Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.     

Some observations regarding the solvent effect in the electrochemical chlorination of c-hexene

The changes in the voltammetric curves upon addition of olefine, chloride ions and water were studied in the electrochemical chlorination of cyclohexene in nitromethane solution. Relation of the changes observed to the donation of solvent was discussed.     

Supercritical carbon dioxide extraction of okra (Hibiscus esculentus L) seeds

Pilot‐scale supercritical fluid extraction of okra seeds was carried out, using carbon dioxide as solvent, at temperatures of 40, 50 and 60 °C and pressures of 150, 300 and 450 bar. Laboratory‐scale Soxhlet extraction of the ground seeds was carried out with ethanol and n‐hexane. The yields of supercritical fluid extraction and n‐hexane Soxhlet extractions were similar. The ethanol Soxhlet extraction gave the highest yield, but the concentrations of β‐sitosterol and tocopherols in this extract were lower than in the supercritical fluid extraction product. The fatty acid profiles of the extracts were determined, and a high unsaturated/saturated ratio was observed. The fatty acid compositions were only slightly different for oils obtained by the different extraction methods. Copyright © 2005 Society of Chemical Industry     

Degradation of condensed tannins by rumen microbes exposed to quebracho tannins (QT) in rumen simulation technique (RUSITEC) and effects of QT on fermentative processes in the RUSITEC

In an earlier study it was observed that the rumen microbes from cattle which had never consumed tannin-containing diets do not have enzymes for degrading condensed tannins. In this study, rumen microbes were exposed to small amounts of quebracho tannins for 8 days using a rumen simulation technique (RUSITEC). The levels maintained in the RUSITEC were 0.1, 0.2 and 0.4 mg of the spray-dried quebracho powder (SDQT) per ml of the medium. After 8 days of tannin exposure, the liquor containing ‘exposed/adapted’ microbes from RUSITEC was incubated for 40 h without and with purified tannins of quebracho and Dichostachys cinerea leaves in in vitro gas method. There was no degradation of condensed tannins. The enzymes for degradation of condensed tannins were not induced in rumen microbes by exposure to different concentrations of tannins for 8 days in the RUSITEC. In the RUSITEC, SDQT significantly reduced the number of total protozoa, entodiniomorphs and holotrichs; effect was higher on holotrichs. There was no significant change in the levels of short chain fatty acid but the molar proportion of propionate was significantly higher and of butyrate significantly lower at 0.4 mg SDQT ml^?1. Significantly lower levels of ammonia in the medium was also observed on injection of tannins. Microbial mass production, calculated using ¹⁵N incorporation, was similar at 0.1 and 0.2 mg SDQT ml^?1 but significantly lower (13%) at 0.4 mg SDQT ml^?1. The dry matter digestibility of the feed (80% hay and 20% barley) was not significantly affected by SDQT.     

Preparation and application of highly porous aerogel-based bioactive materials in dentistry

In this study, the possibility of preparation and application of highly porous silica aerogel-based bioactive materials are presented. The aerogel was combined with hydroxyapatite and p.tricalcium phosphate as bioactive and osteoinductive agents. The porosity of aerogels was in themesoporous region with a maximum pore diameter of 7,4 and 12.7 nm for the composite materials. The newly developed bioactive materials were characterized by scant electron microscopy. The in vitro biological effect of these modified surfaces was also tested on SAOS-2 osteogenic sarcoma cells by confocal laser scanning microscopy.     

Module-Based Reinforcement Learning: Experiments with a Real Robot

The behavior of reinforcement learning (RL) algorithms is best understood in completely observable, discrete-time controlled Markov chains with finite state and action spaces. In contrast, robot-learning domains are inherently continuous both in time and space, and moreover are partially observable. Here we suggest a systematic approach to solve such problems in which the available qualitative and quantitative knowledge is used to reduce the complexity of learning task. The steps of the design process are to:i) decompose the task into subtasks using the qualitative knowledge at hand; ii) design local controllers to solve the subtasks using the available quantitative knowledge and iii) learn a coordination of these controllers by means of reinforcement learning. It is argued that the approach enables fast, semi-automatic, but still high quality robot-control as no fine-tuning of the local controllers is needed. The approach was verified on a non-trivial real-life robot task. Several RL algorithms were compared by ANOVA and it was found that the model-based approach worked significantly better than the model-free approach. The learnt switching strategy performed comparably to a handcrafted version. Moreover, the learnt strategy seemed to exploit certain properties of the environment which were not foreseen in advance, thus supporting the view that adaptive algorithms are advantageous to non-adaptive ones in complex environments.     

Time- and concentration-dependence of the growth-promoting activity of insulin and histamine in Tetrahymena. Application of the MTT-method for the determination of cell proliferation in a protozoan model

The unicellular ciliate Tetrahymena pyriformis was treated with different concentrations of insulin or histamine and at different time points the cell density was measured, using a tetrazolium-based semiautomated colorimetric assay (MTT). The assay was suitable to determine the rate of cell proliferation of Tetrahymena. Insulin in each concentration significantly elevated the cell count up to 3 h. After that, it was neutral or its effect was insignificant. Histamine at 10(-5) M concentration diminished cell count at 3, 5, 7 and 24 h. At 10(-6) M concentration there was no difference and at 10(-7) M concentration it enhanced cell division up to 5 h, after that there being no difference. The two hormones have cell division promoting activity for cells of higher animals and the experiments demonstrate this effect already at a unicellular level.     

The effects of ceramide and its analogues on the secretion of the mucocyst content of Tetrahymena

Monoclonal antibody to Geodia lectin is bound by the mucocyst content of Tetrahymena. By using this (fluorescent-labelled) antibody and confocal microscopy, the actual state of the mucocyst (position, resting, filling or extruding) can be studied. Treatment with C2 ceramide and non-hydroxy fatty acid caused a rapid depletion of mucocyst material. Another ceramide analogue, psychosine caused fusion of mucocysts. In these cases--in contrast to the controls--the contractile vacuole was filled with mucocyst material and this was seen in the tubules in contact with the contractile vacuoles. Hydroxy fatty acid ceramide, sphingomyelin and sphingosine-1-phosphate were ineffective. As the former materials influence also the cytoskeleton, while the latter do not, the cytoskeleton is presumed to have a mediatory effect. Neither the connection of contractile vacuoles with tubular structures nor mucocyst fusion have been described before.