Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Up-regulation of Skp2 after prostate cancer cell adhesion to basement membranes results in BRCA2 degradation and cell proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.     

Adiponectin oligomerization state and adiponectin receptors airway expression in chronic obstructive pulmonary disease

Adiponectin (Acrp30) shows several beneficial properties and circulates as different oligomers. The role of Acrp30 in lung is not fully clear, but a link with chronic obstructive pulmonary disease (COPD) has been highlighted. In this study, we analyzed the anthropometrical and biochemical features and evaluated total Acrp30 levels of a COPD cohort without metabolic complications compared to healthy controls. In addition, being the oligomerization state critical for its biological activities, we characterized the pattern of Acrp30 circulating oligomers focusing on the high molecular weight (HMW) oligomers to verify whether it correlates to COPD. Finally, we investigated AdipoR1 and AdipoR2 expression in lung from COPD. Interestingly, we found for the first time that the oligomerization state of Acrp30 is altered in COPD; particularly, we observed that the higher levels of Acrp30 are associated with a significant and specific increase of HMW. In addition, we demonstrated the presence of AdipoRs with a lower expression of AdipoR2 compared to AdipoR1. In conclusion, we demonstrated that in COPD, the higher levels of Acrp30 are associated with the significantly increase of HMW representing the most biologically active forms. The important role of Acrp30 in pathophysiological conditions of lung is supported also by the modulation of AdipoRs with the down regulation of AdipoR2. The low expression of AdipoR2 could suggest a specific role of this receptor, mainly implicated in Acrp30 effects on inflammation and oxidative stress. Thus, total Acrp30, HMW and its receptors could be considered critical targets to improve diagnostic and therapeutic strategies for lung diseases.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells Involves Cytochrome c Release from Mitochondria and Mitochondrial Shuttle Impairment

To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were investigated: the electron flow via respiratory chain from certain substrates to oxygen and the transfer of extramitochondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mitochondria was found to occur in glutamate-treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH-b5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3-phosphate/dihydroxyacetone phosphate and malate/oxaloacetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired under glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate-treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time-dependent, an index of the progressive damage of the cell.     

Organization of Patient Management and Fungal Epidemiology in Cystic Fibrosis

The achievement of a better life for cystic fibrosis (CF) patients is mainly caused by a better management and infection control over the last three decades. Herein, we want to summarize the cornerstones for an effective management of CF patients and to give an overview of the knowledge about the fungal epidemiology in this clinical context in Europe. Data from a retrospective analysis encompassing 66,616 samples from 3235 CF patients followed-up in 9 CF centers from different European countries are shown.     

Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia‐positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib‐sensitive KCL22S cells versus imatinib‐resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin‐mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr‐Abl activity. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000030.