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Exogenous ubiquitin (UB) plays a protective role in [beta]-adrenergic receptor-stimulated and ischemia/reperfusion (I/R)-induced myocardial remodeling. Here, we report that UB treatment inhibits hypoxia/reoxygenation (H/R)-induced apoptosis in adult rat ventricular myocytes (ARVMs). The activation of Akt was elevated, whereas the activation of glycogen synthase kinase-3[beta] was reduced in UB-treated cells post-H/R. The level of oxidative stress was lower, whereas the number of ARVMs with polarized mitochondria was significantly greater in the UB-treated samples. ARVMs express CXCR4 with majority of CXCR4 localized in the membrane fraction. CXCR4 antagonism using AMD3100, and siRNA-mediated knockdown of CXCR4 negated the protective effects of UB. Two mutated UB proteins (unable to bind CXCR4) had no effect on H/R-induced apoptosis, activation of Akt and GSK-3[beta], or oxidative stress. UB treatment enhanced mitochondrial biogenesis, and inhibition of mitochondrial fission using mdivi1 inhibited H/R-induced apoptosis. Ex vivo, UB treatment significantly decreased infarct size and improved functional recovery of the heart following global I/R. Activation of caspase-9, a key player of the mitochondrial death pathway, was significantly lower in UB-treated hearts post-I/R. UB, most likely acting via CXCR4, plays a protective role in H/R-induced myocyte apoptosis and myocardial I/R injury via modulation of mitochondrial homeostasis and the mitochondrial death pathway of apoptosis. Key words: apoptosis, CXCR4, heart, myocytes, ubiquitin. L'ubiquitine (UB) exogene joue un role protecteur dans le remodelage myocardique stimule par le [beta]-AR et induit par l'ischemie/reperfusion (I/R). Les auteurs rapportent ici qu'un traitement avec l'UB inhibe l'apoptose induite par l'hypoxie/reoxygenation (H/R) dans les myocytes ventriculaires de rats adultes (ARVM, adult rat ventricular myocytes). L'activation d'Akt etait plus elevee alors que l'activation de la GSK-3[beta] etait plus faible chez les cellules traitees a l'UB apres H/R. Le stress oxydant etait plus faible alors que le nombre d'ARVM a mitochondries polarisees etait significativement plus eleve dans les echantillons traites a l'UB. Les ARVM expriment le CXCR4, la majorite du CXCR4 etant localise dans la fraction membranaire. L'antagonisme du CXCR4 par l'AMD3100 et le knockdown de CXCR4 realise par un ARNsi annulaient les effets protecteurs de l'UB. Deux formes mutees de l'UB (incapables de se lier au CXCR4) n'avaient pas d'effet sur l'apoptose induite par H/R, l'activation d'Akt ou de la GSK-3[beta] ou le stress oxydant. Le traitement a l'UB augmentait la biogenese mitochondriale et l'inhibition de la fission mitochondriale par mdivi1 inhibait l'apoptose induite par H/R. Ex vivo, le traitement a l'UB reduisait significativement la taille de l'infarctus et ameliorait la recuperation fonctionnelle du cc[epsilon]ur a la suite d'une I/R globale. L'activation de la caspase-9, un acteur cle de la voie mitochondriale de mort cellulaire, etait significativement plus faible dans les cc[epsilon]urs traites a l'UB apres I/R. L'UB, qui agit fort probablement par l'intermediaire du CXCR4, joue un role protecteur dans l'apoptose des myocytes induite par H/R et dans le dommage d'I/R du myocarde, par la modulation de l'homeostasie mitochondriale et la voie mitochondriale de mort cellulaire par apoptose. Mots-cles: apoptose, CXCR4, cc[epsilon]ur, myocytes, ubiquitine., Introduction Coronary heart disease is a leading cause of death worldwide. The effects of coronary heart disease are generally correlated with myocardial ischemia/reperfusion (I/R) injury (Powers et al. 2007; Hausenloy [...]

The purpose of this study was to explore the link between speech and reading to oneself among struggling readers in secondary schools. The researchers examined the extent to which adolescent struggling readers used various vocal and subvocal behaviors, such as lip movement, mumbling, whispering, and oral reading during individual reading activities. The target population was students in middle and high school identified as struggling with reading skills. The treatment was the individual administration of a norm-referenced measure of reading accompanied by direct observations. Results revealed that fewer than half of the students read silently without lip movement or vocalization. Half of the struggling readers primarily subvocalized, reading silently or moving their lips with no sound, while forty-two percent of the students changed their manifested behaviors as they read to themselves. Vocal and subvocal reading patterns were similar across genders. The findings suggest that many struggling adolescent readers have not fully developed the skill to read silently. (Contains 1 figure and 3 tables.)

Discusses a study concerning the effective use of Supplemental Instruction (SI) in 90 developmental mathematics courses at a southern state university in spring, summer, and fall 2000. Reports that, based on student outcomes, SI may have made a positive difference in performance and retention rates for the students. (Contains 16 references.) (AUTH/NB)

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1[alpha] and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to ~70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 [micro]mol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1. ethyl 3,4-dihyroxybenzoate; heme oxygenase; hepatic ischemia-reperfusion; mitochondrial permeability transition; prolyl hydroxylase inhibitor

These studies explore the consequences of activating the prolyl hydroxylase (PHD) [O.sub.2]-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broadspectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in [O.sub.2] consumption and a 300% increase in lactic acid production under basal conditions. This indicates a ~75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed [O.sub.2] consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase [O.sub.2] consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHItreated cardiomyocytes. Silencing of hypoxia-inducible factor-l[alpha] (HIF-l[alpha]) expression restored the ability of uncoupled PHI-treated myocytes to increase [O.sub.2] consumption; however, basal [O.sub.2] uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively 'clamped' through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the [O.sub.2]-sensing pathway is sufficient to actively 'clamp' [O.sub.2] consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration. mitochondrial membrane potential; hibernation; cardioprotection

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (I[C.sub.50] = 26.1 [+ or -] 0.2 and 27.5 [+ or -] 0.5 [micro]M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (I[C.sub.50] = 32.3 [+ or -] 0.9 and 31.6 [+ or -] 1.3 [micro]M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the [Mg.sup.2+]-ATPase activity of the unphosphorylated (I[C.sub.50] = 12.6 [+ or -] 1.6 and 4.3 [+ or -] 0.5 [micro]M for gizzard and bovine stomach, respectively) and phosphorylated (I[C.sub.50] = 15.0 [+ or -] 0.6 [micro]M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the [Mg.sup.2+]-ATPase activities of gizzard myosin II fragments, heavy meromyosin (I[C.sub.50] = 14.4 [+ or -] 1.6 [micro]M) and subfragment 1 (I[C.sub.50] = 5.5 [+ or -] 0.4 [micro]M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-Dglucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential ([DELTA][[PSI].sub.mito]) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, [DELTA][[PSI].sub.mito] is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse [DELTA][[PSI].sub.mito] in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of [DELTA][[PSI].sub.mito] in PHI-treated myocytes, whereas a role for the 'reverse mode' operation of the [F.sub.1][F.sub.o]-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only ~15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult. fumarate; hibernation; cardioprotection; anaplerotic

Abstract: The effects of changes in the expression levels of h1 calponin (CaP) on actin cytoskeletal organization were studied in control and phorbol-ester-treated A7r5 smooth muscle cells. Protein association and [...]

Molecular mechanisms underlying migration of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (SPC) were analyzed in light of the hypothesis that remodeling of the actin cytoskeleton should be involved. After SPC stimulation, mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38) and p42/44 MAPK (p42/44), were found to be phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44. Pertussis toxin (PTX), a [G.sub.i] protein inhibitor, induced an inhibitory effect on p38 phosphorylation and VSMC migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed migration. Complete inhibition was achieved with the addition of SB-203580. After SPC stimulation, the actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated filopodia, i.e., magunapodia. The peripheral actin bundles consisted of [alpha]- and [beta]-actin, but magunapodia consisted exclusively of [beta]-actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphological data confirmed the regulation of actin remodeling and suggest that VSMCs migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway. arteriosclerosis; cytoskeletal remodeling doi:10.1152/ajpheart.00901.2005