Activation of endothelial nitric oxide synthase by proanthocyanidin-rich fraction from Croton celtidifolius (Euphorbiaceae): involvement of extracellular calcium influx in rat thoracic aorta

The present study investigates the mechanisms related to the endogenous nitric oxide synthase (eNOS) activation in the relaxant effects of a proanthocyanidin-rich fraction (PRF), obtained from Croton celtidifolius Baill barks, in rat thoracic aorta rings with endothelium. In vessels pre-contracted with phenylephrine (Phe), PRF (0.1 - 100 microg/mL) induced a concentration-dependent relaxation. This effect was significantly reduced by endothelium denudation, by N(omega)-nitro-L-arginine, and by 1H[1,2,3]oxadiazolo[4,3-alpha]quinoxalin. However, the vasorelaxant effect was not altered by indomethacin, atropine, tetraethylammonium, and charybdotoxin plus apamin. In thoracic aorta rings pre-contracted with phorbol-12,13-dibuyrate, PRF also induced a concentration-dependent relaxation. The PRF-induced relaxation disappeared in the absence of extracellular calcium in the medium and decreased significantly in the presence of lanthanum. A sulfhydryl alkylating agent, N-ethylmaleimide, and a phospholipase C (PLC) blocker, neomycin, significantly decreased PRF-induced vasorelaxation. In vessels pre-contracted with Phe, the PRF-induced vasorelaxant effect was not altered by quinacrine and ONO-RS-082, genistein and thyrphostin A-23, GF109203, and pertussis toxin and cholera toxin. The results suggest that the PRF-induced vasorelaxant effect is endothelium-dependent and involves the NO/cGMP pathway. We hypothesize that the activation of eNOS is due to an increase of intracellular calcium derived from PLC activation and an N-ethylmaleimide sensitive pathway.     

Electrocatalytic activity of 4-nitrophthalonitrile-modified electrode for the l-glutathione detection

The present work describes the substantial electrocatalytic activity of (NC)2C6H3-NHOH/(NC)2C6H3-NO redox couple-modified electrode toward the low voltage detection of l-glutathione (GSH), in neutral medium, at an applied potential of 0.4V versus Ag/AgCl. After optimizing the operational conditions, the sensor provided a linear response range for GSH from 8.0 up to 83.0 micromol L(-1) with sensitivity, detection and quantification limits of 54nA L micromol(-1), 2.7 micromol L(-1) and 8.0 micromol L(-1), respectively. The proposed sensor presented higher sensitivity when compared to other modified electrodes described in the literature and showed a stable response for at least 100 successive determinations. The repeatability of the measurements with the same sensor and different sensors, evaluated in terms of relative standard deviation, were 4.1 and 5.0%, respectively, for n=10. The developed sensor was applied for GSH determination in yeast extract and the results were statistically the same with those obtained by the comparative method described in the literature at a confidence level of 95%.     

New approach reveals CD28 and IFNG gene interaction in the susceptibility to cervical cancer

Cervical cancer is a complex disease with multiple environmental and genetic determinants. In this study, we sought an association between polymorphisms in immune response genes and cervical cancer using both single-locus and multi-locus analysis approaches. A total of 14 single nucleotide polymorphisms (SNPs) distributed in CD28, CTLA4, ICOS, PDCD1, FAS, TNFA, IL6, IFNG, TGFB1 and IL10 genes were determined in patients and healthy individuals from three independent case/control sets. The first two sets comprised White individuals (one group with 82 cases and 85 controls, the other with 83 cases and 85 controls) and the third was constituted by non-white individuals (64 cases and 75 controls). The multi-locus analysis revealed higher frequencies in cancer patients of three three-genotype combinations [CD28+17(TT)/IFNG+874(AA)/TNFA-308(GG), CD28+17(TT)/IFN+847(AA)/PDCD1+7785(CT), and CD28 +17(TT)/IFNG+874(AA)/ICOS+1564(TT)] (P < 0.01, Monte Carlo simulation). We hypothesized that this two-genotype [CD28(TT) and IFNG(AA)] combination could have a major contribution to the observed association. To address this question, we analyzed the frequency of the CD28(TT), IFNG(AA) genotype combination in the three groups combined, and observed its increase in patients (P = 0.0011 by Fisher's exact test). The contribution of a third polymorphism did not reach statistical significance (P = 0.1). Further analysis suggested that gene-gene interaction between CD28 and IFNG might contribute to susceptibility to cervical cancer. Our results showed an epistatic effect between CD28 and IFNG genes in susceptibility to cervical cancer, a finding that might be relevant for a better understanding of the disease pathogenesis. In addition, the novel analytical approach herein proposed might be useful for increasing the statistical power of future genome-wide multi-locus studies.     

Iron Deficiency in Vegetarian and Omnivorous Individuals: Analysis of 1340 Individuals

The objective of this study was to evaluate the serum levels of ferritin and the prevalence of iron deficiency in vegan and omnivorous individuals by taking into account the presence of elements that cause an elevation of ferritin levels, such as increased homeostatic model assessment of insulin resistance (HOMA-IR), body mass index (BMI), and high-sensitivity C-reactive protein (hs-CRP) values. The parameters were evaluated in 1340 individuals, i.e., 422 men and 225 women who do not menstruate and 693 women who do menstruate, based on omnivorous or vegetarian eating habits. The progressive increase in BMI, HOMA-IR, and inflammation caused an elevation in ferritin concentration, regardless of the eating habits in the groups studied. In the overall sample, omnivores had a higher prevalence of obesity, higher ferritin levels, and a lower prevalence of iron deficiency (ferritin < 30 ng/mL). However, after the exclusion of individuals with inflammation (with overweight/obesity and elevated hs-CRP levels), the actual iron deficiency was assessed and was not higher among vegetarians, except in women with regular menstrual cycles. Our data show that nutritional status and inflammation levels affect ferritin levels and may interfere with the correct diagnosis of iron deficiency in both vegetarian and omnivorous individuals. Compared to vegetarians, women who do not menstruate and men had the same prevalence of iron deficiency when following an omnivorous diet.     

Functional aspects of the human salivary cystatins in the oral environment

OBJECTIVES: The aim of this study was to investigate the cysteine protease inhibitory properties of the human salivary cystatins S, SA and SN in order to identify potential in vivo target cysteine proteases which may include those involved in periodontal tissue destruction. In addition, the potential role of the salivary cystatins with respect to the tooth mineral balance and pellicle formation was also investigated.METHODS: Salivary cystatins S major, S minor, SA, SAT (a truncated form of SA) and SN were purified from human submandibular sublingual saliva. Sensitive fluorometric assays were used to test the inhibitory action of each purified form of salivary cystatin against a variety of cysteine proteases and to determine whether pH affected their inhibitory activity towards the well-characterized cysteine protease papain. Their potential role in the mineral balance of the tooth was assessed by the measurement of calcium binding and the rate of binding to carbonated apatite (CAP). RESULTS AND CONCLUSIONS: Salivary cystatin SN was found to inhibit the human lysosomal cathepsins B, H and L and salivary cystatin SA was found to inhibit human lysosomal cathepsin L in vitro. These proteases are involved in periodontal tissue destruction and these data suggest that salivary cystatins SA and SN are involved in the control of the proteolytic events in vivo. Salivary cystatin S was not an inhibitor of the cysteine proteases tested suggesting that its primary role is not as a cysteine protease inhibitor. However, S was able to bind more calcium and bind more rapidly to CAP than SA or SN, suggesting that its primary role in the oral environment is likely to be the involvement with the mineral balance of the tooth.     

T cells in pancreatic cancer stroma

Pancreatic ductal adenocarcinoma (PDAC) is a highly devastating disease with a dismal 5-year survival rate. PDAC has a complex tumour microenvironment; characterised by a robust desmoplastic stroma, extensive infiltration of immunesuppressive cells such as immature myeloid cells, tumour-associated macrophages, neutrophils and regulatory T cells, and the presence of exhausted and senescent T cells. The cross-talk between cells in this fibrotic tumour establishes an immune-privileged microenvironment that supports tumour cell escape from immune-surveillance, disease progression and spread to distant organs. PDAC tumours, considered to be non-immunogenic or cold, express low mutation burden, low infiltration of CD8⁺ cytotoxic lymphocytes that are localised along the invasive margin of the tumour border in the surrounding fibrotic tissue, and often display an exhausted phenotype. Here, we review the role of T cells in pancreatic cancer, examine the complex interactions of these crucial effector units within pancreatic cancer stroma and shed light on the increasingly attractive use of T cells as therapy.