Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications

Cellular responses to high glucose are numerous and varied but ultimately result in functional changes and, often, cell death. High glucose induces oxidative and nitrosative stress in many cell types causing the generation of species such as superoxide, nitric oxide and peroxynitrite and their derivatives. The role of these species in high glucose-mediated apoptotic cell death is relevant to the complications of diabetes such as neuropathy, nephropathy and cardiovascular disease. High glucose causes activation of several proteins involved in apoptotic cell death, including members of the caspase and Bcl-2 families. These events and the relationship between high glucose-induced oxidative stress and apoptosis are discussed here with reference to additional regulators of apoptosis such as the mitogen-activated protein kinases (MAPKs) and cell-cycle regulators.     

Multiple proteins and phosphorylations regulate Saccharomycescerevisiae Cdc24p localization

Targeting of Saccharomyces cerevisiae Cdc24p to polarized growth sites is essential for its function. Localization of GFP-tagged Cdc24 proteins or fragments was assayed in deletion mutants of Cdc24p-interacting proteins. The boi2Δ, ent2Δ, and hua1Δ mutants showed localization defects. The tos2Δ skg6Δ double mutant displayed aberrant pre-anaphase localization to the mother-bud neck region. The same aberrant pattern was seen when potential phosphorylation sites Ser697, Thr704, and Tyr200 were mutated. The S697A mutation also resulted in phosphorylation defects in vivo. These data support roles for Boi2p, Ent2p, Hua1p, Tos2p, and for Cdc24p phosphorylation in targeting Cdc24p to growth sites.     

Holliday junction resolution: Regulation in space and time

Holliday junctions (HJs) can be formed between sister chromatids or homologous chromosomes during the recombinational repair of DNA lesions. A variety of pathways act upon HJs to remove them from DNA, in events that are critical for appropriate chromosome segregation. Despite the identification and characterization of multiple enzymes involved in HJ processing, the cellular mechanisms that regulate and implement pathway usage have only just started to be delineated. A conserved network of core cell-cycle kinases and phosphatases modulate HJ metabolism by exerting spatial and temporal control over the activities of two structure-selective nucleases: yeast Mus81-Mms4 (human MUS81-EME1) and Yen1 (human GEN1). These regulatory cycles operate to establish the sequential activation of HJ processing enzymes, implementing a hierarchy in pathway usage that ensure the elimination of chromosomal interactions which would otherwise interfere with chromosome segregation. Mus81-Mms4/EME1 and Yen1/GEN1 emerge to define a special class of enzymes, evolved to satisfy the cellular need of safeguarding the completion of DNA repair when on the verge of chromosome segregation.     

New cerebellar phenotypes in YAC transgenic mouse in vivo library of human Down syndrome critical region-1

Down syndrome (DS) is the most frequent genetic cause of mental retardation (MR) associated with neurological alterations. To allow a genetic dissection of DS phenotype, we studied eight transgenic mouse lines carrying YACs containing human DNA fragments covering DS critical region (DCR-1), as an in vivo library. Herein, we found an increased brain size in the 152F7-mice containing DYRK1A gene. We also identified a new cerebellar alteration in two independent lines carrying 230E8-YAC. These mice showed significant elongation of the cerebellar antero-posterior axis (p < 0.001), determined by increased length of rostral folia of the vermis (lobule II-V, p < 0.0001; lobule VI, p < 0.001). In addition, we identified a major neurological defect in culmen and declivus lobules in the 230E8-mice. We analyzed P30, P12, and P9 stages and detected high significant increased lengths of anterior lobules (II-VI) of 230E8-mice at P30 and P12 (lobule II-V, p < 0.0001; lobule VI, p < 0.05), but not at P9, indicating that this new phenotype appears between P9 and P12. Interestingly, 230E8-mice also present increased cortical cell density and mild learning defects. 230E8-YAC contains seven genes, some of which could be potentially responsible for this phenotype. Between them, we proposed DOPEY2 as potential candidate gene for these cerebellar alterations considering its high expression in the brain and that its homologous genes in yeast, Caenorhabditis elegans and Drosophila are involved in morphogenesis, suggesting a conserved role of DOPEY2 as a patterning gene.     

New benzothieno[3,2-d]-1,2,3-triazines with antiproliferative activity: Synthesis,spectroscopic studies,and biological activity

New benzothieno[3,2-d]-1,2,3-triazines, together with precursors triazenylbenzo[b]thiophenes, were designed, synthesized and screened as anticancer agents. The structural features of these compounds prompted us to investigate their DNA binding capability through UV–vis absorption titrations, circular dichroism, and viscometry, pointing out the occurrence of groove-binding. The derivative 3-(4-methoxy-phenyl)benzothieno[3,2-d]-1,2,3-triazin-4(3H)-one showed the highest antiproliferative effect against HeLa cells and was also tested in cell cycle perturbation experiments. The obtained results assessed for the first time the anticancer activity of benzothieno[3,2-d]-1,2,3-triazine nucleus, and we related it to its DNA-binding properties.     

Hexane/ethanol extract of Glycyrrhiza uralensis and its active compound isoangustone A induce G1 cycle arrest in DU145 human prostate and 4T1 murine mammary cancer cells

Although licorice is known to exert anticarcinogenic effects, it contains large quantities of glycyrrhizin (GL), which causes severe hypertension. We have previously demonstrated that the hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) contains no detectable GL and suppresses doxorubicin-induced apoptosis in H9c2 rat cardiac myoblasts. The principal objective of this study was to determine whether and by what mechanism HEGU and its active component, isoangustone A, inhibit cell-cycle progression in DU145 human prostate and 4T1 mouse breast cancer cells. HEGU and isoangustone A dose-dependently decreased DNA synthesis and induced G1 phase arrest in both DU145 and 4T1 cells. HEGU and isoangustone A reduced the levels of CDK2 and CDK4 as well as cyclin A and cyclin D1 proteins, and also induced a decrease in CDK2 activity. The addition of HEGU to drinking water significantly suppressed the orthotopic growth of 4T1 allografts and the expression of the proliferating nuclear cell antigen, CDK2 and CDK4 proteins in the tumor tissues. These results demonstrate the potential of HEGU containing isoangustone A as an antitumor agent.     

Value of the cell cycle arrest biomarkers in the diagnosis of pregnancy-related acute kidney injury

Background: Pregnancy-related acute kidney injury (PRAKI) is still a common serious problem in developing countries. Insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor metalloproteinases-2 (TIMP-2) can identify critically ill patients at risk for the development of severe AKI. Aim: To identify main causes and timing of PRAKI and to study the G1 cell cycle arrest biomarkers in cases diagnosed with (PRAKI) as a diagnostic tool. Methods: 80 pregnant women diagnosed with PRAKI were recruited from a single hospital as well as 30 age-matched pregnant women with normal pregnancy participated in the present study. A urine specimen was collected from all study participants with established AKI within 24 h of ICU admission to measure [TIMP-2]*[IGFBP7]. Results: The incidence of PRAKI was 1.1%. The most common cause of PRAKI is pre-eclampsia/eclampsia spectrum (61%). Most of the cases occur in the third trimester (60%) and postpartum period (23%). At a cutoff 0.33 ng/ml, the estimated sensitivity and specificity of urinary [TIMP-2]*[IGFBP7] in predicting PRAKI is 100% (95% CI) with NPV and PPV are 100%. Conclusion: Urinary [TIMP-2]*[IGFBP7] serves as a sensitive and specific biomarker in the diagnosis of PRAKI.     

Oligomeric Aip2p/Dld2p modifies the protein conformation of both properly folded and misfolded substrates in vitro

Oligomeric actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377, Biochem. Biophys. Res. Commun. 295 (2002) 910] exhibits the unique grapple-like structure with an ATP-dependent opening [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which is required for the F-actin conformation modifying activity in vitro and in vivo [Biochem. Biophys. Res. Commun. 319 (2004) 78]. To further investigate the molecular nature of oligomeric Aip2p/Dld2p, the substrate specificity of its binding and protein conformation modifying activity was examined. In the presence of 1mM ATP or AMP-PNP, oligomeric Aip2p/Dld2p bound to all substrates so far examined, and modified the conformation of actin, DNase I, the mature form of invertase, prepro-alpha-factor, pro-alpha-factor, and mitochondrial superoxide dismutase, as determined by the trypsin susceptibility assay. Of note, the activity could modify even the conformation of pathogenic highly aggregated polypeptides, such as recombinant prion protein in beta-sheet form, alpha-synuclein, and amyloid beta (1-42) in the presence of ATP. The in vivo protein conformation modifying activity, however, depends on the growth stage; the most significant substrate modification activity was observed in yeast cells at the log phase, suggesting the presence of a cofactor/s in yeast cells, where F-actin is supposed to be a major target in vivo. These data further support our previous notion that the oligomeric Aip2p/Dld2p may belong to an unusual class of molecular chaperones [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which can target both properly folded and misfolded proteins in an ATP-dependent manner in vitro.