SMAD inhibition attenuates epithelial to mesenchymal transition by primary keratinocytes in vitro

Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype and contribute directly to fibrotic disease. Recent studies support a role for EMT in cutaneous fibrotic diseases including scleroderma and hypertrophic scarring, though there is limited data on the cytokines and signalling mechanisms regulating cutaneous EMT. We investigated the ability of TGF-β and TNF-α, both over-expressed in cutaneous scleroderma and central mediators of EMT in other epithelial cell types, to induce EMT in primary keratinocytes and studied the signalling mechanisms regulating this process. TGF-β induced EMT in normal human epidermal keratinocytes (NHEK cells) and this process was enhanced by TNF-α. EMT was characterised by changes in morphology, proteome (down-regulation of E-cadherin and Zo-1, and up-regulation of vimentin and fibronectin), MMP secretion and COL1α1 mRNA expression. TGF-β and TNF-α in combination activated SMAD and p38 signalling in NHEK cells. P38 inhibition with SB203580 partially attenuated EMT, whereas SMAD inhibition using SB431542 significantly inhibited EMT and also reversed established EMT. These data highlight the retained plasticity of adult keratinocytes and support further studies of EMT in clinically relevant in vivo models of cutaneous fibrosis, and investigation of SMAD inhibition as a potential therapeutic intervention. This article is protected by copyright. All rights reserved.

The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.

Inhibition of ribosome biogenesis induces apoptosis in p53-/- cells

The nucleolus is an important region of the nucleus that acts as the main site of rRNA transcription by RNA polymerase I (Pol-I), and one of the most prominent morphological markers of proliferative and invasive cancers is increased nucleolar size. Increases in Pol-I transcription leads to increased levels of ribosome biogenesis that are able to fuel rapid cell growth and proliferation due to increased ribosome numbers. Therefore Pol-I transcription seems a viable target for the development of anticancer therapeutics as abrogation of Pol-I transcription leads to cessation of cell growth and eventual cell death. We have confirmed that ellipticine compound, 9-Hydroxyellipticine (9HE), is an efficient inhibitor of Pol-I transcription in vitro and in p53+/+ and -/- cell lines in vivo. Short-term treatments (≤24 h) with micromolar concentrations of 9HE leads to decreased cell viability and proliferation, and leads to activation of caspases 3, 8 and 9, indicating that both intrinsic and extrinsic cell death mechanisms are activated upon Pol-I inhibition. Reactive oxygen species levels were also studied following short and long term treatments with 9HE and there was a 2/3-fold increase in cellular ROS levels. Long-term 9HE treatment (≥24 h) leads to cellular senescence as indicated by increased cellular morphology and senescence associated β-galactosidase staining, and this senescence is not accompanied by induction of autophagy. Following 24 h treatment there is also accumulation of cells in the G0/G1 phase of the cell cycle and qPCR analysis of cell cycle regulators shows down-regulation of G1/S transition associated cyclins. These data show that Pol-I transcription is a viable target for the development of novel chemotherapeutics, although further delineation of the cell death pathways remains. To further elucidate the mechanism, the role of mitochondrial death signals will be investigated by determination of cytochrome c release and regulation of Bcl2 family member proteins.

Étude des impacts écologiques du dynamisme spatio-temporel des habitats naturels sur la faune menacée du Complexe Zones Humides Mahavavy-Kinkony, Madagascar.

This study of the Mahavavy-Kinkony Wetland Complex (MKWC) assesses the impacts of habitat change on the resident globally threatened fauna. Located in Boeny Region, northwest Madagascar, the Complex encompasses a range of habitats including freshwater lakes, rivers, marshes, mangrove forests, and deciduous forest. Spatial modelling and analysis tools were used to (i) identify the important habitats for selected, threatened fauna, (ii) assess their change from 1950 to 2005, (iii) detect the causes of change, (iv) simulate changes to 2050 and (v) evaluate the impacts of change. The approach for prioritising potential habitats for threatened species used ecological science techniques assisted by the decision support software Marxan. Nineteen species were analysed: nine birds, three primates, three fish, three bats and one reptile. Based on knowledge of local land use, supervised classification of Landsat images from 2005 was used to classify the land use of the Complex. Simulations of land use change to 2050 were carried out based on the Land Change Modeler module in Idrisi Andes with the neural network algorithm. Changes in land use at site level have occurred over time but they are not significant. However, reductions in the extent of reed marshes at Lake Kinkony and forests at Tsiombikibo and Marofandroboka directly threaten the species that depend on these habitats. Long term change monitoring is recommended for the Mahavavy Delta, in order to evaluate the predictions through time. The future change of Andohaomby forest is of great concern and conservation actions are recommended as a high priority. Abnormal physicochemical properties were detected in lake Kinkony due to erosion of the four watersheds to the south, therefore an anti-erosion management plan is required for these watersheds. Among the species of global conservation concern, Sakalava rail (Amaurornis olivieri), Crowned sifaka (Propithecus coronatus) and dambabe (Paretroplus dambabe) are estimated the most affected, but at the site level Decken’s sifaka (Propithecus deckeni), kotsovato (Paretroplus kieneri) and Madagascan big-headed turtle (Erymnochelys madagascariensis) are also threatened. Local enforcement of national legislation on hunting means that MKWC is among the sites where the flying fox (Pteropus rufus) and Madagascan rousette (Rousettus madagascariensis) are well protected. Ecological restoration, ecological research and actions to reduce anthropogenic pressures are recommended.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Stabilisation of MAD2 expression through inhibition of miR-433; A novel mechanism for restoring chemo-responsiveness in ovarian tumours

Epithelial ovarian carcinoma (EOC) is characterised by late diagnosis and recurrences, both of which contribute to the high morbidity and mortality of this cancer. Unfortunately, EOC has an innate susceptibility to become chemo-resistant. Specifically, up to 30% of patients may not respond to current standard chemotherapy (paclitaxel and platinum in combination) and of those who have an initial response, some patients relapse within a few months. Therefore, in order to improve patient outcome it is crucial to establish what factors influence a patients' individualised response to chemotherapy. We analysed MAD2 protein expression in a patient cohort of 35 ovarian tumours and a panel of 5 ovarian cancer cell lines. We have demonstrated that low nuclear MAD2 expression intensity was significantly associated with chemo-resistant ovarian tumours (p=0.0136). Moreover, in vitro studies of the 5 ovarian cancer cell lines revealed that reduced MAD2 expression was associated with paclitaxel resistance. In silico analysis identified a putative miR-433 binding domain in the MAD2 3′UTR and expression profiling of miR-433 in the ovarian cancer cell lines showed that low MAD2 protein expression was associated with high miR-433 levels. In vitro over-expression of miR-433 attenuated MAD2 protein expression with a concomitant increase in cellular resistance to paclitaxel. Over-expression of a morpholino oligonucleotide that blocks miR-433 binding to MAD2 3′UTR stabilised MAD2 protein expression and protects from miR-433 induced degradation. Furthermore, miR-433 expression analysis in 35 ovarian tumour samples revealed that high miR-433 expression was associated with advanced stage presentations (p=0.0236). In conclusion, ovarian tumours that display low nuclear MAD2 intensity are chemo-resistant and stabilising MAD2 expression by antagonising miR-433 activity is a potential mechanism for restoring chemo-responsiveness in these tumours.