Cytokinins (CKs) play a crucial role in many physiological and developmental processes at the levels of individual plant components (cells, tissues, and organs) and by coordinating activities across these parts. High-resolution measurements of intracellular CKs in different plant tissues can therefore provide insights into their metabolism and mode of action. Here, we applied fluorescence-activated cell sorting of green fluorescent protein (GFP)-marked cell types, combined with solid-phase microextraction and an ultra-high-sensitivity mass spectrometry (MS) method for analysis of CK biosynthesis and homeostasis at cellular resolution. This method was validated by series of control experiments, establishing that protoplast isolation and cell sorting procedures did not greatly alter endogenous CK levels. The MS-based method facilitated the quantification of all the well known CK isoprenoid metabolites in four different transgenic Arabidopsis thaliana lines expressing GFP in specific cell populations within the primary root apex. Our results revealed the presence of a CK gradient within the Arabidopsis root tip, with a concentration maximum in the lateral root cap, columella, columella initials, and quiescent center cells. This distribution, when compared with previously published auxin gradients, implies that the well known antagonistic interactions between the two hormone groups are cell type specific.

• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• biologický transport MeSH
• cytokininy metabolismus   MeSH
• extrakce na pevné fázi MeSH
• kořeny rostlin cytologie   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• meristém metabolismus   MeSH
• metabolom MeSH
• miniaturizace MeSH
• orgánová specificita MeSH
• protoplasty metabolismus   MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• kyseliny indoloctové MeSH
• zelené fluorescenční proteiny MeSH

p73, a member of the p53 family, exhibits activities similar to those of p53, including the ability to induce growth arrest and apoptosis. p73 influences chemotherapeutic responses in human cancer patients, in association with p53. Alternative splicing of the TP73 gene produces many p73 C- and N-terminal isoforms, which vary in their transcriptional activity towards p53-responsive promoters. In this paper, we show that the C-terminal spliced isoforms of the p73 protein differ in their DNA-binding capacity, but this is not an accurate predictor of transcriptional activity. In different p53-null cell lines, p73beta induces either mitochondrial-associated or death receptor-mediated apoptosis, and these differences are reflected in different gene expression profiles. In addition, p73 induces cell cycle arrest and p21(WAF1) expression in H1299 cells, but not in Saos-2. This data shows that TAp73 isoforms act differently depending on the tumour cell background, and have important implications for p73-mediated therapeutic responses in individual human cancer patients.

• MeSH
• aktivace transkripce MeSH
• alternativní sestřih MeSH
• apoptóza fyziologie   MeSH
• buněčné linie MeSH
• buněčný cyklus fyziologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• genetická transkripce MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• protein p73 MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• signální transdukce fyziologie   MeSH
• stanovení celkové genové exprese MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• transkripční faktory MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• jaderné proteiny MeSH
• nádorové supresorové proteiny MeSH
• nádorový supresorový protein p53 MeSH
• protein - isoformy MeSH
• protein p73 MeSH
• TP53 protein, human MeSH Prohlížeč
• TP63 protein, human MeSH Prohlížeč
• TP73 protein, human MeSH Prohlížeč
• trans-aktivátory MeSH
• transkripční faktory MeSH

Our previously-obtained impressive results of highly increased C2C12 mouse myoblast adhesion to amine plasma polymers (PPs) motivated current detailed studies of cell resistance to trypsinization, cell proliferation, motility, and the rate of attachment carried out for fibroblasts (LF), keratinocytes (HaCaT), rat vascular smooth muscle cells (VSMC), and endothelial cells (HUVEC, HSVEC, and CPAE) on three different amine PPs. We demonstrated the striking difference in the resistance to trypsin treatment between endothelial and non-endothelial cells. The increased resistance observed for the non-endothelial cell types was accompanied by an increased rate of cellular attachment, even though spontaneous migration was comparable to the control, i.e., to the standard cultivation surface. As demonstrated on LF fibroblasts, the resistance to trypsin was similar in serum-supplemented and serum-free media, i.e., medium without cell adhesion-mediating proteins. The increased cell adhesion was also confirmed for LF cells by an independent technique, single-cell force spectroscopy. This method, as well as the cell attachment rate, proved the difference among the plasma polymers with different amounts of amine groups, but other investigated techniques could not reveal the differences in the cell behaviour on different amine PPs. Based on all the results, the increased resistance to trypsinization of C2C12, LF, HaCaT, and VSMC cells on amine PPs can be explained most probably by a non-specific cell adhesion such as electrostatic interaction between the cells and amine groups on the material surface, rather than by the receptor-mediated adhesion through serum-derived proteins adsorbed on the PPs.

• MeSH
• aminy chemie   MeSH
• buněčná adheze účinky léků   MeSH
• buněčné linie MeSH
• lidé MeSH
• plazmové plyny chemie   MeSH
• polymery chemie   farmakologie   MeSH
• povrchové vlastnosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminy MeSH
• plazmové plyny MeSH
• polymery MeSH

We describe a method combining fluorescence-activated cell sorting (FACS) with one-step miniaturized isolation and accurate quantification of cytokinins (CKs) using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure these phytohormones in specific cell types of Arabidopsis thaliana roots. The methodology provides information of unprecedented resolution about spatial distributions of CKs, and thus should facilitate attempts to elucidate regulatory networks involved in root developmental processes.

• Klíčová slova
• Arabidopsis, Cytokinins, Fluorescence-activated cell sorting (FACS), Liquid chromatography–mass spectrometry (LC-MS), Protoplasts, Roots, Solid-phase extraction (SPE),
• MeSH
• Arabidopsis metabolismus   MeSH
• cytokininy metabolismus   MeSH
• genové regulační sítě fyziologie   MeSH
• kořeny rostlin metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• průtoková cytometrie metody   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH

TP73 is a member of the TP53 gene family and produces N- and C-terminal protein isoforms through alternative promoters, alternative translation initiation and alternative splicing. Most notably, p73 protein isoforms may either contain a p53-like transactivation domain (TAp73 isoforms) or lack this domain (ΔTAp73 isoforms) and these variants have opposing or independent functions. To date, there is a lack of well-characterised isoform-specific p73 antibodies. Here, we produced polyclonal and monoclonal antibodies to N-terminal p73 variants and the C-terminal p73α isoform, the most common variant in human tissues. These reagents show that TAp73 is a marker of multiciliated epithelial cells, while ΔTAp73 is a marker of non-proliferative basal/reserve cells in squamous epithelium. We were unable to detect ΔNp73 variant proteins, in keeping with recent data that this is a minor form in human tissues. Most cervical squamous cell carcinomas (79%) express p73α, and the distribution of staining in basal cells correlated with lower tumour grade. TAp73 was found in 17% of these tumours, with a random distribution and no association with clinicopathological features. These data indicate roles for ΔTAp73 in maintaining a non-proliferative state of undifferentiated squamous epithelial cells and for TAp73 in the production of differentiated multiciliated cells.

• Klíčová slova
• Cervical cancer, Endometrium, Fallopian tube, Multiciliated cells, Squamous epithelial stem cells, p73 isoforms,
• MeSH
• epitelové buňky metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• nádorové buněčné linie MeSH
• nádory děložního čípku metabolismus   patologie   genetika   MeSH
• nádory metabolismus   patologie   genetika   MeSH
• protein - isoformy * metabolismus   genetika   MeSH
• protein p73 * metabolismus   genetika   MeSH
• spinocelulární karcinom metabolismus   patologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• delta Np73 protein, human MeSH Prohlížeč
• monoklonální protilátky MeSH
• protein - isoformy * MeSH
• protein p73 * MeSH
• TP73 protein, human MeSH Prohlížeč

BACKGROUND: Sarcosine is a widely discussed oncometabolite of prostate cells. Although several reports described connections between sarcosine and various phenotypic changes of prostate cancer (PCa) cells, there is still a lack of insights on the complex phenomena of its effects on gene expression patterns, particularly in non-malignant and non-metastatic cells. METHODS: To shed more light on this phenomenon, we performed parallel microarray profiling of RNA isolated from non-malignant (PNT1A), malignant (22Rv1), and metastatic (PC-3) prostate cell lines treated with sarcosine. Microarray results were experimentally verified using semi-quantitative-RT-PCR, clonogenic assay, through testing of the susceptibility of cells pre-incubated with sarcosine to anticancer agents with different modes of actions (inhibitors of topoisomerase II, DNA cross-linking agent, antimicrotubule agent and inhibitor of histone deacetylases) and by evaluation of activation of executioner caspases 3/7. RESULTS: We identified that irrespective of the cell type, sarcosine stimulates up-regulation of distinct sets of genes involved in cell cycle and mitosis, while down-regulates expression of genes driving apoptosis. Moreover, it was found that in all cell types, sarcosine had pronounced stimulatory effects on clonogenicity. Except of an inhibitor of histone deacetylase valproic acid, efficiency of all agents was significantly (P < 0.05) decreased in sarcosine pre-incubated cells. CONCLUSIONS: Our comparative study brings evidence that sarcosine affects not only metastatic PCa cells, but also their malignant and non-malignant counterparts and induces very similar changes in cells behavior, but via distinct cell-type specific targets.

• Klíčová slova
• cell cycle, chemoresistance, microarray, prostate cancer, sarcosine,
• MeSH
• apoptóza fyziologie   MeSH
• lidé MeSH
• metastázy nádorů MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové proteiny klasifikace   metabolismus   MeSH
• nádory prostaty * metabolismus   patologie   MeSH
• prostata * metabolismus   patologie   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• sarkosin metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorové biomarkery MeSH
• nádorové proteiny MeSH
• sarkosin MeSH

Nitro-fatty acids (NFAs) are endogenous lipid mediators capable of post-translational modifications of selected regulatory proteins. Here, we investigated the anti-cancerous effects of nitro-oleic acid (NO2OA) and its combination with gamma irradiation on different cancer cell lines. The effects of NO2OA on cell death, cell cycle distribution, or expression of p21 and cyclin D1 proteins were analyzed in cancer (A-549, HT-29 and FaDu) or normal cell lines (HGF, HFF-1). Dose enhancement ratio at 50 % survival fraction (DERIC50) was calculated for samples pre-treated with NO2OA followed by gamma irradiation. NO2OA suppressed viability and induced apoptotic cell death. These effects were cell line specific but not in general selective for cancer cells. HT-29 cell line exerted higher sensitivity toward NO2OA treatment among cancer cell lines tested: induction of cell cycle arrest in the G2/M phase was associated with an increase in p21 and a decrease in cyclin D1 expression. Pre-treatment of HT-29 cells with NO2OA prior irradiation showed a significantly increased DERIC50, demonstrating radiosensitizing effects. In conclusion, NO2OA exhibited potential for combined chemoradiotherapy. Our results encourage the development of new NFAs with improved features for cancer chemoradiation.

• Klíčová slova
• apoptosis, cancer, cell cycle, gamma irradiation, nitro-fatty acids, radiosensitization,
• MeSH
• buňky HT-29 MeSH
• cyklin D1 * MeSH
• dusíkaté sloučeniny * MeSH
• kyseliny olejové MeSH
• lidé MeSH
• mastné kyseliny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CXA-10 MeSH Prohlížeč
• cyklin D1 * MeSH
• dusíkaté sloučeniny * MeSH
• kyseliny olejové MeSH
• mastné kyseliny MeSH

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed 'bottom-up' forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the 'ground truth' for the development of new tools for tackling the inverse problem-estimation of neuronal activity from multimodal non-invasive imaging data.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.

• Klíčová slova
• BOLD fMRI, CMRO2, cerebral blood flow, magnetoencephalography, neurometabolic, neurovascular,
• MeSH
• krysa rodu Rattus MeSH
• kyslík metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční tomografie přístrojové vybavení   metody   MeSH
• mapování mozku přístrojové vybavení   metody   MeSH
• modely neurologické MeSH
• mozkový krevní oběh MeSH
• myši MeSH
• neurony fyziologie   MeSH
• somatosenzorické korové centrum fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kyslík MeSH

• MeSH
• cytotoxické testy imunologické MeSH
• experimentální nádory imunologie   MeSH
• histokompatibilní antigeny MeSH
• kultivované buňky MeSH
• makrofágy imunologie   MeSH
• myši MeSH
• T-lymfocyty imunologie   MeSH
• viry imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• histokompatibilní antigeny MeSH

Mutual interactions between adipocytes and immune cells in white adipose tissue (WAT) are involved in modulation of lipid metabolism in the tissue and also in response to omega-3 polyunsaturated fatty acids (PUFA), which counteract adverse effects of obesity. This complex interplay depends in part on in situ formed anti- as well as pro-inflammatory lipid mediators, but cell types engaged in the synthesis of the specific mediators need to be better characterized. We used tissue fractionation and metabolipidomic analysis to identify cells producing lipid mediators in epididymal WAT of mice fed for 5 weeks obesogenic high-fat diet (lipid content 35% wt/wt), which was supplemented or not by omega-3 PUFA (4.3 mg eicosapentaenoic acid and 14.7 mg docosahexaenoic acid per g of diet). Our results demonstrate selective increase in levels of anti-inflammatory lipid mediators in WAT in response to omega-3, reflecting either their association with adipocytes (endocannabinoid-related N-docosahexaenoylethanolamine) or with stromal vascular cells (pro-resolving lipid mediator protectin D1). In parallel, tissue levels of obesity-associated pro-inflammatory endocannabinoids were suppressed. Moreover, we show that adipose tissue macrophages (ATMs), which could be isolated using magnetic force from the stromal vascular fraction, are not the major producers of protectin D1 and that omega-3 PUFA lowered lipid load in ATMs while promoting their less-inflammatory phenotype. Taken together, these results further document specific roles of various cell types in WAT in control of WAT inflammation and metabolism and they suggest that also other cells but ATMs are engaged in production of pro-resolving lipid mediators in response to omega-3 PUFA.

• Klíčová slova
• Adipose tissue macrophages, Lipid mediators, Lipidomics, Omega-3 PUFA, Protectin D1,
• MeSH
• antiflogistika aplikace a dávkování   MeSH
• aplikace orální MeSH
• bílá tuková tkáň cytologie   účinky léků   imunologie   MeSH
• buňky stromatu účinky léků   imunologie   MeSH
• imunologické faktory imunologie   MeSH
• kultivované buňky MeSH
• makrofágy cytologie   účinky léků   imunologie   MeSH
• metabolismus lipidů účinky léků   imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• omega-3 mastné kyseliny aplikace a dávkování   MeSH
• potravní doplňky MeSH
• tukové buňky účinky léků   enzymologie   imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiflogistika MeSH
• imunologické faktory MeSH
• omega-3 mastné kyseliny MeSH