Protein identification by MALDI-TOF-MS peptide mapping: a new strategy

A new strategy for identifying proteins by MALDI-TOF-MS peptide mapping is reported. In contrast to current approaches, the strategy does not rely on a good relative or absolute mass accuracy as the criterion that discriminates false positive results. The protein sequence database is first searched for all proteins that match a minimum five of the submitted masses within the maximum expected relative errors when the default or externally determined calibration constants are used, for instance, +/-500 ppm. Typically, this search retrieves many thousand candidate sequences. Assuming initially that each of these is the correct protein, the relative errors of the matching peptide masses are calculated for each candidate sequence. Linear regression analysis is then performed of the calculated relative errors as a function of m/z for each candidate sequence, and the standard deviation to the regression is used to distinguish the correct sequence among the candidates. We show that this parameter is independent of whether the mass spectrometric data were internally or externally calibrated. The result is a search engine that renders internal spectrum calibration unnecessary and adapts to the quality of the raw data without user interference. This is made possible by a dynamic scoring algorithm, which takes into account the number of matching peptide masses, the percentage of the protein's sequence covered by these peptides and, as new parameter, the determined standard deviation. The lower the standard deviation, the less cleavage peptides are required for identification and vice versa. Performance of the new strategy is demonstrated and discussed. All necessary computing has been implemented in a computer program, free access to which is provided in the Internet.     

Process-Centered Software Engineering Environments, A Brief History and Future Challenges

Software engineering environments have a history of about two decades. Early environments provided support for small fragments of the software process (usually focusing on programming-in-the small). Then there was a trend towards support for more complete software processes (from early phases like requirements analysis and design down to testing and configuration management). Ten years ago the notion of process-centered software engineering environments initiated a new field in software engineering: software process research. The key idea is to use a model of a software process as input parameter for a software engineering environment. The environment is supposed to behave in accordance to the process model. Some aspects of this vision became true, others turned out to be of little practicability. In this article, we discuss the history of software engineering environments with a particular focus on process-centered software engineering environments (PCSEEs). We discuss the notion of distributed software processes (as one of the most substantial current trends in software process research) and we motivate the notion of a software process middleware which serves as basis of real-world software processes spread over various sites. In addition, we discuss some other trends in the software process research arena.     

A probabilistic model for binaural sound localization.

This paper proposes a biologically inspired and technically implemented sound localization system to robustly estimate the position of a sound source in the frontal azimuthal half-plane. For localization, binaural cues are extracted using cochleagrams generated by a cochlear model that serve as input to the system. The basic idea of the model is to separately measure interaural time differences and interaural level differences for a number of frequencies and process these measurements as a whole. This leads to two-dimensional frequency versus time-delay representations of binaural cues, so-called activity maps. A probabilistic evaluation is presented to estimate the position of a sound source over time based on these activity maps. Learned reference maps for different azimuthal positions are integrated into the computation to gain time-dependent discrete conditional probabilities. At every timestep these probabilities are combined over frequencies and binaural cues to estimate the sound source position. In addition, they are propagated over time to improve position estimation. This leads to a system that is able to localize audible signals, for example human speech signals, even in reverberating environments.     

Reaction behaviour of monomeric β-ketoesters

Summary Polymerizable enamines were synthesized by the reaction of 2-acetoacetoxyethyl methacrylate (AAEMA) with various aliphatic mono- and diamines. The enamines were characterized by elemental analyses, IR,¹H NMR and¹³C NMR spectroscopy. Radical polymerization of synthesized enamines yielded polymers with pendant enamine groups which were also prepared by the reaction of poly(AAEMA) with the corresponding amines.     

Superplasticity in eutectoid steel

A eutectoid steel exhibited abnormally large tensile extensibility in three quite dissimilar circumstances: i) micrograin plasticity, defined by a strain-rate sensitivity exponent m = 0.42, was found at 716°C for straining the ferrite-cementite aggregate at the rate è = 4.4 x 10^-3 min^-1, giving 133 pct elongation; ii) at a much greater strain rate, ε = 25 min^-1, superplasticity appeared in austenite strained at 927°C, giving 142 pct elongation; iii) a new type of transformation plasticity was predicted and experimentally verified: 490 pct elongation resulted from thermal cycling 21 times across Ae₁. Plastic stability analysis distinguishes it from micrograin plasticity by showing that it owes to strain-hard ening during plastically stable flow; hence, there are no restrictions ε or m. Furthermore, it is not necessary to perform the straining during the transformation, since the strain-hardening capacity can be regenerated by thermal cycling through the phase transformation if the transformation serves to recover the flow stress. Additional work showed that straining during austenitizing fails to increase m above pretransformation levels. Formerly Graduate Student, Syracuse University, Syracuse, N. Y.     

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.     

Aerosol lidar intercomparison in the framework of the EARLINET project. 1. Instruments

In the framework of the European Aerosol Research Lidar Network to Establish an Aerosol Climatology (EARLINET), 19 aerosol lidar systems from 11 European countries were compared. Aerosol extinction or backscatter coefficient profiles were measured by at least two systems for each comparison. Aerosol extinction coefficients were derived from Raman lidar measurements in the UV (351 or 355 nm), and aerosol backscatter profiles were calculated from pure elastic backscatter measurements at 351 or 355, 532, or 1064 nm. The results were compared for height ranges with high and low aerosol content. Some systems were additionally compared with sunphotometers and starphotometers. Predefined maximum deviations were used for quality control of the results. Lidar systems with results outside those limits could not meet the quality assurance criterion. The algorithms for deriving aerosol backscatter profiles from elastic lidar measurements were tested separately, and the results are described in Part 2 of this series of papers [Appl. Opt. 43, 977-989 (2004)]. In the end, all systems were quality assured, although some had to be modified to improve their performance. Typical deviations between aerosol backscatter profiles were 10% in the planetary boundary layer and 0.1 x 10(-6) m(-1) sr(-1) in the free troposphere.     

Thermofraktographie zur Schnellanalyse von Kunststoffen. 4. Mitt.: Identifizierung der Basiskomponenten von Epoxidharzen

Thermofractography is suitable for the rapid identification of the basic components of epoxy resins. 2–3 mg of the polymer sample are being heated in the temperature gradient of 200 to 450°C. The thus forming decomposition products are being transferred as a starting line onto the separating thin layer by means of a carrier gas. After development the thermofractogram (TFG) shows in the temperature range of 200 to approx. 350°C the free oligomeres. The resulting phenols and their subsequent products appear on the TFG from 350°C. A cycloaliphatic type does not give phenolic fragments. The degradation mechanisms of epoxy resins are being discussed in regard to the TFG results and are being confirmed by establishing TGA curves. For the rapid evaluation of the thermofractograms of epoxy resins a questioning scheme is given as an evaluation key.     

On-road measurement of particle emission in the exhaust plume of a diesel passenger car

Particle size distributions were measured under real world dilution conditions in the exhaust plume of a diesel passenger car closely followed by a mobile laboratory on a high speed test track. Under carefully controlled conditions the exhaust plume was continuously sampled and analyzed inside the mobile laboratory. Exhaust particle size distribution data were recorded together with exhaust gas concentrations, i.e., CO, CO2, and NO(x), and compared to data obtained from the same vehicle tested on a chassis dynamometer. Good agreement was found for the soot mode particles which occurred at a geometric mean diameter of approximately 50 nm and a total particle emission rate of 10(14) particles km(-1). Using 350 ppm high sulfur fuel and the standard oxidation catalyst a bimodal size distribution with a nucleation mode at 10 nm was observed at car velocities of 100 km h(-1) and 120 km h(-1), respectively. Nucleation mode particles were only present if high sulfur fuel was used with the oxidation catalyst installed. This is in agreement with prior work that these particles are of semivolatile nature and originate from the nucleation of sulfates formed inside the catalyst. Temporal effects of the occurrence of nucleation mode particles during steady-state cruising and the dynamical behavior during acceleration and deceleration were investigated.