Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase is sensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODs used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [14C]acetate incorporation into [14C]cholesterol and by an increased [125I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.     

Respiratory function in cybrid cell lines carrying European mtDNA haplogroups: implications for Leber's hereditary optic neuropathy

The possibility that some combinations of mtDNA polymorphisms, previously associated with Leber's hereditary optic neuropathy (LHON), may affect mitochondrial respiratory function was tested in osteosarcoma-derived transmitochondrial cytoplasmic hybrids (cybrids). In this cellular system, in the presence of the same nuclear background, different exogenous mtDNAs are used to repopulate a parental cell line previously devoid of its original mtDNA. No detectable differences in multiple parameters exploring respiratory function were observed when mtDNAs belonging to European haplogroups X, H, T and J were used. Different possible explanations for the previously established association between haplogroup J and LHON 11778/ND4 and 14484/ND6 pathogenic mutations are discussed, including the unconventional proposal that mtDNA haplogroup J may exert a protective rather than detrimental effect.     

Latent profile analysis in frontotemporal lobar degeneration and related disorders: clinical presentation and SPECT functional correlates

Background  

Frontotemporal Lobar Degeneration (FTLD) thus recently renamed, refers to a spectrum of heterogeneous conditions. This same heterogeneity of presentation represents the major methodological limit for the correct evaluation of clinical designation and brain functional correlates. At present, no study has investigated clinical clusters due to specific cognitive and behavioural disturbances beyond current clinical criteria.     

Neuroendocrine (Merkel-cell) carcinoma of the skin. Fine needle aspiration cytology and clinicopathologic study of a case

Neuroendocrine (Merkel-cell) carcinoma of the skin, a neoplasm usually found in elderly individuals, is often locally aggressive and has the potential to metastasize to lymph nodes and cause death. Microscopically the neoplasm is a small-cell carcinoma that involves primarily the dermis. We report a case of such a neoplasm in a 61-year-old female, stressing the possible contribution of fine needle aspiration cytology in the recognition of this neoplasm and its differentiation from other small-cell neoplasms that may involve the skin.     

A comparison on effects of normalisations in the detection of differentially expressed genes

Background  

Various normalisation techniques have been developed in the context of microarray analysis to try to correct expression measurements for experimental bias and random fluctuations. Major techniques include: total intensity normalisation; intensity dependent normalisation; and variance stabilising normalisation. The aim of this paper is to discuss the impact of normalisation techniques for two-channel array technology on the process of identification of differentially expressed genes.     

INCREASE OF BRAIN TRYPTOPHAN BY ELECTROCONVULSIVE SHOCK IN RATS

—ECS markedly increased tryptophan and 5-hydroxyindoleacetic acid levels in brain. Brain serotonin and plasma tryptophan levels were unaffected.     

Effects of 5-Fluorouracil on Morphology,Cell Cycle,Proliferation, Apoptosis,Autophagy and ROS Production in Endothelial Cells and Cardiomyocytes

Antimetabolites are a class of effective anticancer drugs interfering in essential biochemical processes. 5-Fluorouracil (5-FU) and its prodrug Capecitabine are widely used in the treatment of several solid tumors (gastro-intestinal, gynecological, head and neck, breast carcinomas). Therapy with fluoropyrimidines is associated with a wide range of adverse effects, including diarrhea, dehydration, abdominal pain, nausea, stomatitis, and hand-foot syndrome. Among the 5-FU side effects, increasing attention is given to cardiovascular toxicities induced at different levels and intensities. Since the mechanisms related to 5-FU-induced cardiotoxicity are still unclear, we examined the effects of 5-FU on primary cell cultures of human cardiomyocytes and endothelial cells, which represent two key components of the cardiovascular system. We analyzed at the cellular and molecular level 5-FU effects on cell proliferation, cell cycle, survival and induction of apoptosis, in an experimental cardioncology approach. We observed autophagic features at the ultrastructural and molecular levels, in particular in 5-FU exposed cardiomyocytes. Reactive oxygen species (ROS) elevation characterized the endothelial response. These responses were prevented by a ROS scavenger. We found induction of a senescent phenotype on both cell types treated with 5-FU. In vivo, in a xenograft model of colon cancer, we showed that 5-FU treatment induced ultrastructural changes in the endothelium of various organs. Taken together, our data suggest that 5-FU can affect, both at the cellular and molecular levels, two key cell types of the cardiovascular system, potentially explaining some manifestations of 5-FU-induced cardiovascular toxicity.     

VDJSeq-Solver: In Silico V(D)J Recombination Detection Tool

In this paper we present VDJSeq-Solver, a methodology and tool to identify clonal lymphocyte populations from paired-end RNA Sequencing reads derived from the sequencing of mRNA neoplastic cells. The tool detects the main clone that characterises the tissue of interest by recognizing the most abundant V(D)J rearrangement among the existing ones in the sample under study. The exact sequence of the clone identified is capable of accounting for the modifications introduced by the enzymatic processes. The proposed tool overcomes limitations of currently available lymphocyte rearrangements recognition methods, working on a single sequence at a time, that are not applicable to high-throughput sequencing data. In this work, VDJSeq-Solver has been applied to correctly detect the main clone and identify its sequence on five Mantle Cell Lymphoma samples; then the tool has been tested on twelve Diffuse Large B-Cell Lymphoma samples. In order to comply with the privacy, ethics and intellectual property policies of the University Hospital and the University of Verona, data is available upon request to ti.rvinu.oeneta@itnetu.otroppus after signing a mandatory Materials Transfer Agreement. VDJSeq-Solver JAVA/Perl/Bash software implementation is free and available at http://eda.polito.it/VDJSeq-Solver/.     

Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2

The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG). For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.