Raúl Vinet

Analysis of aberrant methylation on promoter sequences of tumor suppressor genes and total DNA in sputum samples: a promising tool for early detection of COPD and lung cancer in smokers

BackgroundChronic obstructive pulmonary disease (COPD) is a disorder associated to cigarette smoke and lung cancer (LC). Since epigenetic changes in oncogenes and tumor suppressor genes (TSGs) are clearly important in the development of LC. In this study, we hypothesize that tobacco smokers are susceptible for methylation in the promoter region of TSGs in airway epithelial cells when compared with non-smoker subjects. The purpose of this study was to investigate the usefulness of detection of genes promoter methylation in sputum specimens, as a complementary tool to identify LC biomarkers among smokers with early COPD.MethodsWe determined the amount of DNA in induced sputum from patients with COPD (n = 23), LC (n = 26), as well as in healthy subjects (CTR) (n = 33), using a commercial kit for DNA purification, followed by absorbance measurement at 260 nm. The frequency of CDKN2A, CDH1 and MGMT promoter methylation in the same groups was determined by methylation-specific polymerase chain reaction (MSP). The Fisher’s exact test was employed to compare frequency of results between different groups.ResultsDNA concentration was 7.4 and 5.8 times higher in LC and COPD compared to the (CTR) (p < 0.0001), respectively. Methylation status of CDKN2A and MGMT was significantly higher in COPD and LC patients compared with CTR group (p < 0.0001). Frequency of CDH1 methylation only showed a statistically significant difference between LC patients and CTR group (p < 0.05).ConclusionsWe provide evidence that aberrant methylation of TSGs in samples of induced sputum is a useful tool for early diagnostic of lung diseases (LC and COPD) in smoker subjects.Virtual slidesThe abstract MUST finish with the following text: Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1127865005664160

Catecholamines-evoked cytosolic ca2+rise in endothelial cells from bovine adrenal medulla

The effects of catecholamines on intracellular Ca2+concentrations ([Ca2+]i) in single acutely dissociated bovine adrenal medulla endothelial cells (BAMECs) were measured using the intracellular fluorescent probe Fluo-3 AM. 100 μm epinephrine or norepinephrine induced a biphasic [Ca2+]i rise with an initial peak followed by a delayed phase. 10 μm phenylephrine (α1-adrenergic agonist) caused a [Ca2+]i rise similar to that evoked by catecholamines. The increase in [Ca2+]i induced by 10 μm phenylephrine was reverted by 10 μm phenoxybenzamine (α-adrenergic antagonist). Neither isoproterenol (β-adrenergic agonist) nor clonidine (α2-adrenergic agonist) induced [Ca2+]i rise. The initial peak was insensitive to zero external Ca2+ and it was abolished after Ca2+ internal storages were emptied by 10 mM caffeine. The delayed phase was reduced to near zero by external Ca2+ removal. These results indicate that BAMECs possess α1-adrenergic receptors associated to both the release of caffeine-sensitive intracellular Ca2+ stores and the entry of extracellular Ca2+ We suggest that chromaffin cell secretion may activate BAMECs in vivo through an increase in [Ca2+]i which could induce the secretion of vasoactive factors allowing a rapid entry of hormones into the circulation. (Mol Cell Biochem 000: 000-000,1999)

Characterization of fruit development and potential health benefits of arrayan (Luma apiculata), a native berry of South America

The arrayan berry (Luma apiculata) is a native fruit from South America that belongs to the Myrtaceae family. To elucidate and characterize the developmental process and the potential health benefits of this edible fruit, quality and physiological parameters, along with antioxidant capacity, were evaluated during four clearly defined developmental stages of the fruit in two seasons. Fruit firmness slowly decreases during fruit development, whereas the solid soluble content/titratable acidity ratio (SSC/TA) increases significantly in the final stages of development. The measurement of low respiration rates and low ethylene production during growth and ripening suggested that the arrayan berry should be classified as a non-climacteric fruit. Arrayan berries show a significant increase in their antioxidant capacity from small green to black ripe fruit. FRAP and TEAC assays showed high correlations with total polyphenolic content (TPC) during ripening and high antioxidant capacity at all fruit stages, showing greater values in ripe fruit (FRAP: 24 ± 2 and 28 ± 3 μM FeSO4/gFW; TEAC: 18 ± 2 and 20 ± 1 Eq. Trolox/gFW for each season, respectively) than those observed in the blueberry (FRAP: 10 ± 2 and 19 ± 3 μM FeSO4/gFW; TEAC: 10 ± 2 and 17 ± 3). In addition, bioactive assays using ripe fruit extracts show presence of flavonol and anthocyanins, a high ORAC value (62,500 ± 7000 μmol/gDW) and a concentration-dependent vascular protection under high glucose conditions. The results obtained show that these endemic berry fruits have a promising potential as functional food.

Improving Amphetamine Therapeutic Selectivity: N,N‐dimethyl‐MTA has Dopaminergic Effects and does not Produce Aortic Contraction

Amphetamine derivatives have therapeutic potential in diseases such as attention deficit hyperactivity disorder, narcolepsy and obesity. However, their prolonged use has been associated with cardiovascular toxicity and addiction. In recent years, we have studied the pharmacological effects of amphetamine derivatives such as methylthioamphetamine (MTA) and N,N‐dimethyl‐thioamphetamine, with the aim of improving their therapeutic selectivity. In this work, we show that similarly to MTA, N,N‐dimethyl‐thioamphetamine has effects on the dopamine system, producing a significant increase in extracellular levels of dopamine (as measured by in vivo brain microdialysis) and locomotor activity, which is a behavioural measure of dopaminergic activation. However, unlike MTA, N,N‐dimethyl‐ thioamphetamine does not produce aortic contraction in vitro. Our results show that N,N‐dimethyl‐thioamphetamine is a drug that retains the dopaminergic effects of amphetamine derivatives but exhibits a lower potential for producing cardiovascular side effects.

Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca(2+) dependent exocitosis of vasoactive compounds. Using the Ca(2+) indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca(2+) increase and depolarization of the membrane potential (20.3+/-0.9 mV; n=23). The increase in cytosolic Ca(2+) induced by 10microM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 microM of hexamethonium. On the other hand, the increase in cytosolic Ca(2+) could be depressed by nifedipine (0.01 -100 microM) or withdrawal of extracellular Ca(2+). Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal glands microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca(2+) channels, influx of external Ca(2+) and liberation of vasoactive compounds.

New 1H-Benzo[f]indazole-4,9-diones Conjugated with C-Protected Amino Acids and Other Derivatives: Synthesis and in Vitro Antiproliferative Evaluation.

1H-Benzo[f]indazole-4,9-dione derivatives conjugated with C-protected amino acids (glycine, l-alanine, l-phenylalanine and l-glutamic acid) 6a–l were prepared by chemically modifying the prenyl substituent of 3-methyl-7-(4-methylpent-3-enyl)-1H-benzo[f]indazole-4,9-dione 2 through epoxidation, degradative oxidation, oxidation and N-acyl condensation reactions. The chemical structures of the synthesized compounds were elucidated by analyzing their IR, 1H-NMR and 13C-NMR spectral data together with elemental analysis for carbon, hydrogen and nitrogen. The preliminary in vitro antiproliferative activity of the synthesized derivatives was evaluated on KATO-III and MCF-7 cell lines using a cell proliferation assay. The majority of the derivatives exhibited significant antiproliferative activity with IC50 values ranging from 25.5 to 432.5 μM. These results suggest that 1H-benzo[f]indazole-4,9-dione derivatives are promising molecules to be researched for developing new anticancer agents.

Bradykinin and histamine-induced cytosolic calcium increase in capillary endothelial cells of bovine adrenal medulla

We have assessed the effect of bradykinin and histamine on the cytosolic free calcium concentration ([Ca2+]i) of bovine adrenal medulla capillary endothelial cells (BAMCECs). To measure [Ca2+]i changes in BAMCECs the intracellular fluorescent probe, fluo‐3 AM, was used. Bradykinin (3 µM) produced a transient monophasic increase in [Ca2+]i, which was depressed by B1650 (0.1 µM), a B2‐bradykinin receptor antagonist (D‐Arg‐[Hyp3, Thi5,8, D‐Phe7]‐Bradykinin). Similarly, increase in [Ca2+]i induced by histamine was also depressed by tripolidine (0.1 µM), an H1‐histamine receptor antagonist. [Ca2+]i increase induced by both agonists was unaffected in the absence of extracellular Ca2+ or presence of antagonists of voltage operated Ca2+ channels (VOCCs). Thapsigargin (1 µM) did not abolish the increase of [Ca2+]i produced by bradykinin, but abolished that of histamine. In contrast, caffeine (100 µM), abolished the [Ca2+]i response induced by bradykinin (3 µM), but did not affect the [Ca2+]i increase induced by histamine (100 µM). The results indicate the presence of B2 bradykinin‐ and H1 histamine‐receptors in BAMCECs. Liberation of Ca2+ induced by both agonists occurs through 2 different intracellular mechanisms. While bradykinin activates a sarco(endo) plasmic reticulum (SER) containing a SER Ca2+‐ATPase (SERCA) thapsigargin‐insensitive, histamine activates a SER containing a SERCA thapsigargin‐sensitive. We suggest that the increase in [Ca2+]i induced by bradykinin and histamine could be of physiological relevance, modulating adrenal gland microcirculation.

Monoclonal Gammopathy of Undetermined Significance

Monoclonal gammopathies are a heterogeneous group of disorders characterized by the stable or progressive proliferation of an abnormal clone of plasma cells that continue pro‐ ducing antibodies [4]. But because these immunoglobulin proteins are abnormal and mon‐ oclonal (identical copies of each other), these offer no protection against infections and can damage the kidney. This monoclonal immunoglobulin is called M-protein. Each basic unit is a monomeric immunoglobulin consisting of two heavy chains of the same class and subclass and two light chains of the same type. The heavy chain classes are G, A, M, D, E (gamma, alpha, mu, delta, epsilon), while the light chain types are kappa (κ) and lambda (λ).

Effects of two bisbenzylisoquinoline alkaloids, Antioquine and Tetrandrine, compared to Verapamil in Rat Thoracic Aorta

The objective of this study was to compare two alkaloids (antioquine and tetrandrine) with verapamil; knowing that the smooth muscle respond to KCl and relationships with calcium. The effects of antioquine and tetrandrine, was studied in adults Wistar rat with modified methods used in the determination of aorta contractility and compared with verapamil effect in the same assays. The analysis of the effect of a drug or extract on aortic reactivity included maximal relaxation or maximal contraction (Cmax) (Phase 1). In our results, verapamil induced a blockade of 98.7 ± 0.7% (n = 6) in presence of endothelium and 97.9 ± 4.3% in ausence of endothelium, both in phase 1 and in phase 2 of 47.4 ± 4.1% (n = 6) in aortas in the presence of endothelium and 61.8 ± 1.1% in ausence of endothelium; Tetrandrine assays showed a phase 1 blocking effect of 63.4 ± 5.5 and 47.7 ± 2.9% (with and without endothelium, respectively) and phase 2 of 43.5 ± 6.2 and 28.5 ± 5.7%, (with and without endothelium, respectively). Antioquine presents in phase 1 and phase 2, a blockade that is not significant from the point of view of calcium antagonism. We can conclude that tetrandrine block the movement of calcium from both intracellular and extracellular deposits, with the greatest effect when aortas are in the presence of endothelium.

Bases fisiológicas para una interacción entre las células cromafines y las endoteliales de la glándula adrenal

En este trabajo tratamos de investigar las posibles interacciones entre las celulas endoteliales de la glandula adrenal y aquellas sustancias relacionadas con la secrecion de las celulas cromafines. Para lo anterior, estudiamos el efecto de acetilcolina (ACh), o de catecolaminas (CA) tanto en el nivel de Ca2+ citoplasmatico ([Ca2+]i), como en el potencial de membrana de las celulas endoteliales. Nuestros resultados muestran que tanto la ACh como la nicotina, pero no la muscarina, son capaces de inducir un aumento del [Ca2+]i y una despolarizacion de la membrana plasmatica de las celulas endoteliales. El antagonista nicotinico, hexametonium, bloquea tanto el efecto de la ACh como de la nicotina lo que sugiere la presencia de receptores nicotinicos. Por otra parte, las CA (tanto adrenalina como noradrenalina o agonistas a1-adrenergicos) tambien producen un aumento del [Ca2+]i en las celulas endoteliales aunque no despolarizacion evidente. En este caso, el aumento es bifasico siendo la primera fase de un pico rapido e independiente del Ca2+ extracelular en tanto que la segunda se presenta con oscilaciones y depende tanto de que los canales de Ca2+ no esten bloqueados como de la presencia de ese ion en el medio externo. Dado que se ha demostrado que el aumento del [Ca2+]i en las celulas endoteliales desencadena la secrecion de sustancias vasodilatadoras (prostaciclina y oxido nitrico), proponemos que este seria un mecanismo compensatorio del sistema para contrarestar el enorme efecto vasoconstrictor de las CA secretadas por las celulas cromafines