Effect of lycopene on As2O3 induced oxidative stress in SH-SY5Y cells

It is known that oxidative stress may cause neuronal injury and several experimental models showed that As₂O₃ exposure causes oxidative stress. Lycopene, a carotenoid, has been shown to have protective effect in neurological disease models due to antioxidant activity, but its effect on As₂O₃-induced neurotoxicity is not identified yet. The aim of this study is to investigate the effects of lycopene on As₂O₃-induced neuronal damage and the related mechanisms. Cell viability was determined by the MTT assay. Lycopene was administrated with different concentrations (2, 4, 6 and 8 µM) one hour before 2 µM As₂O₃ exposure in SH-SY5Y human neuroblastoma cells. The anti-oxidant effect of lycopene was determined by measuring superoxide dismutase (SOD), catalase (CAT) hydrogen peroxide (H₂O₂), malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS). MTT results and LDH cytotoxicity analyses showed that pretreatment with 8 µM lycopene significantly improved the toxicity due to As₂O₃ exposure in SH?SY5Y neuroblastoma cells. Pretreatment with lycopene significantly increased the activities of anti?oxidative enzymes as well as total antioxidant status and decreased total oxidative status in As₂O₃ exposed cells. The results of this study indicate that lycopene may be a potent neuroprotective against oxidative stress and could be used to prevent neuronal injury or death in several neurological diseases.

     

ERK1/2 and p38 MAP kinase control MMP-2, MT1-MMP, and TIMP action and affect cell migration: a comparison between mesothelioma and mesothelial cells

Pleural malignant mesothelioma is a locally aggressive tumor of mesothelial cell origin. In other tumor types high expression of matrix metalloproteinase (MMP)-2, together with membrane-type1-MMP (MT1-MMP), and low levels of the tissue inhibitor of MMP (TIMP)-2 have been correlated with aggressive tumor progression and low survival rates. Therefore, we compared the expression and activation of these three factors and their regulation by two mesothelioma associated growth factors, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)-beta1 in six human mesothelioma and one mesothelial cell line. Polymerase chain reaction (PCR), immunoblotting, zymography, and small inhibitory RNAs (siRNA) were used to study gene expression, protein activation, and signal transduction. To proof the relevance of our in vitro data immunohistochemistry was performed in tissue sections. PDGF-BB induced, while TGF-beta1 inhibited cell proliferation. PDGF-BB was a chemoattractant for mesothelial cells, and its effect was increased in the presence of TGF-beta1. TGF-beta1 stimulated the de novo synthesis of pro-MMP-2 in both cell types. Pro-MMP-2 synthesis involved p38 MAP kinase. In cell culture and tissue sections only mesothelial cells expressed MT1-MMP. Migration of mesothelioma cells was dependent on the presence of MT1-MMP. Migration, but not proliferation of mesothelioma cells was inhibited by oleoyl-N-hydroxylamide, TIMP-2, and siRNA for MT1-MMP. Our data suggest that in mesothelioma cells the phosphorylation of p38 MAP kinase is deregulated and is involved in pro-MMP-2 expression. Mesothelioma progression depends on an interaction with mesothelial cells that provide MT1-MMP necessary to activate pro-MMP-2 to facilitate migration through an extracellular matrix (ECM) layer.     

Expression and Functional Characterization of the BNIP3 Protein in Renal Cell Carcinomas

BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) is a BH3-only protein that regulates apoptosis and autophagy. BNIP3 plays also an important role in hypoxia-induced cell response and is regulated by HIF1. Here, we studied a possible association of BNIP3 expression and the prognosis of patients with renal cell carcinomas (RCCs) and examined the functional relevance of BNIP3 in the regulation of cell survival and apoptosis of renal carcinoma cells. BNIP3 expression was determined by immunohistochemistry in RCC tumor tissue samples of 569 patients using a tissue microarray. Functional characterization of BNIP3 in renal carcinoma cells indicates prosurvival effects. In human RCC tumor samples, high cytoplasmic BNIP3 expression was associated with high-grade RCCs and regional lymph node metastasis. BNIP3 expression correlated negatively with disease-specific survival. Multivariate Cox regression analysis retained BNIP3 expression as an independent prognostic factor in patients without distant metastasis. Together, our studies imply that BNIP3 regulates cell survival in RCCs and its expression is an independent prognostic marker in patients with localized RCCs.     

The genera of bacteriophages and their receptors are the major determinants of host range

The host range of phages is a key to understand their impact on bacterial ecology and evolution. Because of the complexity of phage–host interactions, the variables that determine the breadth of a phage host range remain poorly understood. Here, we propose a novel holistic approach to identify the host range determinants of a new collection of phages infecting Salmonella, isolated from animal, environmental and wastewater samples that were able to infect 58 of the 71 Salmonella strains in our collection. By using a set of statistic approaches (non-metric dimensional scaling, Bray–Curtis distance, PERMANOVA), we analysed phenotypic (host range on wild-type and receptor mutants) and genetic data (taxonomic assignment and receptor binding proteins) to evaluate the impact of isolation strain and niche, phage receptor and genus on the host range. Statistical analysis revealed that two phage characteristics influence the host range by explaining the most variance: the receptor by 45% and the genus by 51%. Interestingly, phage genus and receptor in combination explained 79% of the variance, establishing these characteristics as the major determinants of the host range. This study demonstrates the power and the novelty of applying statistical approaches to phenotypic and genetic data to investigate the ecology of phage–host interactions.     

Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.     

Mc2 receptor knockdown modulates differentiation and lipid composition in adipocytes

The melanocortin system is involved in central and peripheral regulation of energy homeostasis. In adipocytes, the melanocortin 2 receptor (MC2R) transmits ACTH-dependent signaling and its expression rises substantially during adipocyte differentiation. An in vitro system of retrovirally expressed shRNA directed against Mc2r mRNA in 3T3-L1 cells was established and effects of Mc2r knockdown (kd) in comparison to cells expressing non-targeting shRNA (control) were explored in differentiated adipocytes. Morphology, gene expression, lipolysis and fatty acid composition were analyzed. While gross morphology was unchanged extractable amount of lipids was reduced to 70-80% in kd cell lines (p<0.01). Moreover, expression changes of Pparγ2, aP2, and Pref1 indicated reduced differentiation in Mc2r kd cells. Intriguingly, not only ACTH, but also norepinephrine stimulated lipolysis were substantially reduced demonstrating functional significance of MC2R for general lipolysis pathway. Analysis of fatty acid composition in triglyceride and phospholipid fractions showed a lowered ratio of C16:1/C16:0 and C18:1/C18:0, but increased concentrations of arachidonic acid upon Mc2r knockdown. Reduction of mono-unsaturated fatty acids (MUFAs) was associated with lower expression of stearoyl-Coenzyme A desaturase 1 and 2 in kd cells (21±8% vs. 100±13%, p=0.01 and 32±3% vs. 100±15%, p=0.046). Conversely, high doses of ACTH resulted in gene expression changes, mirroring Mc2r knockdown (higher Pparγ2, Scd1, Hsl expression). MC2R plays an important role for regular lipolytic function and lipid composition in 3T3-L1 adipocytes. Of interest, desaturase expression was reduced and MUFA content accordingly altered in kd cells.