TOR and PKA signaling pathways converge on the protein kinase Rim15 to control entry into G0

The highly conserved Tor kinases (TOR) and the protein kinase A (PKA) pathway regulate cell proliferation in response to growth factors and/or nutrients. In Saccharomyces cerevisiae, loss of either TOR or PKA causes cells to arrest growth early in G(1) and to enter G(0) by mechanisms that are poorly understood. Here we demonstrate that the protein kinase Rim15 is required for entry into G(0) following inactivation of TOR and/or PKA. Induction of Rim15-dependent G(0) traits requires two discrete processes, i.e., nuclear accumulation of Rim15, which is negatively regulated both by a Sit4-independent TOR effector branch and the protein kinase B (PKB/Akt) homolog Sch9, and release from PKA-mediated inhibition of its protein kinase activity. Thus, Rim15 integrates signals from at least three nutrient-sensory kinases (TOR, PKA, and Sch9) to properly control entry into G(0), a key developmental process in eukaryotic cells.     

Drug delivery patterns for different stenting techniques in coronary bifurcations: a comparative computational study

The treatment of coronary bifurcation lesions represents a challenge for the interventional cardiologists due to the lower rate of procedural success and the higher risk of restenosis. The advent of drug-eluting stents (DES) has dramatically reduced restenosis and consequently the request for re-intervention. The aim of the present work is to provide further insight about the effectiveness of DES by means of a computational study that combines virtual stent implantation, fluid dynamics and drug release for different stenting protocols currently used in the treatment of a coronary artery bifurcation. An explicit dynamic finite element model is developed in order to obtain realistic configurations of the implanted devices used to perform fluid dynamics analysis by means of a previously developed finite element method coupling the blood flow and the intramural plasma filtration in rigid arteries. To efficiently model the drug release, a multiscale strategy is adopted, ranging from lumped parameter model accounting for drug release to fully 3-D models for drug transport to the artery. Differences in drug delivery to the artery are evaluated with respect to local drug dosage. This model allowed to compare alternative stenting configurations (namely the Provisional Side Branch, the Culotte and the Inverted Culotte techniques), thus suggesting guidelines in the treatment of coronary bifurcation lesions and addressing clinical issues such as the effectiveness of drug delivery to lesions in the side branch, as well as the influence of incomplete strut apposition and overlapping stents.     

Nucleo-cytoplasmic transport as a therapeutic target of cancer

Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer.     

Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and evaluation of their phosphodiesterase 4 inhibitory activity

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC₅₀ in the nanomolar range. The ability to inhibit TNF-α release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform.     

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 microM H(2)O(2). This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H(2)O(2) treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H(2)O(2) doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.