Reversible acetonitrile-induced inactivation/activation of thermolysin

Thermolysin is catalytically inactive in mixtures of 10-15 % acetonitrile in aqueous buffer. Unexpectedly, dilution of the inactive enzyme with acetonitrile leads to complete recovery of the catalytic activity in a similar way to dilution with aqueous buffer. Circular dichroism and fluorescence studies of thermolysin in the same solvent mixtures reveal discontinuous changes in the overall secondary and tertiary protein structure that correlate well with the reversible differences in catalytic activity. The spectra on either side of the minimum activity point are different from each other, a fact indicating that the enzyme may be able to access two active conformations which are thermodynamically stable in different solvent environments.     

Silicon temperature measurement by infrared absorption. Fundamentalprocesses and doping effects

The fundamental absorption mechanisms in silicon at 1.30 and 1.55 μm have been investigated in the temperature range of 500-800°C. For lightly doped wafers in this temperature range, the absorption at 1.55 μm is by free carriers. and that at 1.30 μm is predominantly by bandgap absorption. The effect of heavy substrate doping on infrared absorption at an elevated temperature has also been studied, and it was found that doping has little effect below levels of 7×10¹⁷ cm^-3. Above that level, the temperature dependence of free carrier absorption strongly affects the transmission as a function of temperature. The knowledge of the fundamental absorption processes is then used to predict the ultimate temperature ranges over which the technique will be useful     

Migration of single-stranded DNA in polyacrylamide gels during electrophoresis

By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single-stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, mu0, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double-stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single-stranded DNA (>2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single-stranded DNA with pulsed field techniques.     

Biodegradability of nonionic surfactants: Screening test for predicting rate and ultimate biodegradation

Environmental water quality evaluations of raw materials in consumer products occupy a position of critical importance in many industries throughout the world. The rapid growth and diversity of the household detergent market requires continuous consideration of new materials needed to meet the demands of new, improved and modified products. As household cleaning products are normally disposed of as a component of domestic sewage, surface active compounds, including nonionic surfactants, would reach surface waters only as a part of a sewage effluent and would be subject to the same degree of biological treatment as the balance of the waste. For this reason, evaluations of such new materials include an environmental assessment in which biodegradability testing of organic materials is an important first step. Biodegradability characteristics of nonionic surfactants, as a class, are generally more difficult to ascertain because of wide structural diversity and a usual lack of functional groups. Such determinations usually involve intricate and laborious test methods which necessitate development of analytical techniques for each degradation product of a given material. A method has been developed, modified and used in our laboratory, that provides, after reasonable opportunity for biological acclimation, a measure of the rate and degree of ultimate biodegradation (conversion to CO₂ and H₂O). This method, which uses simple equipment, has been used to assess the biodegradability of a wide variety of nonionic surfactants, without necessitating the development of specific analytical methods for each surfactant under consideration. Additionally, this method can be adapted to measure degradation under conditions of anaerobiosis or low temperature.     

KlROM2 encodes an essential GEF homologue in Kluyveromyces lactis

Cellular integrity in yeasts is ensured by a rigid cell wall whose synthesis is controlled by a MAP kinase signal transduction cascade. In Saccharomyces cerevisiae upstream regulatory components of this MAP kinase pathway involve a single protein kinase C, which is regulated in part by interaction with the small GTPase Rho1p. This small G protein is in turn rendered inactive (GDP-bound) or is activated (GTP-bound) by the influence of GTPase activating proteins (GAPs) and the GDP/GTP exchange factors (GEFs), respectively. We report here on the isolation of a gene from Kluyveromyces lactis, KlROM2, which encodes a member of the latter protein family. The nucleotide sequence contains an open reading frame of 1227 amino acids, with an overall identity of 57% to the Rom2 protein of S. cerevisiae. Four conserved sequence motifs could be identified: a RhoGEF domain, a DEP sequence, a CNH domain and a less conserved pleckstrin homology (PH) sequence. Klrom2 null mutants show a lethal phenotype, which indicates that the gene may encode the only functional GEF regulating the cellular integrity pathway in K. lactis. Conditional genomic expression of KlROM2 resulted in sensitivity towards caffeine and Calcofluor white as typical phenotypes of mutants defective in this pathway. Overexpression of KIROM2 from multicopy plasmids under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and in K. lactis. The fact that the lethal phenotype was not prevented in mpk1 deletion mutants indicates that growth inhibition is not simply caused by hyperactivation of the Pkc1p signal transduction pathway.     

On-line coupling of size exclusion chromatography with electrospray ionization-tandem mass spectrometry for the analysis of aquatic fulvic and humic acids

A method was developed for the analysis of humic and fulvic acids by size-exclusion chromatography-electrospray ionization-tandem mass spectrometry using a completely volatile eluent. Humic and fulvic acids were separated into three peaks. These fractions occupied different mass ranges and showed differences in the fine structure of their mass spectra. The low-molecular-weight (LMW) fraction of fulvic acids is most sensitively determined by ESI-MS, and it appears that previous results obtained by infusion-ESI-MS were primarily determined by this fulvic acid fraction. The average molecular weight of this fractions turned out to be lower than that reported from infusion-ESI-MS measurements. Its scan spectra and the product ion spectra of some of its molecular anions perfectly match those previously obtained from whole fulvic acid mixtures. Obviously, a class of well-defined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. With decreasing elution time and increasing molecular weight, detection by ESI-MS loses sensitivity as compared to the parallel UV recording, and the fine structure of the scan spectra becomes increasingly uniform for both fulvic and humic acids. The average molecular weight of the HMW fraction exceeds those values calculated from infusion experiments. Scan spectra and product ion spectra of the high-molecular-weight (HMW) fraction of both the humic and the fulvic acids suggest that the HMW fraction consists of several subunits that originate from the LMW fraction.     

Interactive lectures: Effective teaching and learning in lectures using wireless networks

Among the severe instructional shortcomings of lectures is their lack of interactivity. To overcome these problems, the learning environment of the interactive lecture is introduced, in which students and lecturer interact by using mobile computers in a wireless network. In an experimental study, 44 students participated in interactive and in conventional lecture meetings. A quiz service that allowed question to be posted, students’ answers to be evaluated, and the results to be presented graphically, was implemented on mobile computers. The participants rated the interactive condition better, also reporting higher levels of attention, activity and perceived learning success for this condition. Objective measures indicated a slight but insignificant difference toward better learning results in the interactive condition.     

Classification accuracy is not enough

We argue that an evaluation of system behavior at the level of the music is required to usefully address the fundamental problems of music genre recognition (MGR), and indeed other tasks of music information retrieval, such as autotagging. A recent review of works in MGR since 1995 shows that most (82 %) measure the capacity of a system to recognize genre by its classification accuracy. After reviewing evaluation in MGR, we show that neither classification accuracy, nor recall and precision, nor confusion tables, necessarily reflect the capacity of a system to recognize genre in musical signals. Hence, such figures of merit cannot be used to reliably rank, promote or discount the genre recognition performance of MGR systems if genre recognition (rather than identification by irrelevant confounding factors) is the objective. This motivates the development of a richer experimental toolbox for evaluating any system designed to intelligently extract information from music signals.