Verónica Morales-Tlalpan

Screening of antiproliferative effect of aqueous extracts of plant foods consumed in México on the breast cancer cell line MCF-7

We evaluated the antiproliferative effect of aqueous extracts of 14 plant foods consumed in Mexico on the breast cancer cell line MCF-7. The plant foods used were avocado, black sapote, guava, mango, prickly pear cactus stems (called nopal in Mexico, cooked and raw), papaya, pineapple, four different cultivars of prickly pear fruit, grapes and tomato. β-Carotene, total phenolics and gallic acid contents and the antioxidant capacity, measured by the ferric reducing/antioxidant power and the 2,2-diphenyl-1,1-picrylhydrazyl radical scavenging assays, were analyzed in each aqueous extract. Only the papaya extract had a significant antiproliferative effect measured with the methylthiazolydiphenyl-tetrazolium bromide assay. We did not notice a relationship between the total phenolic content and the antioxidant capacity with antiproliferative effect. It is suggested that each extract of plant food has a unique combination of the quantity and quality of phytochemicals that could determine its biological activity. Besides, papaya represents a very interesting fruit to explore its antineoplastic activities.

Modulation of the maitotoxin response by intracellular and extracellular cations.

The aim of the present study was to characterize the role played by intracellular and extracellular calcium and sodium on the maitotoxin (MTX) response in Chinese hamster ovary (CHO) cells. The results presented here indicated that: (1) MTX activates calcium and sodium influx in a concentration-dependent manner; (2) extracellular calcium is required for the sodium influx; (3) removal of the extracellular sodium did not prevent the MTX-induced calcium influx; (4) elevation in the intracellular calcium concentration potentiates the MTX response; and (5) MTX, at the concentrations tested, did not compromise cell viability.

Characterization of the maitotoxin-activated cationic current from human skin fibroblasts

The maitotoxin (MTX)‐induced cationic current (Imtx) from human skin fibroblasts was characterized using the patch‐clamp technique in whole‐cell configuration. Under resting conditions (absence of MTX), the main current observed is produced by an outwardly rectifying K+ channel which is inhibited by 1 mm TEA. The current reversal potential was −86 mV (n= 12). MTX (500 pm) activated a current with a linear current–voltage relationship and a reversal potential of −10 mV (n= 10). Replacing the extracellular Na+ and K+ with N‐methyl‐d‐glucamine (NMDG) caused a shift of the reversal potential to a value below −100 mV, indicating that Na+ and K+, but not NMDG, carry Imtx. Further ion selectivity experiments showed that Ca2+ carries Imtx also. The resulting permeability sequence obtained with the Goldman–Hodgkin–Katz equation yielded Na+ (1) ≈ K+ (1) > Ca2+ (0.87). The Imtx activation time course reflected the changes in intracellular Ca2+ and Na+ measured with the fluorescent indicators fura‐2 and SBFI, respectively, suggesting that the activation of Imtx brings about an increment in intracellular Ca2+ and Na+. Reducing the extracellular Ca2+ concentration below 1.8 mm prevented the activation of Imtx and the increment in intracellular Na+ induced by MTX. Mn2+ and Mg2+ could not replace Ca2+, but Ba2+ could replace Ca2+. MTX activation of current in 10 mm Ba2+ was approximately 50 % of that induced in the presence of 1.8 mm Ca2+. When 5 mm of the Ca2+ chelator BAPTA was included in the patch pipette, MTX either failed to activate the current or induced a small current (less than 15 % of the control), indicating that intracellular Ca2+ is also required for the activation of Imtx. Intracellular Ba2+ can replace Ca2+ as an activator of Imtx. However, in the presence of 10 mm Ba2+ the activation by MTX of the current was 50 % less than the activation with nm concentrations of free intracellular Ca2+.

Ryanodine-Sensitive Intracellular Ca2+ Channels in Rat Suprachiasmatic Nuclei Are Required for Circadian Clock Control of Behavior

Electrophysiological and calcium mobilization experiments have suggested that the intracellular calcium release channel ryanodine receptors (RyRs) are involved in the circadian rhythmicity of the suprachiasmatic nucleus (SCN). In the present report the authors provide behavioral evidence that RyRs play a specific and major role in the output of the molecular circadian clock in SCN neurons. They measured the circadian rhythm of drinking and locomotor behaviors in dim red light before, during, and after administration of an activator (ryanodine 0.1 µM) or an inhibitor (ryanodine 100 µM) of the RyRs. Drugs were delivered directly into the SCN by cannulas connected to osmotic minipumps. Control treatments included administration of artificial cerebrospinal fluid, KCl (20 mM), tetrodotoxin (1 µM), and anysomicin (5 µg/µl). Activation of RyRs induced a significant shortening of the endogenous period, whereas inhibition of these Ca2+ release channels disrupted the circadian rhythmicity. After the pharmacological treatments the period of rhythmicity returned to basal values and the phase of activity onset was predicted from a line projected from the activity onset of basal recordings. These results indicate that changes in overt rhythms induced by both doses of ryanodine did not involve an alteration in the clock mechanism. The authors conclude that circadian modulation of RyRs is a key element of the output pathway from the molecular circadian clock in SCN neurons in rats.

Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca2+ mobilization

BackgroundGranulosa cells play an important endocrine role in folliculogenesis. They mobilize Ca2+ from intracellular stores by a coordinated action between 1,4,5 inositol trisphosphate and ryanodine receptors (IP3R and RyR). The aim of this study was to explore the isoforms of IP3Rs expressed in mouse C57BL/6 NHsd granulosa cells, characterizing their intranuclear localization and the relation with other Ca2+-handling proteins.MethodsOvarian tissue and granulosa cells were analyzed by multiphotonic and confocal microscopy to determine the intracellular presence of IP3R types 1, 2 and 3, RyR, thapsigargin-sensitive Ca2+-ATPase, and endomembranes. Cellular fractionation and Western blot assays were also used to further confirm the nuclear occurrence of the three IP3R isoforms. Free nuclear and cytosolic Ca2+ concentrations were measured using Fluo-4 AM by confocal microscopy.ResultsBy using antibodies and specific fluorophores, was shown that granulosa cells endomembranes contain three isoforms of IP3R, the RyR, and the thapsigargin-sensitive Ca2+-ATPase (SERCA). Interestingly, all these proteins were also detected in the nuclear envelope and in well-defined intranuclear structures. Microsomal membranes depicted characteristic bands of the 3 types of IP3R, but also variants of lower molecular weight. Analysis of nuclear membranes and nucleoplasmic fraction confirmed the nuclear localization of the IP3R types 1, 2 and 3. We demonstrated ATP-induced Ca2+ transients in the nuclear and cytoplasmic compartments. Remarkably, the inhibitory effect on ATP-induced Ca2+ mobilization of brefeldin A was more accentuated in the cytoplasm than in the nucleus.ConclusionThese findings provide evidence that granulosa cells, including nuclei, express the Ca2+-handling proteins that allow Ca2+ mobilization. All three IP3R were also detected in ovarian slices, including the nuclei of granulosa cells, suggesting that these cells use the three IP3R in situ to achieve their physiological responses.

Recognition and activation of ryanodine receptors by purines.

Ryanodine receptor (RyR) is a tetrameric, high molecular weight protein that functions as a calcium release channel. It plays a key role in phenomena such as signal transduction, excitation-contraction and excitation-secretion coupling. Hyperthermia maligna, central core disease and myocardial infarction have been related with RyR dysfunction. RyR is present as three isoforms in vertebrates: RyR 1 mainly localized in skeletal muscle, RyR 2 in cardiac muscle, and RyR 3 in nervous system. RyR is regulated by a number of physiological and pharmacological factors. Main physiological modulators: calcium, kinases and phosphatases, redox state and energy charge. Main pharmacological regulators: caffeine, dantrolene, ruthenium red, heavy metals and ryanodine. Purines have to do with both, physiological and pharmacological regulation of the RyR activity. So far, the mechanisms of RyR activation by ATP and caffeine have been described in detail using [3H]-ryanodine binding assays and unitary channel activity recorded in planar lipid bilayers. However, some questions remain to be addressed and are at present aim of active scrutiny: How many sites for purines are present in the RyR? Is the same site recognized by nucleotides and methylxanthines? What differences exist among the interaction between RyR and purine bases, nucleosides and nucleotides? Are the phosphate groups important for the recognition of nucleotides? Is the sugar moiety important for the recognition of nucleosides? The review article will examine the most recent specialized literature about the mechanism of activation of RyR by purines with emphasis on reports with approaches of structure-function and structure-activation.