Emerging and re-emerging viral infections in Europe

Emerging viral infections are becoming a serious problem in Europe in the recent years. This is particularly true for severe acute respiratory syndrome (SARS), West Nile virus (WNV) disease, Toscana virus (TOSV) disease, and potentially for avian influenza virus (H5N1). In contrast, emergence or re-emergence of severe viral infections, including tick borne encephalitis virus, and viral haemorrhagic fever caused by Hantavirus and dengue virus have been frequently reported in several European countries. Laboratory diagnosis of these viral infections based on viral isolation or detection by immune electron microscopy, immunoassay and polymerase chain reaction (PCR) has dramatically improved in the recent years, and SARS represents a good example of a diagnostic approach to emerging viral infections. Finally, old and new promising agents are in the pipeline of pharmaceutical companies to treat emerging viral infections. However only prevention based on large epidemiological studies, and research and development of new vaccines may be able to control and eventually eradicate these deadly viral infections.     

Reactive oxygen species plasmatic levels in ischemic stroke

Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion, and the antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. The aim of this study was to investigate the status of oxidative stress, NO and ONOO⁻ levels in patients with atherothrombotic and lacunar acute ischemic stroke and iNOS, eNOS and nitrotyrosine expression in the same patients. Plasma ONOO⁻ levels were significantly higher in patients than in controls while NO decreases in patients in respect to controls. Densitometric analysis of bands indicated that iNOS and N-Tyr protein levels were significantly higher in patients in respect to controls. This study has highlighted a significant NO decrease in our patients compared with controls and this is most probably due to the increased expression of inducible NO synthase by the effect of thrombotic attack. In fact, the constitutive NO isoforms, which produce small amounts of NO, are beneficial, while activation of the inducible isoform of NO, which produces much more NO, causes injury, being its toxicity greatly enhanced by generation of peroxynitrite. The significant ONOO⁻ increase observed in our patients, compared to controls, is most probably due to reaction of NO with O₂^·−. These findings suggest that free radical production and oxidative stress in ischemic stroke might have a major role in the pathogenesis of ischemic brain injury. Peroxynitrite might be the main marker of brain damage and neurological impairment in acute ischemic stroke.     

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.     

Role of D-Limonene in Autophagy Induced by Bergamot Essential Oil in SH-SY5Y Neuroblastoma Cells

Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005–0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70^S6K (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125–750 µM) and linalyl acetate (62.5–375 µM), were individually tested at concentrations comparable to those found in 0.005–0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by d-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by d-limonene.     

Early senescence in heterozygous ABCA1 mutation skin fibroblasts: A gene dosage effect beyond HDL deficiency?

Purpose

Homozygous ABCA1 gene mutation causes Tangier disease (TD). The effects reported in heterozygous state regard plasma HDL, cell cholesterol efflux and coronary artery disease. We investigated whether in vitro replicative skin fibroblast senescence shown in TD proband (Hom), his father (Het), and in a healthy control might be induced in a “gene-dosage way”.

Methods

Senescence was evaluated by staining test for β-Galactosidase and telomere length (TL) on fibroblast DNA at different replicative stages. ABCG1 and LDLR (low density lipoprotein receptor) gene expression was also evaluated.

Results

Hom cells showed early senescent morphology and reduced growth at all passages in vitro. The cell positive percentage for β-Galactosidase test was highly increased in Hom compared to Het cells at late replicative status (66.1% vs 41.3% respectively). TL was significantly shorter at high stage either in Hom (p < 0.0001) or in Het (p < 0.005). At early replication cycles ABCG1 gene expression was about 3-fold higher in Hom compared to Het cells (0.44 vs 0.14 arbitrary unit).

Conclusions

ABCA1 gene mutation may have “gene-dosage way” effect on in vitro fibroblast senescence. Furthermore, increased ABCG1 and LDLR gene expression could highlight a role of ABCA1 on cytoskeleton regulation associated to cell cholesterol metabolism.