Arthur A. Nery

DNA and RNA Aptamers: From Tools for Basic Research Towards Therapeutic Applications

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.

Human mesenchymal stem cells: From immunophenotyping by flow cytometry to clinical applications

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 patients 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.

Neural differentiation of rat aorta pericyte cells

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.

Recognition of biomarkers and cell‐specific molecular signatures: Aptamers as capture agents

RNA and DNA aptamers developed by systematic evolution of ligands by exponential enrichment (SELEX) have turned into important tools in diagnostics, research, and therapeutics. Unlike antibodies, high-affinity and specific aptamers identified through an in vitro selection process can be chemically modified to gain nuclease resistances in biological fluids and to extend their bioavailability in animals. Aptamers can be raised against virtually any target including those which are toxic or do not elicit any immune response in animals. They can be developed in automated processes against various protein targets and then easily modified by attaching fluorescence reporters, nanoparticles or biotin moieties, rival antibodies in high-throughput proteomics and cell separations. In this review, we will discuss the high competence of aptamers in recognizing biomarkers and molecular signatures of cell surfaces, and how these unique features can be exploited for the identification and isolation of cancer, stem cells and even detection of parasite-infected cells.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO‐1 allowing precise discrimination between mono‐ and multinucleated forms of P. falciparum‐infected red blood cell. The use of YOYO‐1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont‐stage parasites were about 10‐fold greater than those of ring‐trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N‐acetyl‐serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non‐treated control cultures. YOYO‐1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds.

The monoterpene (–)‐carvone: A novel agonist of TRPV1 channels

(–)‐Carvone is an antinociceptive monoterpene found as the main active constituent of essential oils obtained from plants of the genus Mentha. Here, we have investigated the pharmacology of this monoterpene in dorsal root ganglia (DRG) neurons and TRPV1‐expressing HEK293 cells. (–)‐carvone at pharmacological active concentrations did not reveal significant cytotoxicity to the cells used in this study, as investigated by neutral red and propidium iodide flow cytometry assays. In calcium imaging experiments 1 mM (–)‐carvone increased the cytosolic calcium levels in DRG neurons from 120.6 ± 5.0 nM (basal) to 310.7 ± 23.1 nM (P < 0.05). These effects were completely abolished when neurons were preincubated with calcium‐free bath solution or ruthenium‐red (5 µM) and capsazepine (10 µM), suggesting the possibility of TRPV1 channel‐activation by (–)‐carvone. Activity of (–)‐carvone on TRPV1 channels was further investigated in HEK293 cells expressing recombinant human TRPV1 channels revealing dose‐dependent calcium transients with an EC50 of 1.3 ± 0.2 mM (Hill coefficient = 2.5). In conclusion, we show for the first time the ability of (–)‐carvone to induce increases in cytosolic calcium concentration through TRPV1 activation.

Neuronal differentiation involves a shift from glucose oxidation to fermentation

Energy metabolism in the adult brain consumes large quantities of glucose, but little is known to date regarding how glucose metabolism changes during neuronal differentiation, a process that is highly demanding energetically. We studied changes in glucose metabolism during neuronal differentiation of P19 mouse embryonal carcinoma cells, E14Tg2A embryonic stem cells as well as during brain development of BLC57 mice. In all these models, we find that neurogenesis is accompanied by a shift from oxidative to fermentative glucose metabolism. This shift is accompanied by both a decrease in mitochondrial enzymatic activities and mitochondrial uncoupling. In keeping with this finding, we also observe that differentiation does not require oxidative metabolism, as indicated by experiments demonstrating that the process is preserved in cells treated with the ATP synthase inhibitor oligomycin. Overall, we provide evidence that neuronal differentiation involves a shift from oxidative to fermentative metabolism, and that oxidative phosphorylation is not essential for this process.

Mode of Cembranoid Action on Embryonic Muscle Acetylcholine Receptor

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H‐1 cells was studied by using whole‐cell and single‐channel patch‐clamp current measurements. With whole‐cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 μM EUAC with 50 μM, 100 μM, or 500 μM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 μM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01–40 μM) obtained with 100 μM and 500 μM CCh displayed slope coefficients, nH, significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single‐channel studies was that EUAC did not affect single‐channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open‐channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed‐channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.

A novel physiological property of snake bradykinin-potentiating peptides-reversion of MK-801 inhibition of nicotinic acetylcholine receptors.

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [<ENWPHPQIPP] is independent of somatic ACE inhibition. On the other hand, nicotinic acetylcholine receptors expressed in blood vessels have been related to blood pressure regulation. Therefore, we have studied the effects of BPP-10c on acetylcholine receptor function in the PC12 pheochromocytoma cell line, which following induction to neuronal differentiation expresses most of the nicotinic receptor subtypes. BPP-10c did not induce receptor-mediated ion flux, nor potentiated carbamoylcholine-provoked receptor activity as determined by whole-cell recording. This peptide, however, alleviated MK-801-induced inhibition of nicotinic acetylcholine receptor activity. Although more data are needed for understanding the mechanism of the BPP-10c effect on nicotinic receptor activity and its relationship with the anti-hypertensive activity, this work reveals possible therapeutic applications for BPP-10c in establishing normal acetylcholine receptor activity.

Kinin-B1 and B2 receptor activity in proliferation and neural phenotype determination of mouse embryonic stem cells

The kinins bradykinin and des‐arg9‐bradykinin cleaved from kininogen precursors by kallikreins exert their biological actions by stimulating kinin‐B2 and B1 receptors, respectively. In vitro models of neural differentiation such as P19 embryonal carcinoma cells and neural progenitor cells have suggested the involvement of B2 receptors in neural differentiation and phenotype determination; however, the involvement of B1 receptors in these processes has not been established. Here, we show that B1 and B2 receptors are differentially expressed in mouse embryonic E14Tg2A stem cells undergoing neural differentiation. Proliferation and differentiation assays, performed in the presence of receptor subtype‐selective agonists and antagonists, revealed that B1 receptor activity is required for the proliferation of embryonic and differentiating cells as well as for neuronal maturation at later stages of differentiation, while the B2 receptor acts on neural phenotype choice, promoting neurogenesis over gliogenesis. Besides the elucidation of bradykinin functions in an in vitro model reflecting early embryogenesis and neurogenesis, this study contributes to the understanding of B1 receptor functions in this process.