Effects of growth factors and basement membrane proteins on the phenotype of U-373 MG glioblastoma cells as determined by the expression of intermediate filament proteins

Various growth factors and basement membrane proteins have been implicated in the pathobiology of astrocytomas. The goal of this study was to determine the relative contribution of these two factors in modulating the phenotype of U-373 MG glioblastoma cells as determined by the expression of the intermediate filament proteins glial fibrillary acidic protein, vimentin, and nestin. For these determinations, cells plated in serum-free medium were treated either with growth factors binding to tyrosine kinase receptors including transforming growth factor-alpha, epidermal growth factor, platelet-derived growth factor-AA, basic fibroblast growth factor, and insulin-like growth factor-1 or with basement membrane proteins including collagen IV, laminin, and fibronectin. The changes in the expression levels of intermediate filament proteins in response to these treatments were analyzed by quantitation of immunoblots. The results demonstrate that collagen IV and growth factors binding to tyrosine kinase receptors decrease the glial fibrillary acidic protein content of U-373 MG cells. Growth factors binding to tyrosine kinase receptors also decrease the vimentin content of these cells but do not affect their nestin content. On the other hand, basement membrane proteins decrease the nestin content of U-373 MG cells but do not affect their vimentin content. The significance of these results with respect to the role played by different factors in modulating the phenotype of neoplastic astrocytes during tumor progression is discussed.     

Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites

Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo[a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for high-throughput in vitro genotoxicity screening.     

Synergistic metabolic toxicity screening using microsome/DNA electrochemiluminescent arrays and nanoreactors

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time reflects relative DNA damage rates. "Toxic hits" uncovered by the array were studied in structural detail by using enzyme/DNA films on silica nanospheres as "nanoreactors" to provide nucleobase adducts from reactive metabolites. The utility of this synergistic approach was demonstrated by estimating relative DNA damage rates of three mutagenic N-nitroso compounds and styrene. Relative enzyme turnover rates for these compounds using ECL arrays and LC-UV-MS correlated well with TD 50 values for liver tumor formation in rats. Combining ECL array and nanoreactor/LC-MS technologies has the potential for rapid, high-throughput, cost-effective screening for reactive metabolites and provides chemical structure information that is complementary to conventional toxicity bioassays.     

Synthesis and characterization of azo‐based methacrylate polymers with methoxy and nitro end groups for nonlinear optical applications

One new and three already described azobased methacrylate monomers with methoxy and nitro end groups and spacer length 2 and 6 were synthesized. These monomers were copolymerized with methyl methacrylate and the monomers as well as copolymers were characterized by classical spectroscopy techniques (FTIR, NMR, and UV‐VIS), gel permeation chromatography (GPC), elemental analysis and thermal analysis (TGA and DSC). The glass transition temperature of the polymers was found to be above room temperature and thermal decomposition temperatures above 100°C. All the polymers were amorphous in nature and formed excellent homogeneous films with good optical transparency. The polymer films coated on indium tin oxide glass slides were poled and their order parameters were calculated to check the stability of oriented dipoles. Few samples were also studied for their second harmonic generation properties. Temporal stability, checked up to 120 h at room temperature, was found to be quite satisfactory. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 3497–3504, 2007     

Charge derivatization of peptides for analysis by mass spectrometry

The analysis of peptide derivatives by fast atom bombardment, liquid secondary-ionization mass spectrometry, plasma desorption, electrospray ionization, and matrix-assisted laser desorption/ionization is reviewed. The fragmentation patterns of peptides and of charge-derivatized peptides are compared, and the proposed fragment ion structures are summarized. A variety of derivatization approaches and the distinguishing features of mass spectra produced from these derivatives are described. The most promising derivatization approaches are evaluated, and the strengths and limitations of these approaches are discussed.     

Modeling path duration distributions in MANETs and their impact on reactive routing protocols

We develop a detailed approach to study how mobility impacts the performance of reactive mobile ad hoc network routing protocols. In particular, we examine how the statistics of path durations including probability density functions vary with the parameters such as the mobility model, relative speed, number of hops, and radio range. We find that at low speeds, certain mobility models may induce multimodal distributions that reflect the characteristics of the spatial map, mobility constraints and the communicating traffic pattern. However, this paper suggests that at moderate and high velocities the exponential distribution with appropriate parameterizations is a good approximation of the path duration distribution for a range of mobility models. Analytically, we show that the reciprocal of the average path duration has a strong linear relationship with the throughput and overhead of dynamic source routing (DSR), which is also confirmed by simulation results. In addition, we show how the mathematical expression obtained for the path duration distribution can also be used to prove that the nonpropagating cache hit ratio in DSR is independent of velocity for the freeway mobility model. These two case studies illustrate how various aspects of protocol performance can be analyzed with respect to a number of significant parameters including the statistics of link and path durations.     

Homology modeling of human sialidase enzymes NEU1, NEU3 and NEU4 based on the crystal structure of NEU2: hints for the design of selective NEU3 inhibitors

Four types of human sialidases have been cloned and characterized at the molecular level. They are classified according to their major intracellular location as intralysomal (NEU1), cytosolic (NEU2), plasma membrane (NEU3) and lysosomal or mitochondrial membrane (NEU4) associated sialidases. These human isoforms are distinct from each other in their enzymatic properties as well as their substrate specificity. Altered expression of sialidases has been correlated with malignant transformation of cells and different sialidases have been known to behave differently during carcinogenesis. Particularly, increased expression of NEU3 has been implicated in the survival of various cancer cells and also in the development of insulin resistance. In the present study, we have modeled three-dimensional structures of NEU1, NEU3 and NEU4 based on the crystal structure of NEU2 using the homology modeling program MODELER. The best model in each enzyme case was chosen on the basis of various standard protein analysis programs. Predicted structures and the experimental protein-ligand complex of NEU2 were compared to identify similarities and differences among the active sites. The molecular electrostatic potential (MEP) was calculated for the predicted models to identify the differences in charge distribution around the active site and its vicinity. The primary objective of the present work is to identify the structural differences between the different isoforms of human sialidases, namely NEU1, NEU2, NEU3 and NEU4, thus providing a better insight into the differences in the active sites of these enzymes. This can in turn guide us in the better understanding and rationale of the differential substrate recognition and activity, thereby aiding in the structure-based design of selective NEU3 inhibitors.     

Microscale-calibrated modeling of the deformation response of low-carbon martensite

Micropillar compression tests were used to determine the uniaxial compressive stress–strain response of martensite blocks extracted from a low-carbon, fully lath martensitic sheet steel, M190, with the nominal composition C = 0.18, Mn = 0.47, P = 0.007, S = 0.006, Si = 0.18, Al = 0.06, Ti = 0.045, B = 0.0014 and balance Fe (all in wt.%). Specimens with a diameter exceeding ～1 μm and consisting of a single martensite block showed elastic–nearly perfectly plastic behavior with a yield stress of the order 1200 MPa. Similar specimens which contained multiple martensite blocks showed pronounced strain hardening, arising from the geometrical constraint produced by the interface(s). No size dependence of flow stress was observed in micropillars with diameters exceeding 1.0 μm, but a significant scatter in strength and hardening rate was observed in micropillars with smaller diameters. Flow data for micropillars in the size-independent regime were used to determine parameters in a crystal-plasticity-based model of martensite. Full three-dimensional crystal plasticity simulations, with material properties determined from micropillar tests, were then used to predict the macroscopic uniaxial stress–strain behavior of a representative volume element of martensite. The predicted stress–strain behavior was in excellent agreement with experimental measurements, and demonstrates the potential for micropillar tests to determine material parameters for individual phases of a complex microstructure.