Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.

• MeSH
• antigeny povrchové chemie   metabolismus   MeSH
• buňky PC-3 MeSH
• endocytóza MeSH
• fluorescenční spektrometrie metody   MeSH
• glutamátkarboxypeptidasa II chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sondy chemie   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty diagnóza   metabolismus   MeSH
• optické zobrazování metody   MeSH
• proteinové domény MeSH
• transplantace heterologní MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

• MeSH
• biosenzitivní techniky metody   MeSH
• hipokampální oblast CA3 cytologie   MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• kyselina glutamová analýza   chemie   metabolismus   MeSH
• lidé MeSH
• molekulární sondy analýza   chemie   genetika   metabolismus   MeSH
• nervový přenos genetika   fyziologie   MeSH
• optické zobrazování MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the last few years, magnetically labeled cells have been intensively explored, and non-invasive cell tracking and magnetic manipulation methods have been tested in preclinical studies focused on cell transplantation. For clinical applications, it is desirable to know the intracellular pathway of nanoparticles, which can predict their biocompatibility with cells and the long-term imaging properties of labeled cells. Here, we quantified labeling efficiency, localization, and fluorescence properties of Rhodamine derivatized superparamagnetic maghemite nanoparticles (SAMN-R) in mesenchymal stromal cells (MSC). We investigated the stability of SAMN-R in the intracellular space during a long culture (20 days). Analyses were based on advanced confocal microscopy accompanied by atomic absorption spectroscopy (AAS) and magnetic resonance imaging. SAMN-R displayed excellent cellular uptake (24 h of labeling), and no toxicity of SAMN-R labeling was found. 83% of SAMN-R nanoparticles were localized in lysosomes, only 4.8% were found in mitochondria, and no particles were localized in the nucleus. On the basis of the MSC fluorescence measurement every 6 days, we also quantified the continual decrease of SAMN-R fluorescence in the average single MSC during 18 days. An additional set of analyses showed that the intracellular SAMN-R signal decrease was minimally caused by fluorophore degradation or nanoparticles extraction from the cells, main reason is a cell division. The fluorescence of SAMN-R nanoparticles within the cells was detectable minimally for 20 days. These observations indicate that SAMN-R nanoparticles have a potential for application in transplantation medicine.

• MeSH
• dextrany metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• intracelulární prostor metabolismus   MeSH
• lidé MeSH
• magnetické nanočástice chemie   MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• molekulární sondy chemie   metabolismus   MeSH
• molekulární zobrazování metody   MeSH
• rhodaminy chemie   MeSH
• tuková tkáň cytologie   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Insulin, insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively), and their receptors (IR and IGF-1R) are the key elements of a complex hormonal system that is essential for the development and functioning of humans. The C and D domains of IGFs (absent in insulin) likely play important roles in the differential binding of IGF-1 and -2 to IGF-1R and to the isoforms of IR (IR-A and IR-B) and specific activation of these receptors. Here, we attempted to probe the impact of IGF-1 and IGF-2 D domains (DI and DII, respectively) and the IGF-2 C domain (CII) on the receptor specificity of these hormones. For this, we made two types of insulin hybrid analogues: (i) with the C-terminus of the insulin A chain extended by the amino acids from the DI and DII domains and (ii) with the C-terminus of the insulin B chain extended by some amino acids derived from the CII domain. The receptor binding affinities of these analogues and their receptor autophosphorylation potentials were characterized. Our results indicate that the DI domain has a more negative impact than the DII domain does on binding to IR, and that the DI domain Pro-Leu-Lys residues are important factors for a different IR-A versus IR-B binding affinity of IGF-1. We also showed that the additions of amino acids that partially "mimic" the CII domain, to the C-terminus of the insulin B chain, change the binding and autophosphorylation specificity of insulin in favor of the "metabolic" IR-B isoform. This opens new venues for rational enhancement of insulin IR-B specificity by modifications beyond the C-terminus of its B chain.

• MeSH
• embryo savčí cytologie   metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fosforylace MeSH
• hypoglykemika metabolismus   MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• insulinu podobný růstový faktor II metabolismus   MeSH
• inzulin metabolismus   MeSH
• konformace proteinů MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty cytologie   metabolismus   MeSH
• molekulární sondy metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• receptor IGF typ 1 metabolismus   MeSH
• receptor inzulinu metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug-metabolizing enzymes andmany other genes. Radiolabeled ³²P-DNA-probes had been used in studies of PXR-DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.

• MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• molekulární sondy genetika   metabolismus   MeSH
• responzivní elementy * MeSH
• retardační test metody   MeSH
• steroidní receptory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle control. Although not fully understood at present, the functions of the individual members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, responses to genotoxic stress, regulation of cellular differentiation, and senescence. In addition, the seven currently known CKIs qualify as either established or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, we report the isolation and characterization of a panel of 10 mouse monoclonal antibodies (MAbs) that specifically recognize p21WAF1/CIP1 (p21) or the individual members of the INK4 family of CKIs: p15INK4b (p15), p16INK4a (p16), p18INK4c (p18), or p19INK4d (p19). These antibodies are proving to be invaluable molecular probes for analyses of protein abundance, subcellular localization, interacting cellular proteins, and ultimately the function(s) of these cell cycle regulators. Epitopes targeted by the antibodies were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. Individual MAbs of our series recognize distinct pools of the respective CKIs, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Together, these antibodies represent useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivation of these (candidate) tumor suppressors in human malignancies.

• MeSH
• cyklin-dependentní kinasy analýza   antagonisté a inhibitory   metabolismus   MeSH
• cykliny * imunologie   MeSH
• inhibitor p21 cyklin-dependentní kinasy MeSH
• inhibitory enzymů * imunologie   MeSH
• lidé MeSH
• molekulární sondy biosyntéza   chemie   metabolismus   MeSH
• monoklonální protilátky biosyntéza   genetika   metabolismus   MeSH
• multigenová rodina imunologie   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádory prsu enzymologie   chemie   MeSH
• nádory tračníku enzymologie   chemie   MeSH
• orgánová specificita imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH