Opioid Peptide Gene Expression in the Primary Hereditary Cardiomyopathy of the Syrian Hamster II. ROLE OF INTRACELLULAR CALCIUM LOADING

We have previously shown that prodynorphin gene expression was markedly increased in adult myocytes of BIO 14.6 cardiomyopathic hamsters and that nuclear protein kinase C (PKC) may be involved in the induction of this opioid gene. Here we report that the cytosolic Ca2 xv v, b+ concentration was significantly increased in resting and in KCl-depolarized cardiomyopathic myocytes compared with normal cells. In normal and in cardiomyopathic cells, KCl significantly increased prodynorphin mRNA levels and prodynorphin gene transcription. These effects were abolished by the Ca2+ channel blocker verapamil. In control myocytes, the KCl-induced increase in prodynorphin mRNA expression was in part attenuated by chelerythrine or calphostin C, two selective PKC inhibitors. In these cells, KCl induced the translocation of PKC-α into the nucleus, increasing nuclear PKC activity. In resting cardiomyopathic myocytes, the increase in prodynorphin mRNA levels and gene transcription were significantly attenuated by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxy-methylester being completely abolished when the chelating agent was administered in the presence of PKC inhibitors. KCl and the PKC activator 1,2-dioctanoyl-sn-glycerol additively stimulated prodynorphin gene expression both in normal and in cardiomyopathic cells. Therefore, we conclude that PKC activation and intracellular Ca2+ overload may represent the two major signaling mechanisms involved in the induction of the prodynorphin gene in cardiomyopathic cells

Gamma-oryzanol, a main component of rice bran oil: <i>in vitro</i> studies of its antioxidant properties

Since investigations so far carried out provided an unclear picture of the mechanism of the antioxidant action of gamma-oryzanol, the aim of the present work has been the contribution to the elucidation of its the molecular mechanisms by using in vitro, previously well-characterized, experimental models (such as scavenging of DPPH° ROS scavenging, Fe and azocompound triggered lipoperoxidation) that allow us to study the reactions involved in the complex process of lipoperoxidation

The Oxidative state of LDL is the major determinant of anti/prooxidant effect of coffee on Cu²⁺ catalysed peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

The Oxidative State of LDL is the Major Determinant of Anti/Prooxidant Effect of Coffee on Cu2+ Catalysed Peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

L’evoluzione al femminile. Il contributo delle femmineall’evoluzione dell’Homo sapiens

Libri: L’evoluzione al femminile. Il contributo delle femmine all’evoluzione dell’Homo sapiens. Pendragon, Bologna. L’autrice Bruna Tadolini, già docente di Biochimica e Biologia molecolare, Università di Bologna e di Sassari in dialogo con Gina Chianese, docente di Pedagogia Università di Trieste

NAMI-A inhibits the PMA-induced ODC gene expression in ECV304 cells: involvement of PKC/Raf/Mek/ERK signalling pathway

Imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A) is a new compound active against lung metastasis of solid metastasizing tumours. While its in vivo effect has been studied, the molecular insights that underlie its action are largely unknown. Among the possible pathways responsible for malignant transformation, PKC arose as one of the most promising targets for new antineoplastic drugs. We demonstrated the capability of NAMI-A of inhibiting PMA induced-PKC activity in ECV304 in a dose-dependent fashion. Furthermore, NAMI-A through modulation of PKC activity has been proved capable of reducing the phorbol ester induced expression of ornithine decarboxilase (ODC) gene and to abrogate the activation of the Raf/MEK/ERK pathway. Taken together these results suggest that many of the in vivo outcomes of NAMI-A treatment may be the result of a direct action on PKC

The Anti-metastatic agent imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate induces endothelial cell apoptosis by inhibiting the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway

Imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate (NAMI-A) is a new ruthenium compound active against lung metastasis in vivo and tumor cell invasion in vitro. Since angiogenesis was recognized as a key event in the metastasizing process, the manipulation of neo-vessel formation has been developed as a new therapeutic approach. Within this context, a pivotal role for apoptosis in regulating cellular growth has been proposed. In the present study, we exposed to NAMI-A the spontaneously transformed human endothelial cell line ECV304 and assessed a number of apoptosis-related features, including the DNA degradation rate, the activation of caspase-3 protease, the expression of Hsp27, and the release of cytochrome c. Cell treatment with NAMI-A elicited a significant increment in the apoptotic response, as indicated by DNA fragmentation and caspase-3 activation, two classical hallmarks of cellular suicide. Furthermore, NAMI-A was able to down-regulate Hsp27 protein expression and provoke the release of mitochondrial cytochrome c in the cytosol. Here, we analyze the involvement of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signal transduction pathway in the induction of apoptosis elicited by NAMI-A. Such a response was associated with a marked inhibition of MAPK/ERK kinase (MEK) and ERK phosphorylation with a time course and dose dependency overlapping those observed throughout NAMI-A-induced apoptosis. In addition, we report that PD98059, a selective MEK inhibitor, is able to induce apoptosis by itself in the ECV304 cell line. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating an apoptotic event in ECV304

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-<i>myc</i> gene expression and endothelial cell proliferation

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation

PKC/Raf/MEK/ERK signaling pathway modulates native-LDL-induced E2F-1 gene expression and endothelial cell proliferation

Background and objectives: The interactions of low-density lipoprotein (LDL) with the endothelium are thought to play a major role in the development of atherosclerosis. Due to this reason, the molecular sequelae of events resulting from native LDL (N-LDL) interaction with human endothelial cells (HECs) are largely under investigation. Methods and results: Here, we report that the exposure of serum-free HECs to different concentrations of N-LDL-cholesterol (LDL-chol) elicited a time- and dose-dependent induction of DNA synthesis. The exposure of serum-free HECs to N-LDL was able to elicit a time- and dose-dependent increase of protein kinase C (PKC) activity that, along with the activation of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, leads to an increase in E2F-1 gene expression. In addition, the treatment of HECs with N-LDL was also able to induce both E2F-1 gene transcription and protein expression. These N-LDL-aroused responses were dramatically counteracted by PKC inhibition or down regulation. Similarly to what observed for Raf/MEK/ERK activation and E2F-1 gene expression, the inhibition of PKC as well as its down regulation, significantly lowered the DNA synthesis induced by N-LDL in serum-free HECs. Conclusions: These results suggest that the activation of PKC/Raf/MEK/ERK-mediated events controlling E2F-1 gene expression by N-LDL may represent an important mechanism in the regulation of HECs proliferation during normal and pathological processes

Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis

We have recently described a new method to determine physiological thiols, in which the quantification of plasma homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine was achieved after derivatization with 5-iodoacetamidofluorescein. Samples were separated and measured by capillary electrophoresis with laser-induced fluorescence in an uncoated fused-silica capillary, using a phosphate/borate run buffer and the organic base N-Methyl-D-glucamine as effective electrolyte addictive to obtain a baseline peak separation. In this paper, we propose an improvement of our method useful for the analysis of the intracellular thiols in different cultured cells. In particular, we studied run buffer and injection conditions in order to increase the sensitivity of the assay and we found that, by incrementing two times the injected volume and using the water plug before the sample injection, the sensitivity of our previous method was increased by about ten times. To maintain a good resolution between peaks, particularly between homocysteine and the internal standard d-penicillamine, we lengthened the run time by incrementing the concentration of the electrolyte buffer and the organic base d-glucamine and by decreasing the cartridge temperature from 40 to 25°C. After these changes in electrophoretical parameters, cellular thiols were baseline-resolved in less than 14 min instead of 9 min as in our previous method, but the limit of quantification is increased from 50 to 1 nmol/L. This new procedure allows also to measure the intracellular thiols commonly found at low concentration, such as cysteinylglycine, glutamylcysteine, and homocysteine. The new analytical method performance was assessed by measuring the intracellular thiols in three different cell lines, i.e., HUVEC, ECV304, and R1 stem cells