Search

Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells.

The mechanism of inhibition of a recombinant rat P2X(sub 2) receptor is examined by suramin and TNP-ATP to elucidate the inhibition mechanism of suramin and 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and the pharmacological analysis of P2X receptors. The results show that suramin behaves as a noncompetitive antagonist, binding to a different site on the receptor compared to ATP and confirm the competitive inhibition by TNP-ATP.

Background: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells. Materials and methods: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection. Results: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences. Conclusions: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells. [ABSTRACT FROM AUTHOR]

Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus may have 50 to 200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. The novel 2,701 Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. The activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably α3β4 and/or α3β4α5 subtype. According to the nomenclature, the new peptide was designated as α-RgIB. [ABSTRACT FROM AUTHOR]

Modern medicine will unequivocally include regenerative medicine as a major breakthrough in the re-establishment of damaged or lost tissues due to degenerative diseases or injury. In this scenario, millions of patients worldwide can have their quality of life improved by stem cell implantation coupled with endogenous secretion or administration of survival and differentiation promoting factors. Large efforts, relying mostly on flow cytometry and imaging techniques, have been put into cell isolation, immunophenotyping, and studies of differentiation properties of stem cells of diverse origins. Mesenchymal stem cells (MSCs) are particularly relevant for therapy due to their simplicity of isolation. A minimal phenotypic pattern for the identification of MSCs cells requires them to be immunopositive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, and HLA-DR and other surface markers. MSCs identified by their cell surface marker expression pattern can be readily purified from patient's bone marrow and adipose tissues. Following expansion and/or predifferentiation into a desired tissue type, stem cells can be reimplanted for tissue repair in the same patient, virtually eliminating rejection problems. Transplantation of MSCs is subject of almost 200 clinical trials to cure and treat a very broad range of conditions, including bone, heart, and neurodegenerative diseases. Immediate or medium term improvements of clinical symptoms have been reported as results of many clinical studies. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl- D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. © 2011 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X2 receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [α-32P]ATP, whereas suramin did not interfere with [α-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X2 receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X2 receptor inhibition. [ABSTRACT FROM AUTHOR]

Aptamers compete with antibodies in many applications, in which high-affinity and specificity ligands are needed. In this regard, fluorescence-tagged aptamers have gained applications in flow and imaging cytometry for detecting cells expressing distinct antigens. Here we present prospective methods, as a starting point, for using these high-affinity ligands for cytometry applications.

P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.