Docosahexaenoic acid supplementation induces dose and time dependent oxidative changes in C6 glioma cells

In view of the promising use of n-3 polyunsaturated fatty acids (PUFAs) in the prevention and treatment of neurological diseases, it is necessary to ascertain the lack of detrimental oxidative effects. We evaluated short- and long-term effects of 25, 50 and 75 μM docosahexaenoic acid (DHA) supplementation on the oxidative status of C6 glial cells. DHA was incorporated into cells dose and time dependently without any cytotoxic effect. Reactive oxygen species (ROS) level was related to DHA dose and supplementation time. At the lowest dose no significant increase in ROS values was observed at hour 24. Low doses of DHA strengthened the cellular antioxidant defence system as highlighted by a raise in both GPX and catalase activity, and the decreased levels of lipid peroxidation. This effect was pronounced at 24 h of supplementation, almost disappeared at hour 48, while after 72 h an opposite effect was observed: lipid peroxidation increased concomitantly with DHA doses. Therefore, the final effect of DHA on cellular redox status is dependent on dose and time supplementation.     

A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.     

Viral Infection of Human Lung Macrophages Increases PDL1 Expression via IFNβ

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.     

A model for the incorporation of metal from the copper chaperone CCS into Cu,Zn superoxide dismutase.

BACKGROUND: Recent studies have identified the human copper chaperone CCS as the presumed factor responsible for copper incorporation into superoxide dismutase (SOD). A lack of knowledge of the chaperone's three-dimensional structure has prevented understanding of how the copper might be transferred. RESULTS: The three-dimensional structure of CCS was homology modelled using the periplasmic protein from the bacterial mercury-detoxification system and the structure of one subunit of the human SOD dimeric enzyme as templates. On the basis of the three-dimensional model, a mechanism for the transfer of copper from CCS to SOD is proposed that accounts for electrostatic acceptor recognition, copper storage and copper-transfer properties. CONCLUSIONS: The proposed model identifies a path for copper transfer based on the presence of different metal sites characterized by sulphur ligands. Such a model permits the development of strategies able to interfere with copper incorporation in SOD, providing a possible way to prevent or arrest degeneration in the fatal motor neuron disorder amyotrophic lateral sclerosis.     

The yeast DNA polymerase-primase complex: Genes and proteins

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (α) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase α. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein—protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.     

Chromatographic separation of a thymus fraction active on lipidic metabolism

Three protein fractions have been obtained from a thymus homogenate of young calf, by a multistep sequence of procedures.Every fraction was administered by intraperitoneal means to a distinct group of rats previously fed, for 21 days, on a diet with high content of dietary fat and cholesterol.The serum values of cholesterol and triglycerids in these animals show a considerable decrease, if compared with the data obtained from control animals, especially with the third chromatographic fraction.     

Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.