Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.     

How Cancer Cells Invade Bladder Epithelium and Form Tumors: The Mouse Bladder Tumor Model as a Model of Tumor Recurrence in Patients

Non-muscle-invasive bladder cancer is the most common form of bladder cancer. The main problem in managing bladder tumors is the high recurrence after the transurethral resection of bladder tumors (TURBT). Our study aimed to examine the fate of intravesically applied cancer cells as the implantation of cancer cells after TURBT is thought to be a cause of tumor recurrence. We established an orthotopic mouse bladder tumor model with MB49-GFP cancer cells and traced them during the first three days to define their location and contacts with normal urothelial cells. Data were obtained by Western blot, immunolabeling, and light and electron microscopy. We showed that within the first two hours, applied cancer cells adhered to the traumatized epithelium by cell projections containing α3β1 integrin on their tips. Cancer cells then migrated through the epithelium and on day 3, they reached the basal lamina or even penetrated it. In established bladder tumors, E-cadherin and desmoplakin 1/2 were shown as feasible immunohistochemical markers of tumor margins based on the immunolabeling of various junctional proteins. Altogether, these results for the first time illustrate cancer cell implantation in vivo mimicking cellular events of tumor recurrence in bladder cancer patients.     

Electrospinning of polyurethane fibers

A segmented polyurethaneurea based on poly(tetramethylene oxide)glycol, a cycloaliphatic diisocyanate and an unsymmetrical diamine were prepared. Urea content of the copolymer was 35 wt%. Electrospinning behavior of this elastomeric polyurethaneurea copolymer in solution was studied. The effects of electrical field, temperature, conductivity and viscosity of the solution on the electrospinning process and morphology and property of the fibers obtained were investigated. Results of observations made by optical microscope, atomic force microscope and scanning electron microscope were interpreted and compared with literature data available on the electrospinning behavior of other polymeric systems.     

Surface reconstruction for incremental forming

In spite of extensive efforts being made with regard to virtual process optimization technology, the production of prototype parts is still a necessity. With respect to the production of sheet metal parts in low quantities, incremental sheet metal forming (ISMF) is a highly interesting process. ISMF allows the production of complex parts with drastically reduced costs in tooling and machinery compared to conventional processes like deep drawing. However, ISMF, with it’s incremental nature, introduces the need for generating a tool path considering both final geometry and process-induced deviations or constraints. Consequently, for the generation of the tool path a (tool path) surface, with an adequate offset, is necessary. That is why, within the scope of extensive research work at the Institute of Forming Technology and Lightweight Construction (IUL), a special correction module has been developed, determining this offset e.g. depending on the workpiece geometry. This paper presents the algorithm, the application, and the effect on the produced parts. Furthermore, a concept for an extension regarding further constraints like elastic workpiece behavior is presented.     

Analysis of defects on BN nano-structures using high-resolution electron microscopy and density-functional calculations

Cubic boron nitride (c-BN) nucleation takes place on hexagonal boron nitride (h-BN) layers growing perpendicular to the substrate surface during thin film synthesis. Studies focused on the nucleation of the cubic phase suggest the possibility that transient phases and/or defects on these h-BN structures have a role in sp3-bonded cubic phase nucleation. In this study, we have investigated the nature, energetics, and structure of several possible defects on BN basal planes, including point defects, 4-, and 5-fold BN rings, that may possibly match the experimentally observed transient phase fine structure. TEM image observations are used to build approximate atomic models for the proposed structures, and DFT calculations are used to relax these structures while minimizing their respective total energies. These optimized atomic geometries are then used to simulate TEM images, which are compared to the experimentally observed structures. Data from DFT calculations and analysis of simulated images from the proposed atomic structures suggest that 4-fold BN rings are more likely to exist on the transient phase possibly leading to c-BN nucleation.     

Parameter optimization for the Gaussian model of protein folding

Computational models of protein folding and ligand docking are large and complex. Few systematic methods have yet been developed to optimize the parameters in such models. We describe here an iterative parameter optimization strategy that is based on minimizing a structural error measure by descent in parameter space. At the start, we know the ‘correct’ native structure that we want the model to produce, and an initial set of parameters representing the relative strengths of interactions between the amino acids. The parameters are changed systematically until the model native structure converges as closely as possible to the correct native structure. As a test, we apply this parameter optimization method to the recently developed Gaussian model of protein folding: each amino acid is represented as a bead and all bonds, covalent and noncovalent, are represented by Hooke's law springs. We show that even though the Gaussian model has continuous degrees of freedom, parameters can be chosen to cause its ground state to be identical to that of Go-type lattice models, for which the global ground states are known. Parameters for a more realistic protein model can also be obtained to produce structures close to the real native structures in the protein database.     

The electrical and dielectric characterization of the Co/ZnO-Rods/p-Si heterostructure depending on the frequency

Journal of Materials Science: Materials in Electronics - Zinc oxide (ZnO) rods film was fabricated by homemade and simple spray pyrolysis technique on a p-type silicon (Si) substrate, and the film...     

Investigations of ductile damage during the process chains of toothed functional components manufactured by sheet-bulk metal forming

Sheet-bulk metal forming processes combine conventional sheet forming processes with bulk forming of sheet semi-finished parts. In these processes the sheets undergo complex forming histories. Due to in- and out-of-plane material flow and large accumulated plastic strains, the conventional failure prediction methods for sheet metal forming such as forming limit curve fall short. As a remedy, damage models can be applied to model damage evolution during those processes. In this study, damage evolution during the production of two different toothed components from DC04 steel is investigated. In both setups, a deep drawn cup is upset to form a circumferential gearing. However, the two final products have different dimensions and forming histories. Due to combined deep drawing and upsetting processes, the material flow on the cup walls is three-dimensional and non-proportional. In this study, the numerical and experimental investigations for those parts are presented and compared. Damage evolution in the process chains is simulated with a Lemaitre damage criterion. Microstructural analysis by scanning electron microscopy is performed in the regions with high mechanical loading. It is observed that the evolution of voids in terms of void volume fraction is strongly dependent on the deformation path. The comparison of simulation results with microstructural data shows that the void volume fraction decreases in the upsetting stage after an initial increase in the drawing stage. Moreover, the concurrent numerical and microstructural analysis provides evidence that the void volume fraction decreases during compression in sheet-bulk metal forming.