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Benzothiazoles are a part of the molecular structure of a large number of natural products, biocides, drugs, food flavors, and industrial chemicals. They also appear in the environment mainly as a result of their production and use as rubber vulcanization accelerators. A new headspace solid-phase microextraction (HS-SPME) method for analysis of benzothiazole (BTH) in wine is described. This method is fast, inexpensive, and does not require solvents. The detection limit of BTH in wine was 45 ppt with linearity up to 100 ppb. The quantification of BTH is performed by the standard additions method and does not require the use of an internal standard. We have analyzed 12 wines from different grape varieties grown in several regions, using SPME extraction and gas chromatography-mass spectrometry (GC-MS) detection. Under these experimental conditions, benzothiazole was found in all wines analyzed. Concentration levels in samples varied from 0.24 microg/L (Vermentino) to 1.09 microg/L (Franciacorta).

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1. The effects of ethyl alcohol and wine (red and white) on haemostatic parameters and experimental thrombosis were studied in rats; NO was evaluated as a possible mediator of these effects. 2. We found that red wine (12% alcohol) supplementation (8.4 +/- 0.4 ml d-1 in drinking water, for 10 days) induced a marked prolongation of 'template' bleeding time (BT) (258 +/- 13 vs 132 +/- 13 s in controls; P < 0.001), a decrease in platelet adhesion to fibrillar collagen (11.6 +/- 1.0 vs 32.2 +/- 1.3%; P < 0.01) and a reduction in thrombus weight (1.45 +/- 0.33 vs 3.27 +/- 0.39 mg; P < 0.01). 3. Alcohol-free red wine showed an effect similar to red wine. In contrast, neither ethyl alcohol (12%) nor white wine (12% alcohol) affected these systems. 4. All these effects were also observed after red wine i.v. injection (1 ml kg-1 of 1:4 dilution) 15 min before the experiments. 5. The effects of red wine were prevented by the NO inhibitor, N omega nitro-L-arginine-methyl ester (L-NAME). L-arginine, not D-arginine, reversed the effect of L-NAME on red wine infusion. 6. Red wine injection induced a 3 fold increase in total radical-trapping antioxidant parameter values of rat plasma with respect to controls, while white wine and alcohol did not show any effect. 7. Our study provides evidence that red wine modulates primary haemostasis and prevents experimental thrombosis in rats, independently of its alcohol content, by a NO-mediated mechanism.

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Our previous studies are reviewed and at the same time preliminary experimental observation to the topic of endocrine end-points in autoimmune disease is introduced. To this end, we have used rheumatoid arthritis (RA), including synovial fluids and primary cultures of synovial macrophages, as a model system in order to investigate (a) expression and subcellular localization of high-affinity sites of steroid binding in immune effector cells; (b) steroid metabolic profiles in both male and female RA patients, as compared to healthy subjects; and (c) activities of key steroid enzymes that govern intratissue accumulation of sex hormones. In RA tissues and cells, the concurrent evidence for (1) androgen and/or estrogen receptors, (2) high concentrations of biologically active steroids, (3) key enzymes of steroid metabolism, and (4) significant changes of estrogen to androgen ratio, all strongly suggests that individual immune cells, including synovial macrophages, may behave as steroid-sensitive cells, namely, they may represent a target for sex steroids, supporting the hypothesis of a potential endocrine regulation of the immune response also in RA disease. In this respect, definition of several endocrine end-points may have important implications for the treatment of rheumatic disease and other immunological disorders.

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In the present study we have inspected estrogen metabolism in cultured human prostate cancer cells (LNCaP, DU145, PC3), in relation to the expression of mRNAs for different 17 beta hydroxysteroid dehydrogenase (17 beta HSD) enzymes (from 1 to 4). Using an intact cell analysis, we have compared precursor degradation and product formation after incubation of cells with physiological amounts of radioactive E2 or estrone (E1) for 24-72 h and subsequent reverse-phase high performance liquid chromatography analysis. The LNCaP and DU145 cells only partly converted E2 to E1 (26 and 13% at 72 h, respectively), giving rise to an appreciable production of E2 from E1 (nearly 20% in all cases). Conversely, PC3 cells revealed a massive E2 oxidation to E1 (up to 90% by 72 h) and a scant formation of E2 (<2%) from E1. In addition, an appreciable formation of 16 alpha OHE1 was seen in either PC3 (11%) or DU145 (5%) cells. respectively using E2 or E1 as precursor. All three cell lines exhibited marked amounts of 17 beta HSD4 mRNA species, whilst even greater amounts of 17 beta HSD2 transcript were found in PC3 cells only. No mRNA for either 17 beta HSD1 or 17 beta HSD3 could be detected in any cell line. The present evidence indicates that pathways of estrogen metabolism are distinctly governed in prostate cancer cells depending on their endocrine status, being associated with a differential expression of mRNA for different 17 beta HSD enzymes.

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We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 microM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (delta4Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 microM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to delta4Ad. In either cell line, T transformation to delta4Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.

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