With increasing demands on protein analyses in complex biological matrices, the insistence on developing new sample preparation techniques is rising. Recently, we introduced a new displacement electrophoresis technique (epitachophoresis) and instrumentation for preparative concentration and cleaning of DNA samples. This work describes the possibility of applying this device to protein samples. We have developed a method for the epitachophoretic concentration of proteins in a cationic mode and tested it by concentrating and collecting the protein zones from complex biological matrices (urine and growth medium). Under optimized conditions, we have obtained recoveries up to 99%. Furthermore, the applicability of the developed method was proven by concentrating and collecting the cytochrome c zone from a HeLa cell line growth medium, where the protein cytochrome c was released during cell apoptosis.
- MeSH
- cytochromy c MeSH
- HeLa buňky MeSH
- izotachoforéza * metody MeSH
- lidé MeSH
- proteiny MeSH
- tělesné tekutiny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Inhalation of lead oxide nanoparticles (PbO NPs), which are emitted to the environment by high-temperature technological processes, heavily impairs target organs. These nanoparticles pass through the lung barrier and are distributed via the blood into secondary target organs, where they cause numerous pathological alterations. Here, we studied in detail, macrophages as specialized cells involved in the innate and adaptive immune response in selected target organs to unravel their potential involvement in reaction to subchronic PbO NP inhalation. In this context, we also tackled possible alterations in lipid uptake in the lungs and liver, which is usually associated with foam macrophage formation. RESULTS: The histopathological analysis of PbO NP exposed lung revealed serious chronic inflammation of lung tissues. The number of total and foam macrophages was significantly increased in lung, and they contained numerous cholesterol crystals. PbO NP inhalation induced changes in expression of phospholipases C (PLC) as enzymes linked to macrophage-mediated inflammation in lungs. In the liver, the subchronic inhalation of PbO NPs caused predominantly hyperemia, microsteatosis or remodeling of the liver parenchyma, and the number of liver macrophages also significantly was increased. The gene and protein expression of a cholesterol transporter CD36, which is associated with lipid metabolism, was altered in the liver. The amount of selected cholesteryl esters (CE 16:0, CE 18:1, CE 20:4, CE 22:6) in liver tissue was decreased after subchronic PbO NP inhalation, while total and free cholesterol in liver tissue was slightly increased. Gene and protein expression of phospholipase PLCβ1 and receptor CD36 in human hepatocytes were affected also in in vitro experiments after acute PbO NP exposure. No microscopic or serious functional kidney alterations were detected after subchronic PbO NP exposure and CD68 positive cells were present in the physiological mode in its interstitial tissues. CONCLUSION: Our study revealed the association of increased cholesterol and lipid storage in targeted tissues with the alteration of scavenger receptors and phospholipases C after subchronic inhalation of PbO NPs and yet uncovered processes, which can contribute to steatosis in liver after metal nanoparticles exposure.
- MeSH
- cholesterol MeSH
- fosfolipasy typu C * MeSH
- kovové nanočástice * chemie MeSH
- lidé MeSH
- makrofágy MeSH
- olovo MeSH
- oxidy MeSH
- zánět MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.
- MeSH
- Barrettův syndrom * metabolismus MeSH
- chromatografie kapalinová MeSH
- formiáty MeSH
- glycin analýza MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- methanol analýza MeSH
- sliny chemie MeSH
- taurin analýza MeSH
- žlučové kyseliny a soli analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The knowledge of the structure, function, and abundance of specific proteins related to the EMT process is essential for developing effective diagnostic approaches to cancer with the perspective of diagnosis and therapy of malignancies. The success of all-trans retinoic acid (ATRA) differentiation therapy in acute promyelocytic leukemia has stimulated studies in the treatment of other tumors with ATRA. This review will discuss the impact of ATRA use, emphasizing epithelial-mesenchymal transition (EMT) proteins in breast cancer, of which metastasis and recurrence are major causes of death.
- MeSH
- epitelo-mezenchymální tranzice * MeSH
- lidé MeSH
- metastázy nádorů MeSH
- nádorové proteiny agonisté metabolismus MeSH
- nádory prsu metabolismus mortalita patologie MeSH
- receptory kyseliny retinové agonisté metabolismus MeSH
- tretinoin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R3SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared.
- MeSH
- 2D gelová elektroforéza MeSH
- antitumorózní látky farmakologie MeSH
- down regulace MeSH
- epitelo-mezenchymální tranzice účinky léků MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- organocínové sloučeniny farmakologie MeSH
- proteomika metody MeSH
- retinoidní X receptory agonisté metabolismus MeSH
- signální transdukce účinky léků MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tandemová hmotnostní spektrometrie MeSH
- tretinoin farmakologie MeSH
- trialkylcínové sloučeniny farmakologie MeSH
- vimentin metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Changes in glycoprotein content, altered glycosylations, and aberrant glycan structures are increasingly recognized as cancer hallmarks. Because breast cancer is one of the most common causes of cancer deaths in the world, it is highly urgent to find other reliable biomarkers for its initial diagnosis and to learn as much as possible about this disease. In this Review, the applications of lectins to a screening of potential breast cancer biomarkers published during recent years are overviewed. These data provide a deeper insight into the use of modern strategies, technologies, and scientific knowledge in glycoproteomic breast cancer research. Particular attention is concentrated on the use of lectin-based affinity techniques, applied independently or most frequently in combination with mass spectrometry, as an effective tool for the targeting, separation, and reliable identification of glycoprotein molecules. Individual procedures and lectins used in published glycoproteomic studies of breast-cancer-related glycoproteins are discussed. The summarized approaches have the potential for use in diagnostic and predictive applications. Finally, the use of lectins is briefly discussed from the view of their future applications in the analysis of glycoproteins in cancer.
This work aimed to provide, in one isolation and separation step, an overview of the content of proteins with different solubility after treatment with all-trans retinoic acid, which is considered to be an important therapeutic agent, predominantly in acute promyelocytic leukemia. Breast, ovarian, bladder, and skin cancers have been demonstrated to be suppressed by retinoic acid, as well. The bottom-up proteomic strategies were applied for the analysis of proteins extracted from triple-negative breast cancer MDA-MB-231 cells utilizing a commercially manufactured kit. The gel electrophoresis followed by MALDI-TOF MS analysis was used for protein determination. By employing PDQuest™ software, we identified several proteins affected by all-trans retinoic acid. Two proteins, vimentin and CD44, which are associated with the epithelial-mesenchymal transition, were selected for a detailed study. We have found that all-trans retinoic acid results in significantly reduced levels of vimentin and CD44 in both the cytoplasmic and membrane fractions. A significant effect was particularly evident in CD44, where protein level in the cytoplasmic fraction was almost completely suppressed.
Trialkyltins and triaryltins function as nuclear retinoid X receptors (RXR) agonists due to their affinity to the ligand-binding domain of RXR subtypes and function as transcriptional activators. We present the data on combined effects of all-trans retinoic acid (ATRA), retinoic acid receptor (RAR) ligand and tributyltin chloride or triphenyltin chloride (RXR ligands) on protein pattern in MDA-MB-231 cells. Proteomic strategies based on bottom-up method were applied in this study. The total cell proteins were extracted, separated on 2D SDS-PAGE and their characterization was achieved by MALDI-TOF/TOF MS/MS. By employing PDQuest™ software, we identified more than 30 proteins differently affected by the above compounds. For further studies, we selected specific proteins associated either with metabolic pathway (glyceraldehyde-3-phosphate dehydrogenase) or to cellular processes as apoptosis, regulation of gene transcription or epithelial-mesenchymal transition (annexin 5, nucleoside diphosphate kinase B and vimentin). We have found that treatment of MDA-MB-231 cells with triorganotins reduced the expression of studied proteins. Moreover, the treatment of MDA-MB-231 cells with triorganotin compounds together with ATRA resulted in an additional reduction of annexin 5, vimentin and nucleoside diphosphate kinase B. These results demonstrate that RXR/RAR heterodimer may act under this experimental design as permissive heterodimer allowing activation of RXR by triorganotins.
In the present study, we analyzed the cell lysates of human tumour cell lines representing two major clinically different types of breast cancer. Our main goal was to show the differences between them on proteomic level. Gel electrophoresis followed by MALDI-TOF MS analysis was used for proteins determination. Exactly 98 proteins were unequivocally identified and 60 of them were expressed differentially between MDA-MB-231 and MCF-7 cell lines. Among the proteins reported here, some well-known breast cancer markers (e.g., annexin A1, annexin A2 and vimentin) were identified in the MDA-MB-231 cell line and thus we were able to distinguish both cell lines sufficiently.
- MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- nádorové biomarkery metabolismus MeSH
- nádorové proteiny metabolismus MeSH
- nádory prsu metabolismus patologie MeSH
- proteom metabolismus MeSH
- receptory pro estrogeny metabolismus MeSH
- regulace genové exprese u nádorů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND: Within last few years, the occurrence of food allergens and corresponding food allergies has been increasing, therefore research into the individual allergens is required. In the present work, the effect of cereal processing on the amounts of allergenic proteins is studied by modern proteomic-based approaches. The most important wheat and barley allergens are low-molecular-weight (LMW) proteins. Therefore we investigated the relative quantitative changes of these proteins after food technological processing, namely wheat couscous production and barley malting. RESULTS: A comparative study using mass spectrometry in connection with the technique of isobaric tag for relative and absolute quantification (iTRAQ) revealed that the amount of wheat allergenic LMW proteins decreased significantly during couscous production (approximately to 5-26% of their initial content in wheat flour). After barley malting, the amounts of the majority of LMW proteins decreased as well, although to a lesser extent than in the case of wheat/couscous. The level of two allergens even slightly increased. CONCLUSION: Suggested proteomic strategy proved as universal and sensitive method for fast and reliable identification of various cereal allergens and monitoring of their quantitative changes during food processing. Such information is important for consumers who suffer from allergies.
- MeSH
- alergeny škodlivé účinky analýza chemie MeSH
- antigeny rostlinné škodlivé účinky analýza chemie MeSH
- dietní proteiny škodlivé účinky analýza chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- ječmen (rod) chemie MeSH
- jedlá semena škodlivé účinky chemie MeSH
- kontrola potravin metody MeSH
- lidé MeSH
- manipulace s potravinami * MeSH
- molekulová hmotnost MeSH
- peptidové mapování MeSH
- potravinová alergie etiologie prevence a kontrola MeSH
- proteomika MeSH
- pšenice chemie MeSH
- rostlinné proteiny škodlivé účinky analýza chemie MeSH
- semenáček chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Česká republika MeSH
