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Plasma cells (PCs) enrichment from bone marrow samples of multiple myeloma (MM) patients is frequently performed by immunomagnetic separation (magnetic activated cell sorting, MACS) using anti-CD138 MicroBeads. The aim of our work was to find optimal strategy for immunomagnetic separation of PCs and determine optimal algorithm of separation techniques for samples with various percentage of neoplastic cells. From 2007 to 2008, selection of PCs using separation programs Possels and Posseld2 was carried out on 234 bone marrow samples obtained from 208 MM patients. In 2008, an optimal algorithm for separation programs was introduced based on the analysis of the previous experiments. The Possels program is applicable for samples with >10% PCs in the mononuclear fraction, while the Posseld2 program is used for samples with 5–10% PCs in the mononuclear fraction. Median purity of 92.6% for the positive fraction of cells (range 14.5–99.6%) and median recovery of 60.4% (range 25.7–99.5%) were obtained when the Possels program was applied (n = 45). A total of 80% (36/45) of processed samples had purity of >70%. Median purity for the positive fraction of 83.7% (range 14.3–99.7%) and median recovery of 14.3% (range 3.6–50.0%) were achieved using the Posseld2 program (n = 99). A total of 68% (67/99) of processed samples reached >70% purity. This separation strategy enabled us to obtain sufficient amounts of highly purified PCs required for subsequent research purposes. The MACS method has been unsuccessful if the percentage of PCs in the initial sample was

All intracellular and some extracellular proteins are continually degraded and replaced by synthesis of new proteins. Both these processes need to stay in equilibrium since their balance may lead to emergence of diseases. Cells contain many proteolytic systems that ensure highly specific and controlled degradation of proteins. One of these systems is the proteasome, a very complex molecular engine allowing degradation of proteins conjugated to ubiquitin. Since the first isolation of proteasome in 1968, many details about its function have been uncovered. In 2004, Nobel Prize for chemistry was awarded for these discoveries. In our review article, we aimed to summarize information about the mechanism of highly selective degradation of proteins by the ubiquitin proteasome pathway. Individual parts of the paper summarize current knowledge about highly selective degradation of proteins by the ubiquitin proteasome system, mechanisms of protein degradation regulation and bio-logical effects of proteasome inhibitors.

Recently, the term biomarker has become, especially in connection with the term clinical proteomics, one of the most frequent terms in the field of biomedical research. The aim of this work was to select an appropriate pre-fractionation method of blood plasma prior to a subsequent proteomic analysis of low-abundant fraction of proteins by two dimensional gel electrophoresis (2-DE) and mass spectrometry to improve the resolution of 2-DE maps and protein identification.First, we compared two prefractionation methods (MARS versus ProteoMiner) preceding 2-DE analysis using 10 blood plasma samples. Based on the results of the comparative experiments, low-abundant plasma protein fractions from 18 multiple myeloma patients treated with bortezomib were analyzed. Patients were divided into two groups: a group resistant to chemotherapy (9 patients--disease progression, stable disease) and a group with positive clinical response (9 patients--complete and partial remission).Samples prefractioned by ProteoMiner method yielded 2-DE maps with a significantly increased number of detected protein spots, as compared to immunodepletion method MARS (Multiple Affinity Removal System). Between groups of chemoresistant and sensitive patients treated with bortezomib, 15 differently intense spots were revealed by image analysis. These spots were found to correspond to 10 proteins, as confirmed by mass spectrometry. Seven proteins had significantly lower protein level in the group of chemosensitive patients (serum amyloid P, fibrinogen--gamma chain, retinol-binding protein 4, complement factor C4-A, apolipoprotein E, carboxypeptidase N and complement factor H-related protein 1) and 3 proteins showed significantly higher levels of protein (or were only detected) in the group of chemosensitive patients (serum paraoxonase 1, alpha-1-antitrypsin and complement factor B).

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy characterized by the accumulation of monoclonal PCs in the bone marrow. For deeper understanding of the molecular mechanisms involved in the development of this disease, the influence of microenvironment, or the prediction of response of tumor PCs to anti-MM treatment, it is possible to use modern technologies for genomic and proteomic analyses. Due to progress in instrumentation, one of the main tools of proteomic analysis is mass spectrometry in combination with chosen separation techniques. This review will provide a short survey of the most commonly used proteomic techniques and show examples of their applications in MM proteome studies.

This diploma work is engaged in issue of the colorectal carcinoma surgery, which is the second most often tumour in the Czech Republic at men and women. The study was analysed from the data of patients, whose endured elective operation for colorectal carcinoma with laparoscopic or open-technique at the Surgery clinic of the University hospital in Ostrava. Due to important technical progress in recent years innovates this laparoscopic technique in area of abdominal surgery and some of modern instrumentations are routinely used in open surgery too. In statistical analysis are compared demographic and clinical characteristics of patients. To these characteristics belong age, sex, ASA clasification (American Society of Anaestesiologist) and BMI (Body Mass Index). By both types of operation are farther compared data concerned with operation. The analysed data, which are the hospitalization length and the postoperative complications, lead to valuation of postoperative process at the group of laparoscopic operated patients and open operated patients. Results are statistical evaluated, documented in tables and displayed in graphs.

Introduction: Multiple myeloma (MM) is still an incurable disease characterized by clonal expansion of malignant plasma cells. New anticancer drugs further improve prognosis of myeloma patients. Despite promising clinical activity, some patients with MM failed to respond to bortezomib therapy. The aim of this study was to evaluate changes in protein expression of myeloma cell line ARH-77 after bortezomib treatment. Materials and methods: Myeloma cell line ARH-77 was treated with bortezomib (5–20nM) for various periods of time. The proteins contained in total myeloma cell lysate were separated by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and the differentially expressed proteins between the untreated (control) and treated cell lines were excised and identified by mass spectrometry. Results: There were analyzed 94 proteins differentially expressed between treated and control cells; total of 34 protein spots were upregulated: proteins involved in regulation of apoptosis, chaperons/stress related proteins, proteolysis of ubiquitin/protein degradation and cytoskeleton proteins. Sixty protein spots were downregulated: proteins involved in synthesis, regulation of apoptosis, chaperons/stress related proteins, regulation of cell cycle proteins, proteins connected to glycolysis and proteolysis of ubiquitin/protein degradation and antioxidant/redox proteins. Conclusion: Employing optimized proteomic approach we identified 94 proteins with altered expression after bortezomib treatment.