The importance of redox state in liver damage.

Oxidative stress is a major pathogenetic event occurring in several liver disorders ranging from metabolic to proliferative ones, and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. The main sources of ROS are represented by mitochondria and cytocrome P450 enzymes in the hepatocyte, by Kupffer cells and by neutrophils. Cells are provided with efficient molecular strategies to strictly control the intracellular ROS level and to maintain the balance between oxidant and antioxidant molecules. A cellular oxidative stress condition is determined by an imbalance between the generation of ROS and the antioxidant defense capacity of the cell and can affect major cellular components including lipids, proteins and DNA. Proteins are very important signposts of cellular redox status and through their structure/function modulation, ROS can also influence gene expression profile by affecting intracellular signal transduction pathways. While several enzymatic (such as superoxide dismutase, catalase, glutathione peroxidase) and non enzymatic (such as 4-hydroxynonenal, decrease of glutathione, vitamin E, vitamin C, malondialdehyde) markers of chronic oxidative stress in liver are well known, early protein targets of oxidative injury are yet not well defined. Identification of these markers will enable early detection of liver diseases and will allow monitoring the degree of liver damage, the re1 Department of Biomedical Sciences and Technologies, University of Udine, P.le Kolbe 4, 33100 Udine, Italy. 2 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, via Giorgieri 1, 34127 Trieste, Italy. 3 Centro Studi Fegato, AREA Science Park Bldg Q, Campus Basovizza, ss 14, km 163.5, 34012 Trieste, Italy. Address for correspondence

Mitochondrial DNA Repair in Neurodegenerative Diseases and Ageing

Mitochondria are the only organelles, along with the nucleus, that have their own DNA. Mitochondrial DNA (mtDNA) is a double-stranded circular molecule of ~16.5 kbp that can exist in multiple copies within the organelle. Both strands are translated and encode for 22 tRNAs, 2 rRNAs, and 13 proteins. mtDNA molecules are anchored to the inner mitochondrial membrane and, in association with proteins, form a structure called nucleoid, which exerts a structural and protective function. Indeed, mitochondria have evolved mechanisms necessary to protect their DNA from chemical and physical lesions such as DNA repair pathways similar to those present in the nucleus. However, there are mitochondria-specific mechanisms such as rapid mtDNA turnover, fission, fusion, and mitophagy. Nevertheless, mtDNA mutations may be abundant in somatic tissue due mainly to the proximity of the mtDNA to the oxidative phosphorylation (OXPHOS) system and, consequently, to the reactive oxygen species (ROS) formed during ATP production. In this review, we summarise the most common types of mtDNA lesions and mitochondria repair mechanisms. The second part of the review focuses on the physiological role of mtDNA damage in ageing and the effect of mtDNA mutations in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Considering the central role of mitochondria in maintaining cellular homeostasis, the analysis of mitochondrial function is a central point for developing personalised medicine

Mitochondrial apurinic/apyrimidinic endonuclease 1 enhances mtDNA repair contributing to cell proliferation and mitochondrial integrity in early stages of hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is the leading cause of primary liver cancers. Surveillance of individuals at specific risk of developing HCC, early diagnostic markers, and new therapeutic approaches are essential to obtain a reduction in disease-related mortality. Apurinic/apyrimidinic endonuclease 1 (APE1) expression levels and its cytoplasmic localization have been reported to correlate with a lower degree of differentiation and shorter survival rate. The aim of this study is to fully investigate, for the first time, the role of the mitochondrial form of APE1 in HCC. Methods: As a study model, we analyzed samples from a cohort of patients diagnosed with HCC who underwent surgical resection. Mitochondrial APE1 content, expression levels of the mitochondrial import protein Mia40, and mtDNA damage of tumor tissue and distal non-tumor liver of each patient were analyzed. In parallel, we generated a stable HeLa clone for inducible silencing of endogenous APE1 and re-expression of the recombinant shRNA resistant mitochondrially targeted APE1 form (MTS-APE1). We evaluated mtDNA damage, cell growth, and mitochondrial respiration. Results: APE1's cytoplasmic positivity in Grades 1 and 2 HCC patients showed a significantly higher expression of mitochondrial APE1, which accounted for lower levels of mtDNA damage observed in the tumor tissue with respect to the distal area. In the contrast, the cytoplasmic positivity in Grade 3 was not associated with APE1's mitochondrial accumulation even when accounting for the higher number of mtDNA lesions measured. Loss of APE1 expression negatively affected mitochondrial respiration, cell viability, and proliferation as well as levels of mtDNA damage. Remarkably, the phenotype was efficiently rescued in MTS-APE1 clone, where APE1 is present only within the mitochondrial matrix. Conclusions: Our study confirms the prominent role of the mitochondrial form of APE1 in the early stages of HCC development and the relevance of the non-nuclear fraction of APE1 in the disease progression. We have also confirmed overexpression of Mia40 and the role of the MIA pathway in the APE1 import process. Based on our data, inhibition of the APE1 transport by blocking the MIA pathway could represent a new therapeutic approach for reducing mitochondrial metabolism by preventing the efficient repair of mtDNA

PCI Based Read-out Receiver Card in the ALICE DAQ System

The Detector Data Link (DDL) is the high-speed optical link for the ALICE experiment. This link shall transfer the data coming from the detectors at 100 MB/s rate. The main components of the link have been developed: the destination Interface Unit (DIU), the Source Interface Unit (SIU) and the Read-out Receiver Card (RORC). The first RORC version is based on the VME bus. The performance tests show that the maximum VME bandwidth could be reached. Meanwhile the PCI bus became very popular and is used in many platforms. The development of a PCI-based version has been started. The document describes the prototype version in three sections. An overview explains the main purpose of the card: to provide an interface between the DDL and the PCI bus. Acting as a 32bit/33MHz PCI master the card is able to write or read directly to or from the system memory from or to the DDL, respectively. Beside these functions the card can also be used as an autonomous data generator. The card has been designed to be well adapted to applications, which require small software overhead such the high-speed data acquisition systems. The implementation of the firmware will be presented. For the logic design we are using VHDL and schematic draw. Software library routines were written in C and are available on Linux OS. The results of performance measurements will be available to allow the comparison between the VME-RORC and PCI-RORC. In the conclusion the future plans and the idea of the improved (64bit/66MHz) PCI-RORC will be shown

BIOPSY IN COELIAC DISEASE DIAGNOSIS: WHEN IS IT NECESSARY?

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BIOPSY IN COELIAC DISEASE DIAGNOSIS: WHEN IS IT NECESSARY?

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Integration of VICbus, FDL, SCI and Ethernet in the CERN CASCADE data acquisition system

Cascade is a multi-processor real-time data-acquisition system for HEP experiments developed at CERN by the ECP-DS group. Configurations supported today include VMEbus processors running OS-9 and UNIX workstations. The CASCADE data acquisition processes, called stages communicate via links, at present VICbus between VME crates and Ethernet between VMEbus processors and workstations. Work is in progress to introduce new inter-stage links based on the Fast Data Link between VME crates and on SCI for data exchange between SUN stations. The paper gives a short description of the architecture of CASCADE with emphasis on the link aspects. The implementation and current status of the inter-stage links based on VICbus, Ethernet, FDI, and SCI will be described and results on the performances presented

mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with K_d = 1.7 nM. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication

Review and prospects of the CASCADE data acquisition system at CERN

CASCADE, a multi-processor real-time data-acquisition system for HEP experiments developed at CERN by the ECP-DS group, has now been in operation for one year. The current implementation supports configurations based on VMEbus processors running OS-9 and on UNIX workstations interconnected via VICbus or Ethernet. The project is reviewed by describing the main characteristics of the package, the applications in which it has been used, and the results of this experience. The main improvements of 1994, which include a parameterized multi-level event builder, a remote monitoring option and a powerful run control facility, as well as ongoing developments and prospects for 1995, are presented