Analysis of polymorphisms in codons 11, 72 and 248 of TP53 in Brazilian women with breast cancer.

The association between TP53 gene polymorphisms and breast cancer (BC) in Brazilian women is a controversial topic. In this cross-sectional study, we evaluated the association between clinical pathological variables and three polymorphisms (TP53*11, TP53*72, and TP53*248) in BC patients and controls. Genomic DNA was extracted from the blood cells of 393 participants; the cancer-free control subjects were 26-72 years old (41 ± 11.03) and the BC patients were 28-80 years old (51 ± 10.70). We used standard polymerase chain reaction-restriction fragment length polymorphism and confirmed the results by genetic sequencing. In TP53*11, there was 100% homozygous Glu distribution in both groups. TP53*72 showed genotypic distribution: in the control group, there was 16.10% homozygous Pro, and 42.44% heterozygous and 41.46% homozygous Arg; in the BC group, there was 15.43% homozygous Pro, and 42.55% heterozygous and 42.02% homozygous Arg. The relative frequency of each allele was 0.37% for Pro and 0.63% for Arg in the control group, and 0.37% for Pro and 0.63% for Arg in the BC group. The nuclear grade (P = 0.0084) and adapted histological grade (P = 0.0265) were associated with TP53*72. The distribution of the codon 72 genotypes did not deviate from Hardy-Weinberg equilibrium in either group. In TP53*248, there was 100% homozygous Arg distribution in both groups. In codon 72, the Arg allele is the most prevalent in Brazilian women. TP53*72 may be associated with susceptibility to BC, although more studies are required to evaluate the profile of Brazilian women with BC.

The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients.

BACKGROUND AND PURPOSE: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis. EXPERIMENTAL APPROACH: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca(2+) transients by fluorescence and Ca(2+) fluxes by electrophysiological techniques. KEY RESULTS: Canrenone (300-600 micromol L(-1)) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 micromol L(-1)) reduced cell shortening and L-type Ca(2+) channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca(2+) content of the sarcoplasmic reticulum, intracellular Ca(2+) transient amplitude and intracellular diastolic Ca(2+) concentration. However, the time course of [Ca(2+)](i) decline during transients evoked by caffeine was not affected by canrenone. CONCLUSION AND IMPLICATIONS: Canrenone reduced L-type Ca(2+) channel current, amplitude of intracellular Ca(2+) transients and Ca(2+) content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone.

Problems caused by high concentration of ATP on activation of the P2X7 receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2.

Fura-2 is one of the most used fluorophore for measuring intracellular calcium concentration ([Ca2+]i). In mouse bone marrow cell suspensions ATP produces a biphasic effect: till 1 mM, ATP produces increases in [Ca2+]i; from 1 mM on an increase is observed, that is followed by the decrease in the 340/380 nm ratio (R340/380). At high ATP (4 mM) concentration fura-2 leaked from loaded bone marrow cell suspensions. We observed that ATP decreases fluorescence in the absorption and excitation spectra of fura-2, consequently the emitted one is decreased including the isobestic point (360 nm). ATP analogs: BzATP, ATPyS and UTP, but not alphabetaATP, ADP or AMP, promote decrease of fluorescence in the isobestic point of fura-2. The physical/chemical process that reduces the absorption and excitation of fura-2 by ATP is unknown. The P2X7 inhibitors, Mg2+ (5 mM), OxATP (300 microM) and Brilliant Blue (100 nM), blocked the efflux of fura-2 and ATP-induced R340/380 decrease. The J774 cell line and mononuclear cells with a higher expression of P2X7 receptors show the same decrease in R340/380 as that induced by ATP. In the HL-60 cell line, myeloid cells and erythroblasts extracted from bone marrow, such effect does not occur. It is concluded that the use of the fluorescent Ca2+ indicator fura-2 does not allow the correct measurement of [Ca2+]i in these cells in the presence of a higher concentration of ATP which activated the P2X7 receptor.