Mapping of possible prion protein self-interaction domains using peptide arrays

Background  

The common event in transmissible spongiform encephalopathies (TSEs) or prion diseases is the conversion of host-encoded protease sensitive cellular prion protein (PrP^C) into strain dependent isoforms of scrapie associated protease resistant isoform (PrP^Sc) of prion protein (PrP). These processes are determined by similarities as well as strain dependent variations in the PrP structure. Selective self-interaction between PrP molecules is the most probable basis for initiation of these processes, potentially influenced by chaperone molecules, however the mechanisms behind these processes are far from understood. We previously determined that polymorphisms do not affect initial PrP^C to PrP^Sc binding but rather modulate a subsequent step in the conversion process. Determining possible sites of self-interaction could elucidate which amino acid(s) or amino acid sequences contribute to binding and further conversion into other isoforms. To this end, ovine – and bovine PrP peptide-arrays consisting of 15-mer overlapping peptides were probed with recombinant sheep PrP^C fused to maltose binding protein (MBP-PrP).     

Studies on photoinduced effects in pulse-electrodeposited Ag/Hg-1212/CdSe hetero-nanostructures

Metal/superconductor/semiconductor (Ag/Hg-1212/CdSe) hetero-nanostructures have been fabricated using pulse-electrodeposition technique and are characterized by X-ray diffraction (XRD), full-width at half-maximum (FWHM) and scanning electron microscopy (SEM) studies. The junction capacitance of Ag/Hg-1212, Hg-1212/CdSe and Ag/Hg-1212/CdSe heterojunctions is measured in dark and under laser irradiation at room temperature. The nature of the junction formed and built-in-junction potentials were determined. The increase in carrier concentration across the junction due to photo-irradiation has been observed.     

Effect of laser irradiation of C-V characteristics of electrodeposited Ag/Tl-2223/CdSe hetero-nanostructures

One of the innovative technological directions for the high-temperature superconductors has been persued by fabricating the heteroepitaxial multilayer structures such as superconductor-semiconductor heterostructures. In the present investigation, metal/superconductor/semiconductor (Ag/Tl-2223/CdSe) hetero-nanostructures have successfully been fabricated using dc electrodeposition technique and were characterized by X-ray diffraction (XRD), full-width at half-maximum (FWHM) and scanning electron microscopy (SEM) studies. The measurement of junction capacitance as a function of biasing voltage was used for the estimation of junction built-in-potential (V _D) and to study the charge distribution in a heterojunction. The Mott-Schottky plots were measured for each junction in dark and under the photo-irradiation. The effect of laser irradiation on C-V characteristics of hetero-nanostructure has been studied.     

Glycopeptide and Oligosaccharide Libraries

Despite the burgeoning interest in the various biological functions and consequent therapeutic potential of the vast number of oligosaccharides found in nature on glycoproteins and cell surfaces, the development of combinatorial carbohydrate chemistry has not progressed as rapidly as expected. The reason for this imbalance is rooted in the difficulty of oligosaccharide assembly and analysis that renders synthesis a rather cumbersome endeavor. Parallel approaches that generate series of analogous compounds rather than real libraries have therefore typically been used. Since generally low affinity is obtained for interactions between carbohydrate receptors and modified oligosaccharides designed as mimetics of natural carbohydrate ligands, glycopeptides have been explored as alternative mimics. Glycopeptides have been proven in many cases to be superior ligands with higher affinity for a receptor than the natural carbohydrate ligand. High-affinity glycopeptide ligands have been found for several types of receptors including the E-, P-, and L-selectins, toxins, glycohydrolases, bacterial adhesins, and the mannose-6-phosphate receptor. Furthermore, the assembly of glycopeptides is considerably more facile than that of oligosaccharides and the process can be adapted to combinatorial synthesis with either glycosylated amino acid building blocks or by direct glycosylation of peptide templates. The application of the split and combine approach using ladder synthesis has allowed the generation of very large numbers of compounds which could be analyzed and screened for binding of receptors on solid phase. This powerful technique can be used generally for the identification and analysis of the complex interaction between the carbohydrates and their receptors.     

Infrared spectroscopic determination of the degree of charge transfer in complexes of TCNE with methyl-substituted benzenes

The position of the CN stretching frequency in infrared spectra of tetracyanoethylene (TCNE) complexed with methyl-substituted benzenes is used to determine the fraction of charge transferred from the electron donor to the acceptor.     

The formation of the anilinium ion from the ammonium ion in the ammonia chemical ionization of substituted benzenes

Compounds C₆H₅X(X ? F, Cl, Br, NO₂, CN, OCH₃) have been studied under chemical ionization conditions with ammonia as reagent gas. A pulsed electron beam and time resolved ion collection has allowed the determination of the reaction leading to the formation of [C₆H₅NH₃]⁺ (m/z 94). [NH₄]⁺ reacts with C₆H₅X(X ? F, Cl, Br) to yield m/z 94 but C₆H₅X (X ? CN, NO₂) forms this ion only by reactions involving either [NH₃]⁺ or [C₆H₅X]⁺. C₆H₅OCH₃ does not form m/z 94.     

Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry

Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.     

Enzymatic Oxygen Microsensor Based on Bilirubin Oxidase Applied to Microbial Fuel Cells Analysis

A selective oxygen biosensor based on bilirubin oxidase (BOx) was developed. The sensor was used for determining oxygen profiles in a membraneless, single‐chamber microbial fuel cell (SCMFC), fed with raw wastewater. The linear response of the sensor was optimized by a diffusion layer of silica gel. A computer‐controlled stage was used to obtain accurate and precise measurements. Oxygen concentration in biofilms covering electrodes was measured, showing 3 mg L^?1 of O₂ in the bulk solution, decreasing to 0 mg L^?1 in the cathodic biofilm. The MFC generated power in the range of 0–0.08 mW, associated to the oxygen content.