Reactive oxygen species derived from the mitochondrial respiratory chain are not responsible for the basal levels of oxidative base modifications observed in nuclear DNA of Mammalian cells

The mitochondrial electron transport chain (ETC) is the most important source of reactive oxygen species (ROS) in mammalian cells. To assess its relevance to the endogenous generation of oxidative DNA damage in the nucleus, we have compared the background (steady-state) levels of oxidative DNA base modifications sensitive to the repair glycosylase Fpg (mostly 7,8-dihydro-8-oxoguanine) in wild-type HeLa cells and HeLa rho0 cells. The latter are depleted of mitochondrial DNA and therefore are unable to produce ROS in the ETC. Although the levels of ROS measured by flow cytometry and redox-sensitive probes in rho0 cells were only 10-15% those of wild-type cells, steady-state levels of oxidative DNA base modifications were the same as in wild-type cells. Mitochondrial generation of ROS was then stimulated in HeLa wild-type cells using inhibitors interfering with the ETC. Although mitochondrial ROS production was raised up to 6-fold, none of the substances nor their combinations induced additional oxidative base modifications in the nuclear DNA. This was also true for glutathione-depleted cells. The results indicate that the contribution of mitochondria to the endogenously generated background levels of oxidative damage in the nuclear DNA is negligible.     

Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

Background

Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time.

Methods

Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis.

Results

We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed.

Conclusions

Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals.     

Conformational Recognition of an Intrinsically Disordered Protein

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.     

Interaction between dysfunctional connectivity at rest and heroin cues-induced brain responses in male abstinent heroin-dependent individuals

Background

The majority of previous heroin cue-reactivity functional magnetic resonance imaging (fMRI) studies focused on local function impairments, such as inhibitory control, decision-making and stress regulation. Our previous studies have demonstrated that these brain circuits also presented dysfunctional connectivity during the resting state. Yet few studies considered the relevance of resting state dysfunctional connectivity to task-related neural activity in the same chronic heroin user (CHU).

Methodology/Principal Findings

We employed the method of graph theory analysis, which detected the abnormality of brain regions and dysregulation of brain connections at rest between 16 male abstinent chronic heroin users (CHUs) and 16 non-drug users (NDUs). Using a cue-reactivity task, we assessed the relationship between drug-related cue-induced craving activity and the abnormal topological properties of the CHUs'' resting networks. Comparing NDUs'' brain activity to that of CHUs, the intensity of functional connectivity of the medial frontal gyrus (meFG) in patients'' resting state networks was prominently greater and positively correlated with the same region''s neural activity in the heroin-related task; decreased functional connectivity intensity of the anterior cingulate cortex (ACC) in CHUs at rest was associated with more drug-related cue-induced craving activities.

Conclusions

These results may indicate that there exist two brain systems interacting simultaneously in the heroin-addicted brain with regards to a cue-reactivity task. The current study may shed further light on the neural architecture that supports craving responses in heroin dependence.     

The key feature for early migratory processes: Dependence of adhesion,actin bundles,force generation and transmission on filopodia

Migration of cells is one of the most essential prerequisites to form higher organisms and depends on a strongly coordinated sequence of processes. Early migratory events include substrate sensing, adhesion formation, actin bundle assembly and force generation. While substrate sensing was ascribed to filopodia, all other processes were believed to depend mainly on lamellipodia of migrating cells. In this work we show for motile keratinocytes that all processes from substrate sensing to force generation strongly depend on filopodial focal complexes as well as on filopodial actin bundles. In a coordinated step by step process, filopodial focal complexes have to be tightly adhered to the substrate and to filopodial actin bundles to enlarge upon lamellipodial contact forming classical focal adhesions. Lamellipodial actin filaments attached to those focal adhesions originate from filopodia. Upon cell progression, the incorporation of filopodial actin bundles into the lamellipodium goes along with a complete change in actin cross-linker composition from filopodial fascin to lamellipodial α-actinin. α-Actinin in turn is replaced by myosin II and becomes incorporated directly behind the leading edge. Myosin II activity makes this class of actin bundles with their attached FAs the major source of force generation and transmission at the cell front. Furthermore, connection of FAs to force generating actin bundles leads to their stabilization and further enlargement. Consequently, adhesion sites formed independently of filopodia are not connected to detectable actin bundles, transmit weak forces to the substrate and disassemble within a few minutes without having been increased in size.Key words: filopodia, focal complexes, cell migration, focal adhesion, myosin II, force, actin flow, maturation     

Report of a comparative study of DNA damage and repair assays in primary rat hepatocytes with five coded chemicals

We report the results of a collaborative study for the detection of chemical-induced DNA damage in primary cultures of rat hepatocytes. The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory). Interlaboratory standardization was omitted in order to prove the agreement of the assays under routine conditions. Five coded chemicals were tested. For 4 chemicals (2-acetylaminofluorene, thiourea, glycerine and potassium chloride) the UDS data were consistent in all laboratories, thus indicating a high consensus of the test systems applied in the different laboratories. Those 3 chemicals that were not expected to elicit genotoxic activity (thiourea, glycerine, and potassium chloride) yielded negative results in all laboratories. 2-Acetylaminofluorene, a known DNA-damaging agent in hepatocytes, gave strongly positive responses in all laboratories. In contrast, N-nitrosodiphenylamine led to equivocal responses.     

Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.     

Intergeneric hybridization between Psathyrostachys and Hordeum

A total of 38 intergeneric combinations with Hordeum and Psathyrostachys was attempted. Generally the seed set was high, and 23 new intergeneric hybrid combinations were synthesized by the aid of embryo culture technique. All combinations showed complete sterility. The meiotic pairing behaviour in 8 combinations is reported. On the basis of these data it is clear that the N genome in Psathyrostachys is distinct and shows no genomic relationship, neither with the "X" and I nor with the H genomes in Hordeum.