Showing papers by "Elias Castanas published in 2002"

The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton.

Abstract: Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short-time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non-internalizable analog testosterone-BSA increase rapidly the release of prostate-specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non-internalizable fluorescent analog testosterone-BSA-FITC, on whole cells. Binding on these sites is time- and concentration-dependent and specific for testosterone, presenting a KD of 10.9 nM and a number of 144 sites/mg protein (approximately 13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.

A new automated method for the determination of the Total Antioxidant Capacity (TAC) of human plasma, based on the crocin bleaching assay

Abstract: Antioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (poly)aromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. In the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay), based on the crocin bleaching (oxidation) method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L), and performed using an end point measurement. The TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS) test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM), while a diet rich in antioxidants more than doubled this value. The proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and/or exogenous metabolites. The antioxidant capacity of plasma thus calculated can be used as a useful indicator of the antioxidant value of foods and beverages in the daily diet.

Natural antisense RNA inhibits the expression of BCMA, a tumour necrosis factor receptor homologue.

Abstract: BCMA (B-cell maturation) belongs to the tumour necrosis factor receptor gene family, and is specifically expressed in mature B lymphocytes. Antisense BCMA RNA is produced by transcription from the same locus and has typical mRNA features, e.g, polyadenylation, splicing, Kozak consensus sequence and an ORF (p12). To investigate the function of antisense BCMA RNA, we expressed BCMA in cell lines, in the presence of antisense p12 or a mutant lacking the initiation ATG codon (p12-ATG). Overexpression of both p12 and p12-ATG antisense BCMA resulted in a large decrease in the amount of BCMA protein produced, with no change in BCMA RNA levels, indicating that BCMA expression is regulated by antisense BCMA RNA at the translational level. We have also observed slight adenosine modifications, suggestive of the activity of a double-stranded RNA-specific adenosine deaminase. These data suggest that antisense BCMA may operate under physiological conditions using similar antisense-mediated control mechanisms, to inhibit the expression of the BCMA gene.

Rapid effects of 17β-estradiol and progesterone on sheep visceral and parietal pleurae via a nitric oxide pathway

Abstract: We investigated the effects of 17β-estradiol and progesterone on transepithelial electrical resistance (R TE) in sheep visceral and parietal pleurae. Specimens of intact pleurae from adult female s...

Immunologic effects of emdogain in humans: one-year results.

Abstract: Tissue regeneration after therapeutic manipulations is essential in periodontology, oral surgery, and trauma of the periodontal tissues. Local inflammation because of poor oral hygiene also plays a crucial role in the above situations. Local inflammatory reaction, accompanied by the local production of cytokines, profoundly influences bone turnover and regeneration. Several products of low immunogenicity for augmenting tissue regeneration have been recently proposed as boosters of soft and mineralized tissue regeneration. Among them, Emdogain, an amelogenin derivative of porcine origin, has recently been introduced. Clinical results indicate that this product might be a good additive, producing fast tissue regeneration with no apparent clinical side effects. In contrast, very little is known about its in vivo immunologic effects. A previous study showed that Emdogain does not modify the cellular or humoral immune response in vitro. In the present work, performed in 10 patients, only a slight, nonsignificant activation of the immune system occurred during the first year following Emdogain application. Neither cellular immunity nor humoral immune response was significantly modified. In addition, the in vitro response of the patients' lymphocytes to Emdogain was assayed 2 and 12 months postoperative. We did not find any significant specific lymphocyte transformation in the presence of Emdogain, although lymphocytes could be stimulated by nonselective mitogens. These results indicate the immunologic safety of the agent in vivo, at least after 1 year.

Matrix metalloproteinases and their inhibitors in acute viral hepatitis

Abstract: Matrix metalloproteases (MMPs) and their inhibitors are effector molecules involved in extracellular matrix remodelling. The serum profile for these proteolytic enzymes and their inhibitors during acute self-limiting viral hepatitis has not been studied. We therefore determined serum concentrations of MMP-1, MMP-3, MMP-2, MMP-9 and their inhibitors (tissue inhibitors of metalloproteinase) TIMP-1, TIMP-2 and alpha2 macroglobulin (AMG) in the serum of patients during the icteric stage of self-limiting acute viral hepatitis. Transforming growth factor-beta (TGF-beta) and interleukin (IL)-10, two cytokines involved in the regulation of MMPs and TIMPs were also assessed. Nineteen patients (12 men, seven women) with a mean age of 29.9 years (range 16-65 years) participated in the study. Fifteen had hepatitis B virus (HBV, two HCV and two HAV infection. The values of patients were compared with those obtained from 15 blood donor controls (eight men, seven women), mean age 36.2 years (range 18-55 years). Serum levels of TGF-beta, IL-10, MMP-1, MMP-3, MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed by ELISA. MMP-2 and MMP-9 were also measured by a zymogram protease assay. alpha2 macroglobulin (AMG) was measured by nephelometry. Compared with the healthy controls the mean serum concentrations of all MMPs were significantly decreased in the acute hepatitis patients. There was no difference in the serum concentration of TIMP-1 between patients and the controls. Serum levels of TIMP-2 (P < 0001), TGF-beta (P < 0.05), IL-10 (P < 0.001) and AMG (P < 0001) were increased in patients compared to healthy controls. A statistically significant negative correlation by linear regression analysis was found between AMG and MMP-1 (P=0003). The decreased levels of MMPs observed, together with normal and increased levels of TIMP-1 and TIMP-2, may indicate an attempt to limit matrix degradation at this stage of disease resolution. The increased levels of the anti-inflammatory cytokines IL-10 and TGF-beta might be the underlying mechanism responsible for the above effect. AMG inhibition especially for MMP-1 may play an additional important role.

Somatostatin and Opioid Receptors in Mammary Tissue

Abstract: Somatostatin and opioid systems, are the two main inhibitory systems in mammals. Both classes of substances have been identified in normal and malignant mammary gland, as well as their cognitive receptors. They have been implied in the inhibition of cell growth of cancer cells and cell lines, in a dose-dependant and reversible manner. Somatostatin acts through homologous receptors (SSTRs), belonging to five distinct classes (SSTR1-5). We, and others have identified SSTR2 and 3 as been the only SSTRs present in the breast. Furthermore, opioids act through the three classes of opioid receptors (μ,δ,κ).Inthe breast,kappa opioid receptor subtypes(κ1-κ3) are the most widely expressed. We further have shown that opioids, in additionto their binding to opioid receptors, compete for binding to SSTRs. This functional interaction, together with other identified modes of opioid action in the breast (modulation ofsteroid receptors,proteases,secretion,interaction with cytoskeletalelements),will be discussed,takingintoconsiderationalso the possible local production of casomorphins (casein-derived opioids), which are very potent antiproliferative agents.