PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.     

Cyclic and pulse voltammetric study of dopamine at the interface between two immiscible electrolyte solutions

The detection of dopamine by differential pulse voltammetry (DPV) and square wave voltammetry (SWV) at the interface between two immiscible electrolyte solutions (ITIES) has been studied. Voltammetry at the liquid/liquid (water/1,2-dichloroethane) interface provides a simple method for overcoming the major problem associated with dopamine detection by voltammetry at solid electrodes: the co-existence of ascorbate at higher concentrations. Selectivity for dopamine was achieved by the use of dibenzo-18-crown-6 as an ionophore for the facilitated transfer voltammetry of protonated dopamine across the ITIES. Under these conditions, ascorbate is not transferred and hence does not interfere in the ion transfer current for dopamine. By use of DPV and SWV, the lowest concentration detectable can be lowered from ca. 0.1 mM (obtained with cyclic voltammetry) to 2 microM. Evaluation of the effect of some other physiologically important species (acetylcholine, sodium, potassium and ammonium ions) on the dopamine transfer voltammetry has been studied, indicating the need for improved ionophore designs in order to achieve practically useful selectivity.     

Spatio-Temporal Distribution of Freshwater Snail Species in Relation to Migration and Environmental Factors in an Irrigated Area from Morocco

Nine sites were sampled 19 times over 2 years in an irrigation system in Morocco in order to study species abundance in a snail community in relation to environmental parameters (including human activities) and migration (geographic distance) among sites. Each site was made of a sink and the first meters of the downstream canal. The snail community included four species (Bulinus truncatus, Lymnaea truncatula, Mercuria similis and Physa acuta). Strong spatial variation in species occurrence and abundance was detected which might be partly due to variation in water availability. However abundance in sinks and canals in which water availability differs were correlated. There was, as predicted, limited evidence in favor of isolation by distance which might be due to fast water current. Dispersal might therefore be an important factor structuring this community. On the other hand, the temporal variation was much more limited. This is consistent with the analysis of individual size distributions in B. truncatus, since no clear-cut cohorts were detected. The environmental parameters recorded (e.g. temperature, occurrence of macrophytes or cleaning of sinks) were extremely variable in time and space, except temperature. Analyzing their association with species through multidimensional methods indicated that P. acuta is ubiquitous and B. truncatus positively associated with macrophytes. These two species were associated in sinks. Less clear trends were detected for the two other species. Annual cleaning of sinks affected all species, but population recovery was fast in B. truncatus and P. acuta.     

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).     

Genotoxicity of inhaled nanosized TiO(2) in mice

In vitro studies have suggested that nanosized titanium dioxide (TiO(2)) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO(2) genotoxicity exist. Recently, nanosized TiO(2) administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO(2) administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO(2) due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO(2) is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO(2) (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m(3) could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO(2) anatase. Our inhalation exposure resulted in much lower systemic TiO(2) doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO(2) than BAL cells in the earlier intratracheal study.     

Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes

Micronuclei are formed from chromosomes and chromosomal fragments that lag behind in anaphase and are left outside daughter nuclei in telophase. They may also be derived from broken anaphase bridges. Nuclear buds, micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated similarly to micronuclei during nuclear division or in S-phase as a stage in the extrusion of extra DNA, possibly giving rise to micronuclei. To better understand these phenomena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelomeric DNA probes. Such information has not earlier been available for human primary cells. Surprisingly, there appears to be no previous data on the occurrence of telomeres in micronuclei (or buds) of normal human cells in general. Our results suggest that nuclear buds and micronuclei have partly different mechanistic origin. Interstitial DNA without centromere or telomere label was clearly more prevalent in nuclear buds (43%) than in micronuclei (13%). DNA with only telomere label or with both centromere and telomere label was more frequent in micronuclei (62% and 22%, respectively) than in nuclear buds (44% and 10%, respectively). Folate deprivation especially increased the frequency of nuclear buds and micronuclei harboring telomeric DNA and nuclear buds harboring interstitial DNA but also buds and micronuclei with both centromeric and telomeric DNA. According to the model we propose, that micronuclei in binucleate lymphocytes primarily derive from lagging chromosomes and terminal acentric fragments during mitosis. Most nuclear buds, however, are suggested to originate from interstitial or terminal acentric fragments, possibly representing nuclear membrane entrapment of DNA that has been left in cytoplasm after nuclear division or excess DNA that is being extruded from the nucleus.     

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.     

Evaluation of “alperujo” composting based on organic matter degradation, humification and compost quality

The main by-product generated by the Spanish olive oil industry, a wet solid lignocellulosic material called "alperujo" (AL), was evaluated as a composting substrate by using different aeration strategies and bulking agents. The experiments showed that composting performance was mainly influenced by the type of bulking agent added, and by the number of mechanical turnings. The bulking agents tested in this study were cotton waste, grape stalk, a fresh cow bedding and olive leaf; the latter showed the worse performance. Forced ventilation alone was revealed to work inadequately in most of the experiments. The composting process involved a substantial degradation of the organic substrate with average losses of 48.4, 28.6, 53.7 and 57.0% for total organic matter, lignin, cellulose and hemicellulose, respectively. Both organic matter biodegradation and humification were greatly influenced by the lignocellulosic nature of the starting material, which led to low organic matter and nitrogen loss rates and a progressive increase in more humified substances, as revealed by the end-values of the humification indices. The resulting composts were of good quality in terms of nutrient content, stabilised and non-phytotoxic organic matter and low heavy metal content. This demonstrates that composting technology can be used as an alternative treatment method to turn AL into compost that can be used as organic amendments or fertilisers for agricultural systems.