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While a plant cell wall is formed by a complex of various components, including polysaccharides and structural proteins, its composition and representation may vary during cell growth. Currently, plant research targets the proteins participating in wall loosening. Multiple classes of enzymes, including various hemicellulases and cellulases, are required for plant material degradation to achieve the maximum decomposition. Identifying the set of proteins involved in the breakdown of cell-wall polymers is important to understand plant material conversion into suitable products. The objective of this study was to describe a method which can be used to carry out proteomics analysis of complex plant samples and identify enzymes degrading biomass. For this purpose we used proteomic techniques including gel electrophoresis, high pressure liquid chromatography combinated with mass spectrometry followed by data evaluation using databases searching. Results show that more than 50 % of these activities correspond to enzymes with proteolytic function. This study was focused primarily on enzymes able to breakdown the lignocellulosic and hemicellulosic parts that are very important for the material conversion into required products of degradation. [ABSTRACT FROM AUTHOR]

1 The modulatory effects of agonists and antagonists of prejunctional α[sub 2]-adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′ triphosphate (ATP) were measured in endothelium-denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long-duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2 The EFS-evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 µM) and guanethidine (10 µM) in the artery and vein. Desipramine (10 µM), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)-evoked overflow of NA in both artery and vein. EFS-evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS- evoked overflow of ATP was equal in the artery and vein. 3 Stimulation of α[sub 2]-adrenoceptors with clonidine (0.1 µM) and oxymethazoline (0.3 µM) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the α[sub 2]-adrenoceptor agonists in the artery and vein. 4 In artery, blockade of α[sub 2]-adrenoceptors with yohimbine at a concentration of 0.1 µM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 µM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 µM) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 µM) increased the NA... [ABSTRACT FROM AUTHOR]

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all- trans and 9- cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e. g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial–mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1. [ABSTRACT FROM AUTHOR]

Retinoic acid (all- trans and 9- cis ) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all- trans retinoic acid, 9- cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment. [ABSTRACT FROM AUTHOR]