Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall beta-1,3-glucan

Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by beta-mercaptoethanol (beta-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with beta-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys(214)-12aa-Cys(227)-COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys(130)Ser or Cys(197)Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1Delta and gsc2/fks2Delta cells, defective in beta-1,3-glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by beta-ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the beta-1,3-glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2-unprocessed Pir4 form. Pir4 failed to be incorporated in alkali-sensitive linkages involving beta-1,3-glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the beta-1,3-glucan.     

Separation of recombinant human erythropoietin glycoforms by capillary electrophoresis using volatile electrolytes. Assessment of mass spectrometry for the characterization of erythropoietin glycoforms

The separation of the glycoforms of erythropoietin (EPO) by capillary electrophoresis (CE) was recently published as a monograph by the European Pharmacopoeia (European Pharmacopoeia 4 2002, 1316, 1123-1128). Although the experimental CE conditions employed a background electrolyte containing additives suitable for on-line UV-absorption detection, they were not appropriate for on-line mass spectrometry (MS) detection. In this work, an attempt was made to investigate experimental conditions employing volatile electrolyte systems to achieve the separation and characterization of EPO glycoforms using CE and ESI-MS methodologies. The influence of several operating conditions, such as the coating of the internal walls of the capillary as well as the composition, concentration, and the pH of the separation buffer were investigated. The results demonstrated that when the internal walls of the capillaries were permanently coated with Polybrene and a buffer electrolyte containing 400 mM of HAc-NH4Ac (acetic acid-ammonium acetate), pH 4.75, was used, a significantly reproducible separation was achieved for EPO glycoforms. Intact EPO was characterized by two mass spectrometry techniques: electrospray ionization (ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS). The data demonstrated that MALDI-TOF-MS provided a good approximation to an average molecular mass of the EPO molecule. However, it was still necessary to carry out further separation of the intact EPO glycoforms in order to obtain molecular mass information when ESI-MS was used.     

Adaptive value function approximation for continuous-state stochastic dynamic programming

Approximate dynamic programming (ADP) commonly employs value function approximation to numerically solve complex dynamic programming problems. A statistical perspective of value function approximation employs a design and analysis of computer experiments (DACE) approach, where the “computer experiment” yields points on the value function curve. The DACE approach has been used to numerically solve high-dimensional, continuous-state stochastic dynamic programming, and performs two tasks primarily: (1) design of experiments and (2) statistical modeling. The use of design of experiments enables more efficient discretization. However, identifying the appropriate sample size is not straightforward. Furthermore, identifying the appropriate model structure is a well-known problem in the field of statistics. In this paper, we present a sequential method that can adaptively determine both sample size and model structure. Number-theoretic methods (NTM) are used to sequentially grow the experimental design because of their ability to fill the design space. Feed-forward neural networks (NNs) are used for statistical modeling because of their adjustability in structure-complexity . This adaptive value function approximation (AVFA) method must be automated to enable efficient implementation within ADP. An AVFA algorithm is introduced, that increments the size of the state space training data in each sequential step, and for each sample size a successive model search process is performed to find an optimal NN model. The new algorithm is tested on a nine-dimensional inventory forecasting problem.     

Evaluation of the surface urban heat island effect in the city of Madrid by thermal remote sensing

The surface urban heat island (SUHI) effect is defined as the increased surface temperatures in urban areas in contrast to cooler surrounding rural areas. In this article, the evaluation of the SUHI effect in the city of Madrid (Spain) from thermal infrared (TIR) remote-sensing data is presented. The data were obtained from the framework of the Dual-use European Security IR Experiment (DESIREX) campaign that was carried out during June and July 2008 in Madrid. The campaign combined the collection of airborne hyperspectral and in situ measurements. Thirty spectral and spatial high-resolution images were acquired with the Airborne Hyperspectral Scanner (AHS) sensor in a 11, 21, and 4 h UTC scheme. The imagery was used to retrieve the SUHI effect by applying the temperature and emissivity separation (TES) algorithm. The results show a nocturnal SUHI effect with a highest value of 5 K. This maximum value agrees within 1 K with the highest value of the urban heat island (UHI) observed using air temperature data (AT). During the daytime, this situation is reversed and the city becomes a negative heat island.     

Rural electrification systems based on renewable energy: The social dimensions of an innovative technology

Many areas in rural non-Western regions are installing electrification systems based on renewable energy. Although these projects are usually welcome, they sometimes fail. Explanations for failures often cite technical reasons. In this research, partly based on the results of the SOPRA_RE project, we focus on the users, studying the relationships between technology and society. By analysing potential sources of failures, we identify the root of the problem as how a new technology is received by the local society. In this paper we introduce dimensions that we consider to be especially important in the process of technology integration. We defend the importance of combining the technical outlook with the sociological one, based on the idea that the latter is often indispensable as a complementary element of technical explanations of system failures, which in turn provides a better basis for solving them. We use specific examples to bolster our insistence on the need to achieve convergence between the technical and the sociological outlooks.     

Mechanical characteristics of porous hydroxyapatite/oxide composites produced by post-sintering hot isostatic pressing

Porous hydroxyapatite and hydroxyapatite/oxide composites, with 10 wt% addition of Y₂O₃, ZrO₂ or TiO₂ have been prepared by hot isostatic pressing after pressureless sintering, and their phase composition, mechanical properties and microstructure investigated. The quantitative X-ray diffraction analyses reveal that the addition of Y₂O₃ inhibits the hydroxyapatite decomposition completely during the consolidation processes as a result of the Y ion dissolution into the hydroxyapatite lattice. On the contrary, it is found that the addition of ZrO₂ or TiO₂ enhances the decomposition of hydroxyapatite into Ca₃(PO₄)₂, which causes the transformation of the starting oxides. The mechanical characterization of the materials has been accomplished by three-point flexure tests, and nanoindentation and microhardness measurements. Post-sintering hot isostatic pressed pure hydroxyapatite, which resulted in a porous biphasic material with 13.2 wt% β-Ca₃(PO₄)₂, exhibited the best mechanical properties. The failure of hydroxyapatite-10Y₂O₃ during the flexure tests was no catastrophic in contrast to the catastrophic behaviour found in the other materials.     

Identification of the origin of commercial enological tannins by the analysis of monosaccharides and polyalcohols

The monosaccharide and polyalcohol composition of 28-samples of different commercial tannins, including oak wood, grape seed and skin, plant gall, chestnut, quebracho and gambier, has been evaluated by gas chromatography–mass spectrometry after derivatization into their trimethylsilyl ethers. Quercitol was found to be characteristic of oak tannins, whereas gall plant tannins could be differentiated by their content of pinitol. Myo-inositol and arabitol were detected in tannins from quebracho. These polyalcohols, together with muco-inositol and chiro-inositol, were found in tannins from chestnut while bornesitol was found to be characteristic of tannins from gambier. Monosaccharide composition also helped to distinguish among tannin origins: arabinose, xylose, fructose and glucose were quantified in oak, quebracho and chestnut tannins, whereas only fructose and glucose were detected in plant gall and grape tannins. These results imply that the qualitative study of monosaccharides and polyalcohols could help to determine and control the authenticity of enological tannins.