Changes in the proteomes of the hemocytes and fat bodies of the flesh fly Sarcophaga bullata larvae after infection by Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Insects have an efficient self-defense system that is based on innate immunity. Recent findings have disclosed many parallels between human and insect innate immunity, and simultaneously fine differences in the processes between various species have been revealed. Studies on the immune systems of various insect species may uncover the differences in their host defense strategies.</p> <p>Results</p> <p>We analyzed the proteomes of the hemocytes and fat bodies of <it>Sarcophaga bullata </it>larvae after infection by <it>Escherichia coli</it>. The 2-DE gels of the hemocytes and fat bodies of infected larvae were compared with those of aseptically injured larvae. Our analysis included the construction of protein maps of the hemocyte cells and cells from fat bodies, the identification of the changed proteins, in response to infection, using LC-MS/MS, and the estimation of the trends in expression of these proteins at three time points (30 min, 6 hours and 22 hours) after infection. In total, seven changed spots were found in the hemocytes, and four changed spots were found in the fat bodies. Three types of trends in protein expression were observed. Cofilin and transgelin were undetectable at 30 min after infection but were continuously up-regulated in the induced larvae after 22 hours. A prophenoloxidase isoform and lectin subunit α were slightly up-regulated at 30 min after infection, and their protein levels reached the highest points after 6 hours but decreased after 22 hours. T-Complex subunit α, GST, ferritin-like protein and an anterior fat body protein (regucalcin homologue) were down-regulated at 22 hours after infection.</p> <p>Conclusions</p> <p>Many proteins identified in our study corresponded to the proteins identified in other insects. Compared to the former studies performed in insects, we presented 2-D protein maps of the hemocytes and fat bodies and showed the trends in expression of the immune-elicited proteins.</p

Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

<p>Abstract</p> <p>Background</p> <p>Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest.</p> <p>Methods</p> <p>We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots.</p> <p>Results</p> <p>Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics.</p> <p>Conclusions</p> <p>Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.</p

Supplementary material for the article: Macháčková, K.; Chrudinová, M.; Radosavljević, J.; Potalitsyn, P.; Křížková, K.; Fábry, M.; Selicharová, I.; Collinsová, M.; Brzozowski, A. M.; Žáková, L.; et al. Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation. Biochemistry 2018, 57 (16), 2373– 2382. https://doi.org/10.1021/acs.biochem.7b01260

Supplementary material for: [https://doi.org/10.1021/acs.biochem.7b01260]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2135

Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues

<p>Abstract</p> <p>Background</p> <p>Breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences.</p> <p>Methods</p> <p>We prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE) gels in pH range 4–7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package) and data-mining analysis (GUHA). For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed.</p> <p>Results</p> <p>Three protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased.</p> <p>Conclusion</p> <p>We have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.</p