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

Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes

Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs

Abstract Background Lead is well known environmental pollutant, which can cause toxic effects in multiple organ systems. However, the influence of lead oxide nanoparticles, frequently emitted to the environment by high temperature technological processes, is still concealed. Therefore, we investigate lead oxide nanoparticle distribution through the body upon their entry into lungs and determine the microscopic and ultramicroscopic changes caused by the nanoparticles in primary and secondary target organs. Methods Adult female mice (ICR strain) were continuously exposed to lead oxide nanoparticles (PbO-NPs) with an average concentration approximately 106 particles/cm3 for 6 weeks (24 h/day, 7 days/week). At the end of the exposure period, lung, brain, liver, kidney, spleen, and blood were collected for chemical, histological, immunohistochemical and electron microscopic analyses. Results Lead content was found to be the highest in the kidney and lungs, followed by the liver and spleen; the smallest content of lead was found in brain. Nanoparticles were located in all analysed tissues and their highest number was found in the lung and liver. Kidney, spleen and brain contained lower number of nanoparticles, being about the same in all three organs. Lungs of animals exposed to lead oxide nanoparticles exhibited hyperaemia, small areas of atelectasis, alveolar emphysema, focal acute catarrhal bronchiolitis and also haemostasis with presence of siderophages in some animals. Nanoparticles were located in phagosomes or formed clusters within cytoplasmic vesicles. In the liver, lead oxide nanoparticle exposure caused hepatic remodeling with enlargement and hydropic degeneration of hepatocytes, centrilobular hypertrophy of hepatocytes with karyomegaly, areas of hepatic necrosis, occasional periportal inflammation, and extensive accumulation of lipid droplets. Nanoparticles were accumulated within mitochondria and peroxisomes forming aggregates enveloped by an electron-dense mitochondrial matrix. Only in some kidney samples, we observed areas of inflammatory infiltrates around renal corpuscles, tubules or vessels in the cortex. Lead oxide nanoparticles were dispersed in the cytoplasm, but not within cell organelles. There were no significant morphological changes in the spleen as a secondary target organ. Thus, pathological changes correlated with the amount of nanoparticles found in cells rather than with the concentration of lead in a given organ. Conclusions Sub-chronic exposure to lead oxide nanoparticles has profound negative effects at both cellular and tissue levels. Notably, the fate and arrangement of lead oxide nanoparticles were dependent on the type of organs

Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs

Abstract Background Lead is well known environmental pollutant, which can cause toxic effects in multiple organ systems. However, the influence of lead oxide nanoparticles, frequently emitted to the environment by high temperature technological processes, is still concealed. Therefore, we investigate lead oxide nanoparticle distribution through the body upon their entry into lungs and determine the microscopic and ultramicroscopic changes caused by the nanoparticles in primary and secondary target organs. Methods Adult female mice (ICR strain) were continuously exposed to lead oxide nanoparticles (PbO-NPs) with an average concentration approximately 106 particles/cm3 for 6 weeks (24 h/day, 7 days/week). At the end of the exposure period, lung, brain, liver, kidney, spleen, and blood were collected for chemical, histological, immunohistochemical and electron microscopic analyses. Results Lead content was found to be the highest in the kidney and lungs, followed by the liver and spleen; the smallest content of lead was found in brain. Nanoparticles were located in all analysed tissues and their highest number was found in the lung and liver. Kidney, spleen and brain contained lower number of nanoparticles, being about the same in all three organs. Lungs of animals exposed to lead oxide nanoparticles exhibited hyperaemia, small areas of atelectasis, alveolar emphysema, focal acute catarrhal bronchiolitis and also haemostasis with presence of siderophages in some animals. Nanoparticles were located in phagosomes or formed clusters within cytoplasmic vesicles. In the liver, lead oxide nanoparticle exposure caused hepatic remodeling with enlargement and hydropic degeneration of hepatocytes, centrilobular hypertrophy of hepatocytes with karyomegaly, areas of hepatic necrosis, occasional periportal inflammation, and extensive accumulation of lipid droplets. Nanoparticles were accumulated within mitochondria and peroxisomes forming aggregates enveloped by an electron-dense mitochondrial matrix. Only in some kidney samples, we observed areas of inflammatory infiltrates around renal corpuscles, tubules or vessels in the cortex. Lead oxide nanoparticles were dispersed in the cytoplasm, but not within cell organelles. There were no significant morphological changes in the spleen as a secondary target organ. Thus, pathological changes correlated with the amount of nanoparticles found in cells rather than with the concentration of lead in a given organ. Conclusions Sub-chronic exposure to lead oxide nanoparticles has profound negative effects at both cellular and tissue levels. Notably, the fate and arrangement of lead oxide nanoparticles were dependent on the type of organs

Additional file 1: of Sub-chronic inhalation of lead oxide nanoparticles revealed their broad distribution and tissue-specific subcellular localization in target organs

Calculation of deposited dose of PbO nanoparticles in the experiments. Figures S1, S2: The size distribution of nanoparticles with respect to the number of particles per unit volume in inhaled air and STEM images of PbO nanoparticles collected on TEM grids. Figures S3, S4: Weight of organs in the experiments. Figure S5: Effect of lead nanoparticles on spleen following 6 weeks exposure to PbO nanoparticles. Figure S6: Detection of proliferating cells in brain tissue. Tables S1, S2: Lead concentration in organs following 6 weeks exposure in the experiments. Tables S3, S4: Pathological changes in kidney, liver and lung in the experiments. (DOCX 4862 kb