Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites

Protein phosphorylation is one of the most important known posttranslational modifications. Tandem mass spectrometry has become an important tool for mapping out the phosphorylation sites. However, when a peptide generated from the enzymatic or chemical digestion of a phosphoprotein is highly phosphorylated or contains many potential phosphorylation residues, phosphorylation site assignment becomes difficult. Separation and enrichment of phosphopeptides from a digest mixture is desirable and often a critical step for MS/MS-based site determination. In this work, we present a novel open tubular immobilized metal ion affinity chromatography (OT-IMAC) method, which is found to be more effective and reproducible for phosphopeptide enrichment, compared to a commonly used commercial product, Ziptip from Millipore. A strategy based on a combination of OT-IMAC, sequential dual-enzyme digestion, and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry for phosphoprotein characterization is presented. It is shown that MALDI MS/MS with collision-induced dissociation can be very effective in generating fragment ion spectra containing rich structural information, which enables the identification of phosphorylation sites even from highly phosphorylated peptides. The applicability of this method for real world applications is demonstrated in the characterization and identification of phosphorylation sites of a Na(+)/H(+) exchanger fusion protein, His182, which was phosphorylated in vitro using the kinase Erk2.     

Isotropic sequence order learning

In this article, we present an isotropic unsupervised algorithm for temporal sequence learning. No special reward signal is used such that all inputs are completely isotropic. All input signals are bandpass filtered before converging onto a linear output neuron. All synaptic weights change according to the correlation of bandpass-filtered inputs with the derivative of the output. We investigate the algorithm in an open- and a closed-loop condition, the latter being defined by embedding the learning system into a behavioral feedback loop. In the open-loop condition, we find that the linear structure of the algorithm allows analytically calculating the shape of the weight change, which is strictly heterosynaptic and follows the shape of the weight change curves found in spike-time-dependent plasticity. Furthermore, we show that synaptic weights stabilize automatically when no more temporal differences exist between the inputs without additional normalizing measures. In the second part of this study, the algorithm is is placed in an environment that leads to closed sensor-motor loop. To this end, a robot is programmed with a prewired retraction reflex reaction in response to collisions. Through isotropic sequence order (ISO) learning, the robot achieves collision avoidance by learning the correlation between his early range-finder signals and the later occurring collision signal. Synaptic weights stabilize at the end of learning as theoretically predicted. Finally, we discuss the relation of ISO learning with other drive reinforcement models and with the commonly used temporal difference learning algorithm. This study is followed up by a mathematical analysis of the closed-loop situation in the companion article in this issue, "ISO Learning Approximates a Solution to the Inverse-Controller Problem in an Unsupervised Behavioral Paradigm" (pp. 865-884).     

ISO learning approximates a solution to the inverse-controller problem in an unsupervised behavioral paradigm

In "Isotropic Sequence Order Learning" (pp. 831-864 in this issue), we introduced a novel algorithm for temporal sequence learning (ISO learning). Here, we embed this algorithm into a formal nonevaluating (teacher free) environment, which establishes a sensor-motor feedback. The system is initially guided by a fixed reflex reaction, which has the objective disadvantage that it can react only after a disturbance has occurred. ISO learning eliminates this disadvantage by replacing the reflex-loop reactions with earlier anticipatory actions. In this article, we analytically demonstrate that this process can be understood in terms of control theory, showing that the system learns the inverse controller of its own reflex. Thereby, this system is able to learn a simple form of feedforward motor control.     

The Second Futamura Projection for Type-Directed Partial Evaluation

A generating extension of a program specializes the program with respect to part of the input. Applying a partial evaluator to the program trivially yields a generating extension, but specializing the partial evaluator with respect to the program often yields a more efficient one. This specialization can be carried out by the partial evaluator itself; in this case, the process is known as the second Futamura projection.We derive an ML implementation of the second Futamura projection for Type-Directed Partial Evaluation (TDPE). Due to the differences between traditional, syntax-directed partial evaluation and TDPE, this derivation involves several conceptual and technical steps. These include a suitable formulation of the second Futamura projection and techniques for making TDPE amenable to self-application. In the context of the second Futamura projection, we also compare and relate TDPE with conventional off-line partial evaluation.We demonstrate our technique with several examples, including compiler generation for Tiny, a prototypical imperative language.     

Pharmacokinetics of Gadomer-17, a new dendritic magnetic resonance contrast agent

Rationale and objectives: Gadomer-17 is a new magnetic resonance (MR) contrast medium presently in clinical development. It is a dendritic gadolinium (Gd) chelate carrying 24 Gd ions. This study investigated the pharmacokinetic behavior of this contrast medium. Methods: The pharmacokinetics of Gadomer-17 were investigated in different species (rat, rabbit, dog, monkey) for up to 7 days after intravenous (i.v.) injection of 25–100 μmol/kg body weight. In addition, elimination and biodistribution were evaluated after single i.v. injection of Gadomer-17 in rats. Results: After i.v. injection Gadomer-17 distributes almost exclusively within the intravascular space without significant diffusion into the interstitial space. The volume of distribution (Vc) in the initial or α-phase ranged from 0.04 1/kg (rats, rabbits) to 0.06 1 kg (monkeys) and 0.07 1/kg (dogs), which reflects mainly the plasma volume. The blood/plasma concentration profile was found to be biphasic. The volume of distribution at a steady state is clearly smaller than that of other contrast media, which distribute to the extracellular space. After single i.v. injection in rats, the dendritic contrast medium was rapidly and completely eliminated from the body, mainly via glomerular filtration. No long-term accumulation or retention of the nonmetabolized agent was detectable in organs or tissues. Conclusions: Gadomer-17 is a promising new MR contrast medium that has an intravascular distribution and a rapid renal elimination.     

Synthesis of 9,12-Dioxo-10(Z)-dodecenoic acid, a new fatty acid metabolite derived from 9-hydroperoxy-10,12-octadecadienoic acid in lentil seed (Lens culinaris medik.)

The previously unknown linoleic acid peroxidation product 9,12-dioxo-10(Z)-decenoic acid (Z5) was detected in lentil seed fluor (Lens culinaris Medik.) by electron impact mass spectrometry (El-MS) after derivatization with pentafluorobenzyl-hydroxylamine-hydrochloride, methylation of acidic groups with diazomethane, and protection of hydroxylic groups with N-methyl-N-trimethylsilyl-trifluoroacetamide. The structure of the natural product was confirmed by synthesis of Z5, 9,12-dioxo-10(E)-decenoic acid, and derivatives. EI-MS, nuclear magnetic resonance and gas chromatographic data of these compounds and synthetic intermediates are discussed.     

A new removable resist for high aspect ratio applications

A variety of different photo resists are used for fabrication of MEMS. Presently good results were reported for SU-8, a chemically amplified negative tone photoresist. But SU-8 has a disadvantage for some applications in LIGA technique, especially in the X-ray mask fabrication. After processing the finished resist pattern are hardly soluble from the substrate. This paper will briefly describe the current status of the development of the new negative tone photoresist CAR 44 whose big advantage is the easy removableness of the cross linked pattern. This work widely uses the contents of the presentation “A New Removable Resist for High Aspect Ratio Applications” to the High Aspect Ratio Micro Structure Technology workshop HARMST 2005 held in Gyeongyu (Republic of Korea), June 10–13, 2005.     

Experimental Modelling using Low Melting Point Metallic Melts: Relevance for Metallurgical Engineering

This paper describes model experiments with liquid metals of low melting point as an important tool to investigate the flow structure and related transport processes in melt flows being relevant for metallurgical applications. Three examples have been selected to demonstrate the benefit of such experiments, namely the electromagnetic stirring of a metallic melt in a pool, the directional solidification of Al‐Si alloys under the influence of an electromagnetically driven flow and the behaviour of a liquid metal bubble plume if a vertically travelling field is applied. Results of flow measurements are presented and the relevance for metallurgical processes is discussed.     

MARQUIS: GENERATION OF USER-TAILORED MULTILINGUAL AIR QUALITY BULLETINS

Air pollution has a major influence on health. It is thus not surprising that air quality (AQ) increasingly becomes a central issue in the environmental information policy worldwide. The most common way to deliver AQ information is in terms of graphics, tables, pictograms, or color scales that display either the concentrations of the pollutant substances or the corresponding AQ indices. However, all of these presentation modi lack the explanatory dimension; nor can they be easily tailored to the needs of the individual users. MARQUIS is an AQ information generation service that produces user-tailored multilingual bulletins on the major measured and forecasted air pollution substances and their relevance to human health in five European regions. It incorporates modules for the assessment of pollutant time series episodes with respect to their relevance to a given addressee, for planning of the discourse structure of the bulletins and the selection of the adequate presentation mode, and for generation proper. The positive evaluation of the bulletins produced by MARQUIS by users shows that the use of automatic text generation techniques in such a complex and sensitive application is feasible.     

Pyrolysis-field ionization mass spectrometry of modified bagasse fractions

The fractions obtained from bagasse after activation with 17.5% NaOH, alternative alkylation with quarternary ammonium groups and extraction with 80% ethanol, water and 5% NaOH, were analyzed using pyrolysis-field ionization mass spectrometry (Py-FIMS). The proposed degradation products from lignin were mostly symmetric fragments containing two aromatic rings while the compounds formed from polysaccharides were the result of dehydration reactions. From the temperature-resolved Py-FIMS it could be seen that ethanol-lignin starts to degrade at temperatures lower than 180°C before the degradation of the hemicellulose fraction. The lignin markers are eliminated from hemicellulose fractions before the polysaccharide gasification starts. The quarternary ammonium groups are cleaved from the fractions at temperatures lower than 200°C.