Isoeugenodilol inhibits smooth muscle cell proliferation and neointimal thickening after balloon injury via inactivation of ERK1/2 pathway

The purpose of this study was to determine the efficacy and the possible mechanism of action of the synthesized drug isoeugenodilol (a new third-generation β-adrenoceptor blocker) on the growth factor-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a rat carotid arterial balloon injury model. Isoeugenodilol significantly inhibited 10% FBS, 20 ng/ml PDGF-BB, and 20 ng/ml vascular endothelial growth factor (VEGF)-induced proliferation. In accordance with these findings, isoeugenodilol revealed blocking of the FBS-inducible progression through the G₀/G₁ to the S phase of the cell cycle in synchronized cells. Neointimal formation, measured 14 days after injury, was reduced by the oral administration of isoeugenodilol (10 mg/kg/day). In an in vitro assay, isoeugenodilol inhibited the migration of VSMCs stimulated by PDGF-BB. These findings indicate that isoeugenodilol shows an inhibitory potency on neointimal formation due to inhibition of both migration and proliferation of VSMCs. In addition, isoeugenodilol in concentration-dependent manner decreased the levels of phosphorylated ERK1/2 in both VSMCs and balloon-injured carotid arteries. The levels of phosphorylated MEK1/2 and Pyk2 as well as intracellular Ca²⁺ and reactive oxygen species (ROS) were in concentration-dependent manner reduced by isoeugenodilol. Taken together, these results indicate that isoeugenodilol may suppress mitogen-stimulated proliferation and migration partially through inhibiting cellular ROS and calcium, and hence, through activation of the Pyk2-ERK1/2 signal pathway. This suggests that isoeugenodilol has potential for the prevention of atherosclerosis and restenosis.     

The gradient of Gurken, a long-range morphogen, is directly regulated by Cbl-mediated endocytosis

The asymmetric localization of gurken mRNA and post-translational sorting mechanisms are responsible for the polar distribution of Gurken protein in Drosophila. However, endocytosis of Egfr, the receptor for Gurken in the follicle cells, also plays a role in shaping the extracellular gradient of the Gurken morphogen. Previously, we have found that mutation in the Cbl gene caused elevated Egfr signaling along the dorsoventral axis, and resulted in dorsalization phenotypes in embryos and egg shells. Here, we report that overexpression of the Cbl long isoform significantly changed Gurken distribution. Using an HRP-Gurken fusion protein, we demonstrate that internalization of the Gurken-Egfr complex depends on the activity of Cbl. Increased levels of CblL promote the internalization of this complex, leading to the reduction of free ligands. The Gurken-Egfr complex trafficks through the Rab5/Rab7 associated endocytic pathway to the lysosomal degradation compartment for signaling termination. We observe endocytic Gurken not only in the dorsal but also in the ventral follicle cells, which is, to our knowledge, the first visualization of Gurken on the ventral side of egg chambers. Our results show that Gurken travels towards the lateral/posterior of the egg chamber in the absence of Cbl, suggesting that Cbl actively regulates Gurken distribution through promoting endocytosis and subsequent degradation.     

A novel tubulin polymerization inhibitor,MPT0B206, downregulates Bcr-Abl expression and induces apoptosis in imatinib-sensitive and imatinib-resistant CML cells

Imatinib, a Bcr-Abl-specific inhibitor, is effective for treating chronic myeloid leukemia (CML), but drug resistance has emerged for this disease. In this study, we synthesized a novel tubulin polymerization inhibitor, MPT0B206 (N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-formamide), and demonstrated its apoptotic effect and mechanism in imatinib-sensitive K562 and imatinib-resistant K562R CML cells. Western blotting and immunofluorescence microscopy showed that MPT0B206 induced microtubule depolymerization in K562 and K562R cells. MPT0B206 inhibited the growth of these cells in a concentration- and time-dependent manner. It did not affect the viability of normal human umbilical vein endothelial cells. MPT0B206 induced G2/M cell cycle arrest and the appearance of the mitotic marker MPM-2 in K562 and K562R cells, which is associated with the upregulation of cyclin B1 and the dephosphorylation of Cdc2. Treatment of K562 and K562R cells with MPT0B206 induced apoptosis and reduced the protein levels of procaspase-9 and procaspase-3 and increased caspase-3 activity and PARP cleavage. MPT0B206 also reduced the levels of the antiapoptotic proteins Mcl-1 and Bcl-2 and increased the level of the apoptotic protein Bax. Additional experiments showed that MPT0B206 markedly downregulated Bcr-Abl mRNA expression and total and phosphorylated Bcr-Abl protein levels and inhibited the phosphorylation of its downstream proteins STAT5, MAPK, and AKT, and the protein level of c-Myc in K562 and K562R cells. Furthermore, MPT0B206 triggered viability reduction and apoptosis in CML cells carrying T315I-mutated Bcr-Abl. Together, these results suggest that MPT0B206 is a promising alternative for treating imatinib-resistant CML.     

Chemiluminescence analysis of antioxidant capacity for serum albumin isolated from healthy or uremic volunteers

Regular hemodialysis treatment induces an elevation in oxidative stress in patients with end‐stage renal failure, resulting in oxidative damage of the most abundant serum protein, albumin. Oxidation of serum albumin causes depletion of albumin reactive thiols, leading to oxidative modification of serum albumin. The aim of this study was to screen the antioxidant capacity of albumins isolated from uremic patients (HD‐ALB) or healthy volunteers (N‐ALB). From high‐performance liquid chromatography spectra, we observed that one uremic solute binds to HD‐ALB via the formation of disulfide bonds between HD‐ALB and the uremic solute. Furthermore, we found using chemiluminescent analysis that the antioxidant capacities for N‐ALB to scavenge reactive oxygen species including singlet oxygen, hypochlorite and hydrogen peroxide were higher than HD‐ALB. Our results suggest that protein‐bound uremic solute binds to albumin via formation of disulfide bonds, resulting in the depletion of albumin reactive thiols. The depletion of albumin reactive thiols leads to a reduced antioxidant capacity of HD‐ALB, implying postmodification of albumin. This situation may reduce the antioxidant capacity of albumin and increase oxidative stress, resulting in increase in complications related to oxidative damage in uremic patients. Copyright © 2016 John Wiley & Sons, Ltd.     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.     

Intra-aneurysmal flow with helix and mesh stent placement across side-wall aneurysm pore of a straight parent vessel

Pulsatile flow fields in a cerebrovascular side-wall aneurysm model with a wide ostium after stenting are presented in terms of particle tracking velocimetry measurements and flow visualization. Among the stent parameters the shape, helix versus mesh, was selected to study its effect on the changes of intraaneurysmal hemodynamics for the reference of minimally invasive endovascular aneurysm treatment. The blocking ratio of the stents was fixed at 30%. The Womersley number was 3.9 and the mean, peak, and minimal Reynolds numbers based on the bulk average velocity and diameter of the parent vessel were 600, 850, and 300, respectively. Four consecutive flow-rate phases were selected to characterize the intra-aneurysmal flow. The results are characterized in terms of velocity vector field, regional average velocity, and intra-aneurysmal vorticity/circulation/wall shear stress. It is found that the hemodynamic features inside the aneurysm alter markedly with the shape of the stent and the size of the orifice. Both stents investigated induce favorable changes in the intra-aneurysmal flow stasis as well as direction and undulation of wall shear stresses. A comparison of the results of the helix to mesh stent shows that the former is more favorable for endovascular treatment.     

Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations

Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.     

Distribution of interstitial retinol-binding protein (IRBP) in the vertebrates

Immunoblots of interphotoreceptor matrix preparations from 20 species belonging to six vertebrate classes were probed with antibodies against bovine interstitial retinol-binding protein (b-IRBP). Each preparation displayed an immunoreactive protein band. In the Osteichthyes, the apparent Mr of this band was 67,600 +/- 2,700 (mean +/- SD, n = 8). In two of the Osteichthyes, the band was resolved into a closely spaced doublet. Including previously published data for five mammals and one amphibian, species from the other classes (Chondrichthyes, one species; Amphibia, four species; Reptilia, one species; Aves, one species; Mammalia, nine species) had IRBPs with Mr that averaged 2.0 times that of the Osteichthyes, namely 134,200 +/- 8,600 (mean +/- SD, n = 17). Frog IRBP was very similar to mammalian IRBP in terms of its immunohistochemical distribution (determined with rabbit anti-frog IRBP antibodies), its molecular weight (sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography), retinol- and concanavalin A-binding ability, and because it was synthesized and secreted in vitro by the isolated retina but not by the pigmented layers of eye. Goldfish IRBP apparently binds exogenous (3H)-retinol but does not bind concanavalin A and has about half the Mr of frog IRBP. The occurrence of IRBP-like proteins cross-reacting with anti b-IRBP antibodies in the interphotoreceptor matrix of all six major vertebrate classes is consistent with the hypothesis that IRBP is an important element in the vertebrate visual cycle.