The authors investigated the incidence of transferable resistance in bacterial strains resistant to cephalosporins of the first and second generation in the course of two years in materials of the microbiological department of one of the larger district hygiene stations. They recorded the development of the first strains resistant also to cephamandol, although this antibiotic is not used at all in the above area, and in exceptional instances also resistance to cephotaxime. It was revealed that the transferable resistance to cephamandol is due to the presence of an enzyme which hydrolyzes this antibiotic. When using cephalosporins of the first generation, it is important to foresee an increasing incidence of transmitted resistance to these antibiotics and possibly some strains resistant also to cephalosporins of the second generation and in exceptional cases strains resistant to cephalosporins of the third generation.

• MeSH
• antibiotická rezistence genetika   MeSH
• cefalosporiny farmakologie   MeSH
• Enterobacteriaceae účinky léků   genetika   MeSH
• Publikační typ
• časopisecké články MeSH

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.

• MeSH
• 2D gelová elektroforéza MeSH
• adaptorové proteiny vezikulární transportní metabolismus   MeSH
• cytoskelet účinky léků   metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• invazivní růst nádoru MeSH
• jádro snRNP - proteiny metabolismus   MeSH
• kofilin 1 metabolismus   MeSH
• lidé MeSH
• nádorové proteiny metabolismus   MeSH
• nádory prsu farmakoterapie   metabolismus   patologie   MeSH
• pohyb buněk účinky léků   MeSH
• proteiny tepelného šoku HSP27 metabolismus   MeSH
• proteomika * metody   MeSH
• protokoly antitumorózní kombinované chemoterapie farmakologie   MeSH
• tretinoin farmakologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.

• MeSH
• 2D gelová elektroforéza MeSH
• enzymy metabolismus   MeSH
• financování organizované MeSH
• játra enzymologie   fyziologie   metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• metylace MeSH
• močovina metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nemoci jater enzymologie   etiologie   metabolismus   MeSH
• oxidace-redukce MeSH
• přetížení železem chemicky indukované   komplikace   metabolismus   MeSH
• proteomika metody   MeSH
• sloučeniny železa MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stupeň závažnosti nemoci MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

The effect of valproic acid (VA) on protein expression in human T-lymphocytic leukemia cells MOLT-4 was studied. VA is an inhibitor of histonedeacetylases and has a potential use as antitumor agent in leukemia treatment. The authors in this work prove that 4 h long incubation with 2 mmol/l VA causes phosphorylation of histone H2A.X and its colocalization with 53BP1 in nuclear foci. Their co-localization is typical for DSB signaling machinery. These foci were detected in cells after 4 h exposure without increase of Annexin V positive apoptotic cells. Slight increase in apoptosis (Annexin V positivity) after 24 h is accompanied by more intensive increase in phosphorylation of H2A.X and also by formation of nuclear foci containing gammaH2A.X and 53BP1. Treatment of cells with 2 mmol/l VA resulted in induction of apoptosis affecting about 30% of cells after incubation for 72 h. The changes in protein expression were examined after cell incubation with 2 mmol/l VA for 4 h. Proteins were separated by two-dimensional electrophoresis and quantified using image evaluation system. Those exhibiting significant VA-induced abundance alterations were identified by mass spectrometry. Changes in expression of 22 proteins were detected, of which 15 proteins were down-regulated. Proteomic analysis resulted in successful identification of three proteins involving alfa-tubulin 3, tubulin-specific chaperone and heterogeneous nuclear ribonucloprotein F. Expression of seven proteins was up-regulated, including heterogeneous nuclear ribonucloprotein A/B. Identified proteins are related to microtubular system and hnRNP family. Suppression of microtubular proteins and changes of balance among hnRNPs can contribute to proliferation arrest and apoptosis induction.

• MeSH
• 2D gelová elektroforéza MeSH
• annexin A5 metabolismus   MeSH
• apoptóza účinky léků   MeSH
• financování organizované MeSH
• heterogenní jaderné ribonukleoproteiny metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• kyselina valproová farmakologie   MeSH
• leukemie T-buněčná metabolismus   patologie   MeSH
• lidé MeSH
• nádorové proteiny metabolismus   MeSH
• proteom analýza   MeSH
• průtoková cytometrie MeSH
• signální transdukce MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.

• MeSH
• anthrachinony MeSH
• barvicí látky MeSH
• buněčná diferenciace MeSH
• extracelulární matrix metabolismus   MeSH
• imunohistochemie MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• osteokalcin fyziologie   metabolismus   MeSH
• osteonektin metabolismus   MeSH
• osteopontin metabolismus   MeSH
• Sus scrofa MeSH
• tkáňové podpůrné struktury MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

A 2D method was developed for separation of phenolic acids and flavonoids natural antioxidants combining LC with MEKC. The in-capillary preconcentration step was applied for the improvement of the sensitivity of 2D method before the second dimension MEKC analysis. The influence of first dimension LC mobile phase composition on migration times in the second MEKC dimension was evaluated. When gradient elution is applied in the first dimension of 2D LC-MEKC system, increasing concentration of organic solvent in the mobile phase and in fractions transferred from LC influences the electroosmotic flow, partitioning equilibria of samples in micelles and properties of the micelles, which results in shifts of migration times during the consecutive runs in the second MEKC separation dimension. The shifts of migration times caused by the influence of increasing concentration of ACN on MEKC separation in second dimension of 2D LC-MEKC system were compensated by aligning the time axis using electroosmotic flow and micellar marker migration times. The optimized LC-MEKC method was applied on the separation of natural antioxidants in the plant extracts samples.

• MeSH
• acetonitrily chemie   MeSH
• antioxidancia analýza   chemie   izolace a purifikace   MeSH
• chromatografie micelární elektrokinetická kapilární metody   MeSH
• flavonoidy analýza   chemie   izolace a purifikace   MeSH
• hydroxybenzoáty analýza   chemie   izolace a purifikace   MeSH
• rostlinné extrakty chemie   MeSH
• senzitivita a specificita MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Background: Platelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS. Results: 190 spots were found to be significantly different. Of these, 180 spots were successfully identified and correspond to 144 different proteins. Five proteins were found that had not previously been identified in platelets: protein CDV3 homolog, protein ETHE1, protein LZIC, FGFR1 oncogene partner 2, and guanine nucleotide-binding protein subunit beta-5. Using spot expression profile analysis, we found two proteins (WD repeat-containing protein 1 and mitochondrial glycerol-3-phosphate dehydrogenase) that may be part of thrombin specific activation or signal transduction pathway(s). Conclusions: Our results, characterizing the differences within proteins in both activated (by various agonists) and resting platelets, can thus contribute to the basic knowledge of platelets and to the understanding of the function and development of new antiplatelet drugs.

• MeSH
• aktivace trombocytů MeSH
• hemostáza MeSH
• kolagen MeSH
• kyselina arachidonová MeSH
• lidé MeSH
• proteom * agonisté   analýza   MeSH
• thrombin MeSH
• trombocyty fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Blood is a complex biological matrix providing valuable information on nutritional, metabolic, and immune status. The detection of blood biomarkers requires sensitive analytical methods because analytes are at very low concentrations. Peripheral blood monocytes play a crucial role in inflammatory processes, and the metabolites released by monocytes during these processes might serve as important signalling molecules and biomarkers of particular physiological states. Headspace solid-phase microextraction (HS-SPME) combined with two different mass spectrometric platforms, two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (2D-GC/TOF-MS) and one-dimensional gas chromatography coupled to Orbitrap mass spectrometry (GC/Orbitrap-MS), were applied for the investigation of volatile organic compounds (VOCs) produced by human peripheral blood monocytes. An optimized method was subsequently applied for the characterization of changes in VOCs induced by lipopolysaccharides (LPS) and zymosan (ZYM) stimulation. Overall, the 2D-GC/TOF-MS and the 1D-GC/Orbitrap-MS analyses each yielded about 4000 and 400 peaks per sample, respectively. In total, 91 VOCs belonging to eight different chemical classes were identified. The samples were collected in two fractions, conditioned media for monitoring extracellularly secreted molecules and cell pellet samples to determine the intracellular composition of VOCs. Alcohols, ketones, and hydrocarbons were the main chemical classes of the metabolic profile identified in cell fractions. Aldehydes, acids and cyclic compounds were characteristic of the conditioned media fraction. Here we demonstrate that HS-SPME-2D-GC/TOF-MS is more suitable for the identification of specific VOC profiles produced by human monocytes than 1D-GC/Orbitrap-MS. We define the signature of VOCs occurring early after monocyte activation and characterise the signalling compounds released by immune cells into media.

• MeSH
• lidé MeSH
• mikroextrakce na pevné fázi MeSH
• monocyty metabolismus   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí metody   MeSH
• reprodukovatelnost výsledků MeSH
• těkavé organické sloučeniny * analýza   izolace a purifikace   metabolismus   MeSH
• Check Tag
• lidé MeSH
• 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.

• 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

• MeSH
• 2D gelová elektroforéza klasifikace   využití   MeSH
• denaturace nukleových kyselin fyziologie   MeSH
• renaturace nukleových kyselin fyziologie   MeSH
• semena rostlinná růst a vývoj   MeSH