The Metabolic Core and Catalytic Switches Are Fundamental Elements in the Self-Regulation of the Systemic Metabolic Structure of Cells

19 p.

Comparison of the Transcriptional Profiles of Melanocytes from Dark and Light Skinned Individuals under Basal Conditions and Following Ultraviolet-B Irradiation

We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype.

Activation of violaxanthin cycle in darkness is a common response to different abiotic stresses : a case study in Pelvetia canaliculata

Background: In the violaxanthin (V) cycle, V is de-epoxidized to zeaxanthin (Z) when strong light or light combined with other stressors lead to an overexcitation of photosystems. However, plants can also suffer stress in darkness and recent reports have shown that dehydration triggers V-de-epoxidation in the absence of light. In this study, we used the highly stress-tolerant brown alga Pelvetia canaliculata as a model organism, due to its lack of lutein and its non-photochemical quenching independent of the transthylakoidal-ΔpH, to study the triggering of the V-cycle in darkness induced by abiotic stressors. Results: We have shown that besides desiccation, other factors such as immersion, anoxia and high temperature also induced V-de-epoxidation in darkness. This process was reversible once the treatments had ceased (with the exception of heat, which caused lethal damage). Irrespective of the stressor applied, the resulting de-epoxidised xanthophylls correlated with a decrease in Fv/Fm, suggesting a common function in the down-regulation of photosynthetical efficiency. The implication of the redox-state of the plastoquinone-pool and of the differential activity of V-cycle enzymes on V-de-epoxidation in darkness was also examined. Current results suggest that both violaxanthin de-epoxidase (VDE) and zeaxanthin-epoxidase (ZE) have a basal constitutive activity even in darkness, being ZE inhibited under stress. This inhibition leads to Z accumulation. Conclusion: This study demonstrates that V-cycle activity is triggered by several abiotic stressors even when they occur in an absolute absence of light, leading to a decrease in Fv/Fm. This finding provides new insights into an understanding of the regulation mechanism of the V-cycle and of its ecophysiological roles.

The Oxidative State of Chylomicron Remnants Influences Their Modulation of Human Monocyte Activation

8 p.

A Model of Ischemia-Induced Neuroblast Activation in the Adult Subventricular Zone

12 p.

Role of Monoubiquitylation on the Control of IκBα Degradation and NF-κB Activity

9 p.

Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

11 p.

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

[ENG]Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs

New Oncogenic Drivers in Hepatocellular Carcinoma: Role of the RNA Binding Protein Hu antigen R

156 p. : graf.

NMDA modulates oligodendrocyte differentiation of subventricular zone cells through PKC activation

7 p.

Increase in the density of resting microglia precedes neuritic plaque formation and microglial activation in a transgenic model of Alzheimer's disease

The formation of cerebral senile plaques composed of amyloid beta peptide (A beta) is a fundamental feature of Alzheimer's disease (AD). Glial cells and more specifically microglia become reactive in the presence of A beta. In a triple transgenic model of AD (3 x Tg-AD), we found a significant increase in activated microglia at 12 (by 111%) and 18 (by 88%) months of age when compared with non-transgenic (non-Tg) controls. This microglial activation correlated with A beta plaque formation, and the activation in microglia was closely associated with A beta plaques and smaller A beta deposits. We also found a significant increase in the area density of resting microglia in 3 x Tg-AD animals both at plaque-free stage (at 9 months by 105%) and after the development of A plaques (at 12 months by 54% and at 18 months by 131%). Our results show for the first time that the increase in the density of resting microglia precedes both plaque formation and activation of microglia by extracellular A beta accumulation. We suggest that AD pathology triggers a complex microglial reaction: at the initial stages of the disease the number of resting microglia increases, as if in preparation for the ensuing activation in an attempt to fight the extracellular A beta load that is characteristic of the terminal stages of the disease. Cell Death and Disease (2010) 1, e1; doi:10.1038/cddis.2009.2; published online 14 January 2010

Analysing the contribution of ATM/ATR pathway activation in establishing the premature senescence of E2F1/E2F2-/- bone-marrow-derived macrophages

E2F1 and E2F2 transcription factors have an important role during the regulation of cell cycle. In experiments done with E2F1/E2F2 knockout mice, it has been described that bone-marrow-derived macrophages (BMDM) undergo an early rapid proliferation event related to DNA hyper-replication. As a consequence, DNA damage response (DDR) pathway is triggered and E2F1/E2F2 knockout macrophages enter premature senescence related to G2/M phase arrest. The exact mechanism trough which DNA hyper-replication leads to DDR in absence of E2F1 and E2F2 remains undiscovered. To determine whether the ATR/ATM pathway, the master regulator of G2/M checkpoint, might be the surveillance mechanism in order to regulate uncontrolled proliferation in the DKO model, we monitored and analysis biochemical properties of BMDM cultures in the presence of caffeine, a potent inhibitor of ATM/ATR activity. Our results show that the addition of caffeine abolishes premature senescence in DKO BMDM, stimulates γ-H2AX accumulation and decreases Mcm2 expression.

Mechanisms of E2F2-mediated transcriptional repression

In this work we wanted to study the mechanism of E2F2-mediated repression. Our hypothesis is that E2F2 activates the expression of one or more E2F members of the “repressor” subset of the family through the E2F motifs present in their promoters, and those repressor E2F(s) would subsequently repress the target promoters. To address this hypothesis, we focused on E2F7. E2F7 is a repressor that lacks the Rb binding domain, and associates with DNA through E2F binding sites (de Bruin et al., 2003). Furthermore, E2F7 itself is also regulated by E2F motifs on its own promoter, and it has been shown to repress DNA metabolism and replication genes in late S-phase (de Bruin et al., 2003; Westendorp et al., 2012). E2F7, together with E2F8 has been found to form heterodimers, being critical on cell proliferation and development, and both seem to have similar functions (Li et al., 2008). Preliminary results from Zubiaga’s group have indicated that E2F2 activates E2F7 transcription in U2OS cells, suggesting that E2F2’s repressor function could be mediated by E2F7. For this purpose, we focused on studying E2F7’s role on the target genes previously known to be repressed by E2F2: Chk1 and Mcm5. The specific aims for this work were the following: - Confirm that E2F2 induces E2F7 in HEK-293T cells - Assess whether E2F7 acts as a transcriptional repressor on E2F sites - Evaluate the role of E2F7 on E2F2-mediated transcriptional repression of Chk1 and Mcm5.

The promoter of cell growth- and RNA protection-associated SND1 gene is activated by endoplasmic reticulum stress in human hepatoma cells

Background: Staphyloccocal nuclease domain-containing protein 1 (SND1) is involved in the regulation of gene expression and RNA protection. While numerous studies have established that SND1 protein expression is modulated by cellular stresses associated with tumor growth, hypoxia, inflammation, heat- shock and oxidative conditions, little is known about the factors responsible for SND1 expression. Here, we have approached this question by analyzing the transcriptional response of human SND1 gene to pharmacological endoplasmic reticulum (ER) stress in liver cancer cells. Results: We provide first evidence that SND1 promoter activity is increased in human liver cancer cells upon exposure to thapsigargin or tunicamycin or by ectopic expression of ATF6, a crucial transcription factor in the unfolded protein response triggered by ER stress. Deletion analysis of the 5'-flanking region of SND1 promoter identified maximal activation in fragment (-934, +221), which contains most of the predicted ER stress response elements in proximal promoter. Quantitative real- time PCR revealed a near 3 fold increase in SND1 mRNA expression by either of the stress- inducers; whereas SND1 protein was maximally upregulated (3.4-fold) in cells exposed to tunicamycin, a protein glycosylation inhibitor. Conclusion: Promoter activity of the cell growth- and RNA-protection associated SND1 gene is up-regulated by ER stress in human hepatoma cells.

Specific Interaction with Cardiolipin Triggers Functional Activation of Dynamin-Related Protein 1

Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G), bundle signaling element (BSE) and stalk domains. However, instead of the lipid-interacting Pleckstrin Homology (PH) domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.