Time-course variations of oxyradical metabolism, DNA integrity and lysosomal stability in mussels, Mytilus galloprovincialis, during a field translocation experiment

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study, several biomarkers were investigated in marine mussels caged for 4 weeks into an industrialised harbour of north-west Italy. Organisms were collected at different time intervals to better characterise the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants (catalase, glutathione S-transferases, glutathione reductase, total glutathione), the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralise specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidised bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with no variation or increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment. These findings suggest an initial counteractive response of mussels toward the enhanced prooxidant challenge, while antioxidants appeared overwhelmed at longer exposure periods. The hypothesis of reactive oxygen species (ROS) mediated toxicity is supported by the appearance of cell damages (DNA integrity and lysosome membrane stability), which exhibited a progressive enhancement during the course of the experiment with a maximum impairment after 30 days of exposure.     

Sensitization of multidrug resistant human ostesarcoma cells to Apo2 Ligand/TRAIL-induced apoptosis by inhibition of the Akt/PKB kinase

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. The emergence of the multidrug resistance (MDR) phenotype in cancer cells is often associated with the overexpression of P-glycoprotein, encoded by the multidrug resistance gene MDR-1. The administration of some of the most common chemotherapeutic agents to these cells becomes ineffective because of their P-gp-driven efflux from the cell. Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that is considered to induce death of cancer cells but not normal cells. Its powerful apoptotic activity is mediated through its cell surface death domain-containing receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, which in turn spread the signal in the cytosol through the activation of the caspase cascade. The Akt/PKB kinase is an important cell survival protein which is regulated by D3-phosphoinositides. High Akt expression and activity levels are well documented in many types of tumors, which very often show an altered PI3-K/Akt/PTEN pathway. In this study the U2OS human osteosarcoma cell line and its multidrug resistant (MDR) subline that overexpresses MDR-1 gene, MDR-U2OS, have been analyzed for their responsiveness to TRAIL. In conflict with the presence of active DR4 and DR5 receptors in both clones, U2OS cells exhibited only a low responsiveness to TRAIL, while the MDR-U2OS subline did exhibit a marked TRAIL sensitivity. An analysis of the post-receptor events showed that TRAIL responsiveness correlates with a reduced expression of endogenous Akt. In fact, expression in MDR-U2OS cells of a constitutively active Akt strongly decreased their sensitivity to TRAIL. The identification of Akt as a key modulator of TRAIL responsiveness could help to design TRAIL-based combinations for treatment of osteosarcoma. Moreover, the discovery that multidrug resistant osteosarcomas are highly sensitive to TRAIL-induced apoptosis indicates TRAIL as a new candidate for the treatment of multidrug resistant bone malignancies.     

Nitric oxide produced by the enterocyte is involved in the cellular regulation of ion transport

The role of nitric oxide (NO) in the intestinal basal ion transport and under conditions of enterotoxin-induced ion secretion is controversial. Namely it is not clear whether NO enhances or counteracts intestinal ion secretion and whether the effects on transport result from a direct interaction with the enterocyte. The cell origin of NO is also unclear. We have tested the hypothesis that NO produced by the enterocyte directly regulates ion transport processes either in basal condition or in response to cholera toxin-induced secretion. Electrical variables reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers exposed to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester, in the presence or absence of cholera toxin. cAMP concentrations were also measured. NO release was determined by nitrite-nitrate concentration. NO synthase activities were assayed by Western blot analysis. Nomega-nitro-l-arginine methyl ester had a secretory effect, as judged by increased basal short-circuit current and cAMP concentration. It also increased cholera toxin-induced electrical response and cAMP production. Either cholera toxin or the cAMP analog 8-bromo-cAMP induced a rapidly progressive and Ca2+-dependent increase in NO concentration, suggesting a homeostatic up-regulation of the constitutive form of NO synthase. Western blot analysis showed an increase in constitutive NO synthase enzyme isoform. These results indicate that the enterocyte regulates its own ion transport processes, either in basal condition or in the presence of active secretion, through the activation of a constitutive NO synthase-NO pathway, functioning as a braking force of cAMP-induced ion secretion.     

Low urinary kallikrein rats: different sensitivity of verapamil on hypertensive response to central acute cadmium administration

Essential hypertension is a common disease caused by a combination of genetic and environmental factors. Cadmium (Cd), an important environmental pollutant, is able to induce hypertension in humans. In rats, intracerebroventricular (icv) Cd administration causes a sustained increase in arterial blood pressure. The kallikrein-kinin system appears an important regulator of cardiovascular function and rats with low renal excretion of kallikrein differ from normal-kallikrein Wistar rats in their pressor response to icv Cd. To clarify these differences in pressor response, we evaluated the protective effect of a calcium antagonist following the administration of 10 microg Cd icv. Pre-treatment with increasing doses of verapamil (100, 150200 microg) in normal-kallikrein rats produced a blocking of the hypertensive effects of Cd, even at the lower doses. In low-kallikrein rats we observed a dose-dependent inhibition of hypertensive effects at 100 and 150 microg, while at 200 microg there was, paradoxically, an increase in pressor values. Our results suggest that a genetically-determined defect in urinary kallikrein excretion leads to different modulation of brain calcium channels antagonists in the hypertensive response to icv Cd. This different sensitivity of low-kallikrein rats suggests that the hypertensive effect of icv Cd is, at least in part, the result of blocking the calcium channels, but is also sensitive to a new hemodynamic equilibrium, such as that present in low kallikrein rats, and probably intervenes as a modulator at the central level in other as yet not well identified systems, also linked to the hypotensive pathways, which may be activated in certain conditions.     

EPIC-Italy cohorts and multipurpose national surveys. A comparison of some socio-demographic and life-style characteristics

BACKGROUND: EPIC-Italy cohort study recruited subjects who voluntarily accepted to participate in the project. From the self-selected bases of the population sample, some bias could derive in the data interpretation when risk estimation for cancer disease related to life-style factors is the principal concern. Knowledge of the bias related to self-selected sampling is important for better directing the interpretation of the EPIC-Italy study results. METHODS: We investigated the characteristics of volunteer subjects recruited in the EPIC-Italy cohorts and compared them with those of the randomly selected subjects recruited in the Multipurpose ISTAT Surveys realized in the same period (1993-1998) in which the EPIC-cohorts were recruited. RESULTS: We found some differences, and in particular a different attitude towards cigarettes smoking and wine consumption, between the EPIC cohort and the Multipurpose ISTAT Surveys, as well as among geographical areas within the EPIC cohort. CONCLUSIONS: The uneven distribution of some characteristics suggests that the self-selected subjects were characterized by an overall lower consumption of wine and cigarette smoking even when the educational level was considered. This could suggest a generally more healthy life-style among subjects recruited on a volountary bases.     

In situ detection of telomeres by fluorescence in situ hybridization and telomerase activity in glioblastoma multiforme: correlation with p53 status, EGFR, c-myc, MIB1, and Topoisomerase IIalpha protein expression

Aberrations of genes/proteins regulating cell cycle and growth, increased proliferation and telomerase activity (TA) are documentable in glioblastoma multiforme. TA is more frequently detectable in secondary glioblastoma, which is also characterized by p53 mutation/overexpression. Discordant telomere (Te) length values have been reported in glioblastomas with and without TA. In 31 glioblastomas, in which pre-existing astrocytoma was not documented, we compared cases with and without TA for the expression of p53, EGFR, c-Myc, MIB-1 and Topoisomerase IIalpha; p53 mutations were also investigated by SSCP-PCR. Correlations were made with Te parameters [TePs: number (TeNo), length and area] as evaluated by image analysis in interphase nuclei of fluorescence in situ hybridization (FISH)-processed sections. We found no differences in the expression of the proteins evaluated and in TePs, except Te/nuclear area %, which was significantly lower in TA+ cases (p=0.02). TePs were, instead, inversely correlated with TA (p=0.0001). TA was positively correlated with MIB1 staining index in the TA+ cases (p=0.033), which also showed a positive correlation between TeNo and EGFR expression (p=0.042), and a trend towards a negative correlation between TeNo and p53 expression (p=0.05). Tumors overexpressing EGFR had a significantly shorter lifetime (p=0.0001). TeNo seems to be inversely correlated to tumor proliferation and lifetime in glioblastoma multiforme.     

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.     

Allelic variants of natriuretic peptide receptor genes are associated with family history of hypertension and cardiovascular phenotype

OBJECTIVE: Abnormalities in the natriuretic peptide system could play a key role in the genesis of hypertension. We evaluated the associations between a family history of hypertension, cardiovascular phenotype and allelic variants of Npr1 and Npr3, two candidate genes that codify for natriuretic peptide receptors. METHODS: We genotyped 45 young normotensive subjects (19 males, 26.8 +/- 3.7 years) with accurately assessed family history of hypertension (FH+) and 52 (26 males, 26.1 +/- 3.1 years) without (FH-) for the known variants of Npr1 and Npr3 genes, and for a novel length difference (3C/4C) polymorphism at position 15129 in the 3'-untranslated region of the Npr1 gene. Blood pressure, echocardiography and plasma brain natriuretic peptide were assessed. RESULTS: Both the novel Npr1 3C allele (59 versus 33%, P < 0.001) and the 3C/3C genotype (31 versus 8%; P < 0.001) were significantly more frequent in FH+ than in FH-. The inverse distribution of the 4C/4C genotype suggested that a casual association was very unlikely. Moreover, the 3C/3C homozygous had significantly higher systolic blood pressure (121.1 +/- 6.3 versus 115.6 +/- 7.8 mmHg in 4C/4C; P < 0.05) and a longer left ventricular isovolumic relaxation time (67 +/- 10 versus 61 +/- 9 ms; P < 0.05). The Npr3 C(-55) allele variant was also more frequent in FH+ (88 versus 76%, P < 0.05), but was not associated with the cardiovascular phenotype. CONCLUSIONS: The novel Npr1 gene 3C variant and the Npr3 gene C(-55) allele are associated with hypertensive family history. Moreover, the functional Npr1 3C variant, when homozygous, is also associated with higher systolic blood pressure and prolonged ventricular relaxation.     

Individual prediction of functional recovery after coronary revascularization in patients with ischemic cardiomyopathy: the scar-to-biphasic model

Currently, the prediction of improvement of left ventricular (LV) ejection fraction (EF) after revascularization in patients with ischemic cardiomyopathy relies only on viable myocardium extent, whereas both the amount of viable and scar tissue may be important. A model was developed, based on the amount of viable and nonviable myocardium, to predict functional recovery. Viable and scarred myocardium was defined by dobutamine stress echocardiography (DSE) in 108 consecutive patients. LVEF before and 9 to 12 months after revascularization was assessed by radionuclide ventriculography; an improvement of ≥5% was considered significant. In the 1,089 dysfunctional segments (63%), DSE elicited biphasic response in 216 segments (20%), sustained improvement in 205 (19%), worsening in 43 (4%), and no change in 625 (57%). LVEF improved in 39 patients (36%). Only the numbers of biphasic and scar segments were predictors of improvement or no improvement of LVEF (odds ratio 1.5, 95% confidence interval 1.2 to 1.7, p <0.0001 for biphasic segments; odds ratio 0.8, 95% confidence interval 0.7 to 0.9, p <0.0005 for scarred segments). The sustained improvement and worsening pattern were not predictive of improvement or no improvement. A regression function, based on the number of scar and biphasic segments, showed that the likelihood of recovery was 85% in patients with extensive biphasic tissue and no scars and 11% in patients with extensive scars and no biphasic myocardium. Patients with a mixture of scar and biphasic tissue had an intermediate likelihood of improvement (50%). In patients with ischemic cardiomyopathy and a mixture of viable and nonviable tissue, both numbers of viable and nonviable segments should be considered to accurately predict functional recovery after revascularization.