The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

• Klíčová slova
• energy metabolism, gel-based proteomics, nLC-MS/MS, oxidative stress, protracted morphine withdrawal, rat brain cortex, rat hippocampus,
• Publikační typ
• časopisecké články MeSH

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

• Klíčová slova
• 2D electrophoresis, Immunoblotting, Mass spectrometry, Mitochondria, Nitrotyrosine,
• MeSH
• 2D gelová elektroforéza metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• imunoblotting metody   MeSH
• kyselina peroxydusitá chemie   MeSH
• mitochondriální proteiny analýza   metabolismus   MeSH
• skot MeSH
• srdeční mitochondrie chemie   metabolismus   MeSH
• tyrosin analogy a deriváty   analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• kyselina peroxydusitá MeSH
• mitochondriální proteiny MeSH
• tyrosin MeSH

The development of hypoxic pulmonary hypertension is characterized by the structural remodeling of pulmonary arteries. However, the relationship between changes of arterial cells and the extracellular matrix remains unclear. We focused on the evaluation of the non-fibrillar collagen changes in tunica media induced by a four-day exposure to hypoxia and the correlation of these changes with the pulmonary arterial wall structure modifications. We used 20 adult male Wistar rats. The amount and localization of collagen VI, collagen IV, matrix metalloproteinase (MMP) 2, and MMP9 were tested in pulmonary arteries immunohistochemically. Two-dimensional electrophoresis and messenger RNA (mRNA) expression were used for the subsequent comparison of protein changes in arterial tunica media cells (normoxia/hypoxia). Collagen VI was significantly reduced strictly in the tunica media of conduit arteries of hypoxia-exposed rats; however, its mRNA increased. The amount of collagen IV and its mRNA were not altered. We detected a significant increase of MMP9 strictly in the tunica media. In addition, a significantly increased number of MMP9-positive cells surrounded the arteries. MMP2 and the expression of its mRNA were decreased in tunica media. We conclude that the loss of collagen VI is an important step characterizing the remodeling of pulmonary arteries. It could influence the phenotypic status and behavior of smooth muscle cells and modify their proliferation and migration.

• Klíčová slova
• collagen VI, hypoxic pulmonary hypertension, pulmonary arterial remodeling, smooth muscle cells, tunica media,
• Publikační typ
• časopisecké články MeSH

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.

• Klíčová slova
• Heart, Myocardial heterogeneity, Proteomics, Two-dimensional electrophoresis, Ventricle, Ventricular myocardium,
• MeSH
• 2D gelová elektroforéza MeSH
• chaperon hsp60 metabolismus   MeSH
• chromatografie kapalinová MeSH
• dihydrolipoamiddehydrogenasa metabolismus   MeSH
• energetický metabolismus MeSH
• kreatinkinasa, forma MM metabolismus   MeSH
• L-laktátdehydrogenasa metabolismus   MeSH
• lehké řetězce myosinu metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• potkani Wistar MeSH
• proteomika * MeSH
• respirační komplex I metabolismus   MeSH
• srdeční komory enzymologie   metabolismus   MeSH
• svalové proteiny izolace a purifikace   metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chaperon hsp60 MeSH
• dihydrolipoamiddehydrogenasa MeSH
• Hspd1 protein, rat MeSH Prohlížeč
• kreatinkinasa, forma MM MeSH
• L-laktátdehydrogenasa MeSH
• lehké řetězce myosinu MeSH
• mitochondriální proteiny MeSH
• respirační komplex I MeSH
• svalové proteiny MeSH