n-Rpy)2]2+ and [Pt(4,4'-Me2dipy)(2-Rpy)2]2+ (dipy = 2,2'-dipyridine; Me = CH3; n = 2-4; R = H or CH3) with double-helical DNA (calf thymus) and RNA [poly(A).poly(U)] has been conducted. With the exception of [Pt(dipy)(2-Mepy)2]2+, all of the complexes interact strongly, by intercalation, with both nucleic acids giving rise to large changes in the electronic spectra and induced circular dichroism signals; in addition, viscosity experiments on rodlike DNA and RNA show that both biopolymers elongate upon interaction with the complexes. The binding constant values, KB, determined at 25 degrees C, indicate that, at 0.101 M ionic strength, the affinity for poly(A).poly(U) is strongly dependent on the complexes nature, while for DNA it is leveled off. [Pt(dipy)(2-Mepy)2]2+ binds to DNA but does not interact appreciably with poly(A).poly(U).     

Curcumin Loaded Dendrimers Specifically Reduce Viability of Glioblastoma Cell Lines

Glioblastoma (GB) is a deadly and aggressive cancer of the CNS. Even with extensive resection and chemoradiotherapy, patient survival is still only 15 months. To maintain growth and proliferation, cancer cells require a high oxidative state. Curcumin, a well-known anti-inflammatory antioxidant, is a potential candidate for treatment of GB. To facilitate efficient delivery of therapeutic doses of curcumin into cells, we encapsulated the drug in surface-modified polyamidoamine (PAMAM) dendrimers. We studied the in vitro effectiveness of a traditional PAMAM dendrimer (100% amine surface, G4 NH₂), surface-modified dendrimer (10% amine and 90% hydroxyl-G4 90/10-Cys), and curcumin (Cur)-encapsulated dendrimer (G4 90/10-Cys-Cur) on three species of glioblastoma cell lines: mouse-GL261, rat-F98, and human-U87. Using an MTT assay for cell viability, we found that G4 90/10-Cys-Cur reduced viability of all three glioblastoma cell lines compared to non-cancerous control cells. Under similar conditions, unencapsulated curcumin was not effective, while the non-modified dendrimer (G4 NH₂) caused significant death of both cancerous and normal cells. By harnessing and optimizing the components of PAMAM dendrimers, we are providing a promising new route for delivering cancer therapeutics. Our results with curcumin suggest that antioxidants are good candidates for treating glioblastoma.     

Semi-automated, solid-phase extraction procedure for liquid chromatographic determination of papaverine, diltiazem, desipramine and nicardipine in urine

Summary A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure is reported for the assay of papaverine, diltiazem, desipramine and nicardipine in urine. Disposable extraction cartridges (DECs) filled with C18, C8, C2, CH and PH silica-bonded phases were used. The effect on recovery of sample pH, composition of washing and elution solvents and nature of SPE cartridge were evaluated. The selectivity of SPE was examined using spiked urine samples and the PH cartridge gave rise to the cleanest extracts. Phenyl cartridges were conditioned with methanol and acetic acid-sodium acetate buffer. Urine sample was buffered and then applied to the DEC. The washing step was with acetone-water and subsequently with methanol-acetate buffer. The analytes were eluted with methanol-acetate buffer. The extract was evaporated to dryness, reconstituted in mobile phase, and chromatographed on a reversed-phase C18 column with UV detection at 212 nm. Recoveries of the tested compounds from spiked urine samples using the PH cartridge were in all cases>80%. The within-day and between-day repeatabilities were<5% and 9%, respectively.     

A novel interferometric method for contour mapping of optically rough surfaces

A new white light interferometric arrangement for contour mapping of light diffusing but not depolarising surfaces is presented and compared with related electronic speckle and holographic interferometry (ESPI and HI) methods. Using two angles of illumination of the tested surface, and modifying the light beam in the observation pupil it is possible to observe contour fringes with arbitrary sensitivity depending on the geometry of a chosen set-up.     

Improved Gain Characteristics in Er3+-Doped Alumina (Al2O3) Channel Optical Waveguide Amplifiers for WDM Systems

Er3+-doped Al2O3 optical waveguides are analyzed both as single amplifiers and as elements of a 16-channel wavelength division multiplexing transmission system; their performance is compared with that from Er3+-doped SiO2 optical waveguide amplifiers. The amplifier model, based on propagation and population-rate equa tions, includes both uniform and pair-induced up-conversion mechanisms and is solved numerically by combining finite elements and the Runge-Kutta algorithm. The analysis showed that Er3+-doped Al2O3 waveguides exhibit much higher signal gains than Er3+-doped SiO2 waveguides and also that the use of Er3+-doped Al2O3 waveguides reduces the deleterious gain peaking effect, increasing the maximum transmission distance.