Biomedical subjects

Anton Posch

Publications and source records attributed to Anton Posch.

4 recordsLinked to original sources

PubMed2005

Combined effects of gamma radiation and arsenite on the proteome of human TK6 lymphoblastoid cells.

Arsenic present in drinking water and mining environments in some areas has been associated with an increased rate of skin and internal cancers. Contrary to the epidemiological evidence in humans, arsenic does not induce cancer in animal models, but is able to enhance the mutagenicity of other agents. In order to achieve a better understanding of the interaction between arsenic and ionising radiation, an investigation was conducted to detect differences at the proteome level of human TK6 lymphoblastoid cells exposed to these agents. Cells were exposed to either a single dose of 1-Gy 137Cs-gamma-rays or to 1 microM arsenite (As(III)) or to both agents in combination. Two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) were employed for the screening and identification of proteins, respectively. It proved possible to identify seven proteins with significantly affected abundance, three of which showed increased levels and the remaining four showed decreased levels under at least one of the exposure conditions. Following arsenite treatment or irradiation, a significant increase compared with that of the control was observed for glutathione (GSH) transferase omega 1 and proteasome subunit beta type 4 precursor. The combined exposure did not result in an induction of the enzymes. The expression of electron-transfer flavoprotein subunit alpha was found to be enhanced under all three-exposure conditions. Ubiquinol-cytochrome C reductase complex core protein I, adenine phosphoribosyl transferase and endoplasmic reticulum protein hERp29 showed decreased levels after irradiation or arsenite treatment, but not after the combined exposure. The level of serine/threonine protein phosphatase 1 alpha decreased with all treatments. The main conclusions are that both arsenite and gamma-radiation influence the levels of several proteins involved in major metabolic and regulatory pathways, either directly or by triggering the defence mechanisms of the cell. The combined effect of both exposures on the level of some essential proteins such as glutathione transferase, proteasome or serine/threonine phosphatase may contribute to the co-carcinogenic effect of arsenic.

Animals↗

PubMed2004

In-gel digestion of proteins using a solid-phase extraction microplate.

We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it. The initial steps of the in-gel digestion process, comprising destaining, reduction/alkylation, dehydration, and digestion, was carried out in the plate containing 75-microm capillaries. Capillaries containing C18 reversed-phase modified monolithic silica rods of a 200-microm diameter were used for the second plate in which extraction and cleanup of peptides were carried out. Peptides were eluted directly from the solid-phase extraction plate onto the MALDI sample support. The separation of the process into two plates led to increased process stability, without compromising sensitivity, i.e. peptide recovery, making it suitable for true high-throughput protein identification. The handling of proteinases could easily be optimized, and no restrictions were made on chosen pH range through the absence of the solid phase in the initial steps of the protocol. Efficient binding of peptides to the solid phase and subsequent direct elution onto the MALDI sample support led to sensitivities in the attomole range. Performance of the process was demonstrated with tryptic digests of proteins stained with colloidal coomassie blue, silver, and the fluorescent stain SYPRO Ruby.

Animals↗

PubMed2003

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis.

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.

Electrophoresis↗

PubMed2002

Evaluation of patatin as a major cross-reactive allergen in latex-induced potato allergy.

BACKGROUND: Potential cross-reactions between natural rubber latex and fruit/vegetable specific immunoglobulin (Ig)E antibodies have been reported for many years. This study was designed to investigate the molecular basis of acquired food sensitization focusing on the storage protein patatin and the patatin-like latex protein Hev b 7. OBJECTIVE: The amount of potato-specific IgE in the serum of latex-allergic health care workers and children with atopic dermatitis was determined to evaluate cross-reactivity between Hev b 7 and patatin. Additionally, the stability of potato patatin to digestion was investigated. METHODS: Human serum was tested on its reactivity to latex and potato proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis. Latex- and potato-specific IgE concentrations were measured in fluorescence enzyme immunoassays (CAP, Pharmacia, Uppsala, Sweden). Further, potato patatin was chromatographically isolated to perform auto-inhibition tests. Stability of patatin to degradation was determined by digestion in vitro. RESULTS: Patatin was identified as major cross-reactive potato allergen by N-terminal sequencing. Seventy-five percent of the potato-sensitized people reacted with patatin in 1-D immunoblots, and 25% of the positive reactions to Hev b 7 could be blocked by preincubation of the patients' sera with purified potato patatin. Examination of children with atopic dermatitis showed that most sera contained patatin-specific IgE, whereas no Hev b 7-specific IgE was detected. Finally, patatin has been found partially stable to digestion in vitro. CONCLUSIONS: Patatin was identified as a major cross-reactive protein in latex-associated potato allergy and appears to be relevant for atopic dermatitis. Therefore, patatin could be a suitable marker for the determination of potato sensitization, and it may also constitute an important food allergen. Cross-reactivity between Hev b 7 and patatin was restricted to primarily latex-sensitized adults, suggesting a different mechanism of sensitization in children with atopic dermatitis.

Adolescent↗