bluuebunny/crossref_metadata_2025 huggingface dataset:
Aquaporin-9 and urea transporter-A gene deletions affect urea transmembrane passage in murine hepatocytes
Authors: Jelen Sabina , Gena Patrizia , Lebeck Janne , Rojek Aleksandra , Praetorius Jeppe , Frøkiaer Jørgen , Fenton Robert A. , Nielsen Søren , Calamita Giuseppe , Rützler Michael
Published: October 2012

In mammals, the majority of nitrogen from protein degradation is disposed of as urea. Several studies have partly characterized expression of urea transporters (UTs) in hepatocytes, where urea is produced. Nevertheless, the contribution of these proteins to hepatocyte urea permeability ( Purea) and their role in liver physiology remains unknown. The purpose of this study was to biophysically examine hepatocyte urea transport. We hypothesized that the water, glycerol, and urea channel aquaporin-9 (AQP9) is involved in hepatocyte urea release. Stopped-flow light-scattering measurements determined that the urea channel inhibitors phloretin and dimethylurea reduced urea permeability of hepatocyte basolateral membranes by 70 and 40%, respectively. In basolateral membranes isolated from AQP9−/−and UT-A1/3−/−single-knockout and AQP9−/−:UT-A1/3−/−double-knockout mice, Pureawas decreased by 30, 40, and 76%, respectively, compared with AQP9+/−:UT-A1/3+/−mice. However, expression analysis by RT-PCR did not identify known UT-A transcripts in liver. High-protein diet followed by 24-h fasting affected the concentrations of urea and ammonium ions in AQP9−/−mouse liver and plasma without generating an apparent tissue-to-plasma urea gradient. We conclude that AQP9 and unidentified UT-A urea channels constitute primary but redundant urea facilitators in murine hepatocytes.
Macrophages modulate cardiac function in lipotoxic cardiomyopathy
Authors: Schilling Joel D. , Machkovech Heather M. , Kim Alfred H. J. , Schwedwener Reto , Schaffer Jean E.
Published: October 2012

Diabetes is associated with myocardial lipid accumulation and an increased risk of heart failure. Although cardiac myocyte lipid overload is thought to contribute to the pathogenesis of cardiomyopathy in the setting of diabetes, the mechanism(s) through which this occurs is not well understood. Increasingly, inflammation has been recognized as a key pathogenic feature of lipid excess and diabetes. In this study, we sought to investigate the role of inflammatory activation in the pathogenesis of lipotoxic cardiomyopathy using the α-myosin heavy chain promoter-driven long-chain acylCoA synthetase 1 (MHC-ACS) transgenic mouse model. We found that several inflammatory cytokines were upregulated in the myocardium of MHC-ACS mice before the onset of cardiac dysfunction, and this was accompanied by macrophage infiltration. Depletion of macrophages with liposomal clodrolip reduced the cardiac inflammatory response and improved cardiac function. Thus, in this model of lipotoxic cardiac injury, early induction of inflammation and macrophage recruitment contribute to adverse cardiac remodeling. These findings have implications for our understanding of heart failure in the setting of obesity and diabetes.
Interpreting genetic effects through models of cardiac electromechanics
Authors: Niederer S. A. , Land S. , Omholt S. W. , Smith N. P.
Published: October 2012

Multiscale models of cardiac electromechanics are being increasingly focused on understanding how genetic variation and environment underpin multiple disease states. In this paper we review the current state of the art in both the development of specific models and the physiological insights they have produced. This growing research body includes the development of models for capturing the effects of changes in function in both single and multiple proteins in both specific expression systems and in vivo contexts. Finally, the potential for using this approach for ultimately predicting phenotypes from genetic sequence information is discussed.
Exogenous ubiquitin modulates chronic β-adrenergic receptor-stimulated myocardial remodeling: role in Akt activity and matrix metalloproteinase expression
Authors: Daniels Christopher R. , Foster Cerrone R. , Yakoob Sana , Dalal Suman , Joyner William L. , Singh Mahipal , Singh Krishna
Published: October 2012

β-Adrenergic receptor (β-AR) stimulation increases extracellular ubiquitin (UB) levels, and extracellular UB inhibits β-AR-stimulated apoptosis in adult cardiac myocytes. This study investigates the role of exogenous UB in chronic β-AR-stimulated myocardial remodeling. l-Isoproterenol (ISO; 400 μg·kg−1·h−1) was infused in mice in the presence or absence of UB (1 μg·g−1·h−1). Left ventricular (LV) structural and functional remodeling was studied 7 days after infusion. UB infusion enhanced serum UB levels. In most parts, UB alone had no effect on morphometric or functional parameters. Heart weight-to-body weight ratios were increased to a similar extent in the ISO and UB + ISO groups. Echocardiographic analyses showed increased percent fractional shortening, ejection fraction, and LV circumferential stress and fiber-shortening velocity in the ISO group. These parameters were significantly lower in UB + ISO vs. ISO. Isovolumic contraction and relaxation times and ejection time were significantly lower in ISO vs. UB + ISO. The increase in the number of TUNEL-positive myocytes and fibrosis was significantly higher in ISO vs. UB + ISO. Activation of Akt was higher, whereas activation of GSK-3β and JNKs was lower in UB + ISO vs ISO. Expression of MMP-2, MMP-9, and TIMP-2 was higher in UB + ISO vs ISO. In isolated cardiac fibroblasts, UB enhanced expression of MMP-2 and TIMP-2 in the presence of ISO. Neutralizing UB antibodies negated the effects of UB on MMP-2 expression, whereas recombinant UB enhanced MMP-2 expression. UB activated Akt, and inhibition of Akt inhibited UB + ISO-mediated increases in MMP-2 expression. Thus, exogenous UB plays an important role in β-AR-stimulated myocardial remodeling with effects on LV function, fibrosis, and myocyte apoptosis.
Right ventricular arrhythmogenesis in failing human heart: the role of conduction and repolarization remodeling
Authors: Lou Qing , Janks Deborah L. , Holzem Katherine M. , Lang Di , Onal Birce , Ambrosi Christina M. , Fedorov Vadim V. , Wang I-Wen , Efimov Igor R.
Published: October 2012

Increased dispersion of repolarization has been suggested to underlie increased arrhythmogenesis in human heart failure (HF). However, no detailed repolarization mapping data were available to support the presence of increased dispersion of repolarization in failing human heart. In the present study, we aimed to determine the existence of enhanced repolarization dispersion in the right ventricular (RV) endocardium from failing human heart and examine its association with arrhythmia inducibility. RV free wall preparations were dissected from five failing and five nonfailing human hearts, cannulated and coronary perfused. RV endocardium was optically mapped from an ∼6.3 × 6.3 cm2 field of view. Action potential duration (APD), dispersion of APD, and conduction velocity (CV) were quantified for basic cycle lengths (BCL) ranging from 2,000 ms to the functional refractory period. We found that RV APD was significantly prolonged within the failing group compared with the nonfailing group (560 ± 44 vs. 448 ± 39 ms, at BCL = 2,000 ms, P < 0.05). Dispersion of APD was increased in three failing hearts (161 ± 5 vs. 86 ± 19 ms, at BCL = 2,000 ms). APD alternans were induced by rapid pacing in these same three failing hearts. CV was significantly reduced in the failing group compared with the nonfailing group (81 ± 11 vs. 98 ± 8 cm/s, at BCL = 2,000 ms). Arrhythmias could be induced in two failing hearts exhibiting an abnormally steep CV restitution and increased dispersion of repolarization due to APD alternans. Dispersion of repolarization is enhanced across the RV endocardium in the failing human heart. This dispersion, together with APD alternans and abnormal CV restitution, could be responsible for the arrhythmia susceptibility in human HF.
Pulmonary artery smooth muscle cell proliferation and migration in fetal lambs acclimatized to high-altitude long-term hypoxia: role of histone acetylation
Authors: Yang Qiwei , Lu Ziyan , Ramchandran Ramaswamy , Longo Lawrence D. , Raj J. Usha
Published: October 2012

High-altitude long-term hypoxia (LTH) is known to induce pulmonary arterial smooth muscle cell (PASMC) proliferation in the fetus, leading to pulmonary arterial remodeling and pulmonary hypertension of the newborn. The mechanisms underlying these conditions remain enigmatic however. We hypothesized that epigenetic alterations in fetal PASMC induced by high-altitude LTH may play an important role in modulating their proliferation during pulmonary arterial remodeling. To test this hypothesis, we have analyzed epigenetic alterations in the pulmonary vasculature of fetal lambs exposed to high-altitude LTH [pregnant ewes were kept at 3,801 m altitude from ∼40 to 145 days gestation] or to sea level atmosphere. Intrapulmonary arteries were isolated, and fetal PASMC were cultured from both control and LTH fetuses. Compared with controls, in LTH fetus pulmonary arteries measurements of histone acetylation and global DNA methylation demonstrated reduced levels of global histone 4 acetylation and DNA methylation, accompanied by the loss of the cyclin-dependent kinase inhibitor p21. Treatment of LTH fetal PASMCs with histone deacetylase (HDAC) inhibitor trichostatin A decreased their proliferation rate, in part because of altered expression of p21 at both RNA and protein level. In PASMC of LTH fetuses, HDAC inhibition also decreased PDGF-induced cell migration and ERK1/2 activation and modulated global DNA methylation. On the basis of these observations, we propose that epigenetic alterations (reduced histone acetylation and DNA methylation) caused by chronic hypoxia leads to fetal PASMC proliferation and vessel remodeling associated with vascular proliferative disease and that this process is regulated by p21.
Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis
Authors: Naik Payal K. , Bozyk Paul D. , Bentley J. Kelley , Popova Antonia P. , Birch Carolyn M. , Wilke Carol A. , Fry Christopher D. , White Eric S. , Sisson Thomas H. , Tayob Nabihah , Carnemolla Barbara , Orecchia Paola , Flaherty Kevin R. , Hershenson Marc B. , Murray Susan , Martinez Fernando J. , Moore Bethany B. ,   the COMET Investigators
Published: October 2012

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03–2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin−/−) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin−/−mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention.
Chronic effects of pulmonary artery stenosis on hemodynamic and structural development of the lungs
Authors: Razavi Hedi , Stewart Sarah E. , Xu Chengpei , Sawada Hirofumi , Zarafshar Shahrzad Y. , Taylor Charles A. , Rabinovitch Marlene , Feinstein Jeffrey A.
Published: October 2012

Pulmonary artery (PA) stenosis is a difficult obstructive defect to manage since clinicians cannot know a priori which obstructions to treat and when. Prognosis of PA stenosis and its chronic effects on lung development are poorly understood. This study aimed to characterize the hemodynamic and structural effects of PA stenosis during development. Fourteen male Sprague-Dawley rats underwent left PA (LPA) banding at age 21 days, and 13 underwent sham operation. Hemodynamic and structural impacts were studied longitudinally at 20, 36, 52, 100, and 160 days. Chronic LPA banding resulted in a significant reduction in LPA flow ( P < 0.0001) and size of both proximal LPA ( P < 0.0001) and distal LPA ( P < 0.01), as well as a significant increase in flow and size of the right PA ( P < 0.05) throughout development. Flows and sizes adapted such that normal levels of wall shear were restored after banding. At 160 days, LPA banding resulted in a significant decrease in left lung volume and an increase in right lung volume but no significant differences in total lung volume. There was an elevation of proximal LPA pressure as well as right ventricular hypertrophy in the banded animals. The banded lung exhibited arterial disorganization, loss of vessels, and enlargement of its bronchial arteries, whereas the contralateral lung showed signs of vascular pathology. There are consequences on development of both lungs in the presence of an LPA stenosis at young age. These results suggest that early intervention may be necessary to optimize left lung growth and minimize right lung vascular pathology.
Increased ceramide content and NFκB signaling may contribute to the attenuation of anabolic signaling after resistance exercise in aged males
Authors: Rivas Donato A. , Morris Evan P. , Haran Prashanth H. , Pasha Evan P. , Morais Mauricio da Silva , Dolnikowski Gregory G. , Phillips Edward M. , Fielding Roger A.
Published: October 2012

One of the most fundamental adaptive physiological events is the response of skeletal muscle to high-intensity resistance exercise, resulting in increased protein synthesis and ultimately larger muscle mass. However, muscle growth in response to contraction is attenuated in older humans. Impaired contractile-induced muscle growth may contribute to sarcopenia: the age-associated loss of muscle mass and function that is manifested by loss of strength, contractile capacity, and endurance. We hypothesized that the storage of ceramide would be increased in older individuals and this would be associated with increases in NFκB signaling and a decreased anabolic response to exercise. To test this hypothesis we measured ceramides at rest and anabolic and NFκB signaling after an acute bout of high-intensity resistance exercise in young and older males. Using lipidomics analysis we show there was a 156% increase in the accumulation of C16:0-ceramide ( P < 0.05) and a 30% increase in C20:0-ceramide ( P < 0.05) in skeletal muscle with aging, although there was no observable difference in total ceramide. C16:0-ceramide content was negatively correlated ( P = 0.008) with lower leg lean mass. Aging was associated with a ∼60% increase in the phosphorylation of the proinflammatory transcription factor NFκB in the total and nuclear cell fractions ( P < 0.05). Furthermore, there was an attenuated activation of anabolic signaling molecules such as Akt ( P < 0.05), FOXO1 ( P < 0.05), and S6K1 ( P < 0.05) after an acute bout of high-intensity resistance exercise in older males. We conclude that ceramide may have a significant role in the attenuation of contractile-induced skeletal muscle adaptations and atrophy that is observed with aging.
Heat shock protein 70 (Hsp70) inhibits oxidative phosphorylation and compensates ATP balance through enhanced glycolytic activity
Authors: Wang Liangli , Schumann Uwe , Liu Yuefei , Prokopchuk Olga , Steinacker Jürgen M.
Published: October 2012

To address possible effects of heat shock protein 70 (Hsp70) on energy metabolism, we established a cell line expressing different levels of Hsp70 and evaluated changes in glucose and lactate metabolites, as well as ATP levels accordingly. In addition, activities of enzymes involved in glycolysis [phosphofructokinase (PFK) and lactate dehydrogenase (LDH)], Krebs cycle [citric synthase (CS)], and oxidative phosphorylation {NADH dehydrogenase [ complex I (CI)] and ubiquinol:cytochrome-c reductase [ complex III (CIII)]} were analyzed. The results show that both glucose consumption and lactate excretion were elevated significantly in cells expressing increased levels of Hsp70. Simultaneously, the activities of glycolytic enzymes PFK and LDH were increased markedly in cells overexpressing Hsp70. Activities of enzymes CI and CIII, both involved in oxidative phosphorylation, decreased upon increased expression of Hsp70. These findings were supported by nonsignificant reductions of CS activities in cells that overexpressed Hsp70, whereas intracellular ATP levels remained constant over a wide range of Hsp70 expression. In conclusion, overexpression of Hsp70 in HeLa cells results in downregulation of oxidative phosphorylation, in particular, multiprotein CIII, the main source of reactive oxygen species. In exchange, upregulation of the glycolytic pathway compensates for the homeostasis of cellular ATP supply.
Type 2 diabetes impairs tendon repair after injury in a rat model
Authors: Ahmed Aisha S. , Schizas Nikos , Li Jian , Ahmed Mahmood , Östenson C.-G. , Salo Paul , Hewitt Carolyn , Hart David A. , Ackermann Paul W.
Published: October 2012

Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mechanisms, however, by which diabetes alters connective tissue metabolism, especially tendon, are poorly defined. The aim of this study was to determine the effect of type 2 diabetes on the mechanical, histological, and molecular properties of the intact and healing Achilles tendon. The right Achilles tendon was transected in 11 male diabetic Goto-Kakizaki (GK) and 10 age- and sex-matched Wistar control rats, while the left Achilles tendon was left intact. At 2 wk postinjury the intact and injured tendons were assessed by biomechanical testing and histology. The gene expression of collagen I and III, biglycan, versican, MMP-13, and MMP-3 was measured by quantitative RT-PCR, and their protein distribution was studied by immunohistochemistry. Intact tendons exhibited only small differences between the groups. In injured tendons, however, a significantly smaller transverse area and lower stiffness was found in diabetic GK compared with Wistar control rats. This correlated with impaired structural organization of collagen fibers and a reduced expression of collagen I and III in the injured tendons of the diabetic GK compared with Wistar control. Moreover, MMP-3 gene expression was downregulated in the injured diabetic GK tendons compared with injured Wistar controls. Our results indicate that in a rat model of diabetes tendon healing is impaired mainly due to altered expression of collagen and MMPs reflecting decreased degradation of matrix proteins and impaired tissue remodeling. Further our data suggest that therapeutic modulation of collagens or MMPs might be targets for new regenerative approaches in operated, injured, or maybe also degenerative tendon diseases in diabetes.
Pulsed high oxygen induces a hypoxic-like response in human umbilical endothelial cells and in humans
Authors: Cimino F. , Balestra C. , Germonpré P. , De Bels D. , Tillmans F. , Saija A. , Speciale A. , Virgili F.
Published: October 2012

It has been proposed that relative changes of oxygen availability, rather than steady-state hypoxic or hyperoxic conditions, play an important role in hypoxia-inducible factor (HIF) transcriptional effects. According to this hypothesis describing the “normobaric oxygen paradox”, normoxia following a hyperoxic event is sensed by tissues as an oxygen shortage, upregulating HIF-1 activity. With the aim of confirming, at cellular and at functional level, that normoxia following a hyperoxic event is “interpreted” as a hypoxic event, we report a combination of experiments addressing the effects of an intermittent increase of oxygen concentration on HIF-1 levels and the activity level of specific oxygen-modulated proteins in cultured human umbilical vein endothelial cells and the effects of hemoglobin levels after intermittent breathing of normobaric high (100%) and low (15%) oxygen in vivo in humans. Our experiments confirm that, during recovery after hyperoxia, an increase of HIF expression occurs in human umbilical vein endothelial cells, associated with an increase of matrix metalloproteinases activity. These data suggest that endothelial cells “interpret” the return to normoxia after hyperoxia as a hypoxic stimulus. At functional level, our data show that breathing both 15 and 100% oxygen 30 min every other day for a period of 10 days induces an increase of hemoglobin levels in humans. This effect was enhanced after the cessation of the oxygen breathing. These results indicate that a sudden decrease in tissue oxygen tension after hyperoxia may act as a trigger for erythropoietin synthesis, thus corroborating the hypothesis that “relative” hypoxia is a potent stimulator of HIF-mediated gene expressions.
Heat and α1-adrenergic responsiveness in human skeletal muscle feed arteries: the role of nitric oxide
Authors: Ives Stephen J. , Andtbacka Robert H. I. , Kwon Sun Hyung , Shiu Yan-Ting , Ruan Ting , Noyes R. Dirk , Zhang Quan-Jiang , Symons J. David , Richardson Russell S.
Published: October 2012

Increased local temperature exerts a sympatholytic effect on human skeletal muscle feed arteries. We hypothesized that this attenuated α1-adrenergic receptor responsiveness may be due to a temperature-induced increase in nitric oxide (NO) bioavailability, thereby reducing the impact of the α1-adrenergic receptor agonist phenylephrine (PE). Thirteen human skeletal muscle feed arteries were harvested, and wire myography was used to generate PE concentration-response curves at 37°C and 39°C, with and without the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (l-NMMA). A subset of arteries ( n = 4) were exposed to 37°C or 39°C, and the protein content of endothelial NOS (eNOS) and α1-adrenergic receptors was determined by Western blot analysis. Additionally, cultured bovine endothelial cells were exposed to static or shear stress conditions at 37°C and 39°C and assayed for eNOS activation (phosphorylation at Ser1177), eNOS expression, and NO metabolites [nitrate + nitrite (NOx)]. Maximal PE-induced vasocontraction (PEmax) was lower at 39°C than at 37°C [39 ± 10 vs. 84 ± 30% maximal response to 100 mM KCl (KClmax)]. NO blockade restored vasocontraction at 39°C to that achieved at 37°C (80 ± 26% KClmax). Western blot analysis of the feed arteries revealed that heating increased eNOS protein, but not α1-adrenergic receptors. Heating of bovine endothelial cells resulted in greater shear stress-induced eNOS activation and NOx production. Together, these data reveal for the first time that, in human skeletal muscle feed arteries, NO blockade can restore the heat-attenuated α1-adrenergic receptor-mediated vasocontraction and implicate endothelium-derived NO bioavailability as a major contributor to heat-induced sympatholysis. Consequently, these findings highlight the important role of vasodilators in modulating the vascular response to vasoconstrictors.
Effects of aging and exercise training on skeletal muscle blood flow and resistance artery morphology
Authors: Behnke Bradley J. , Ramsey Michael W. , Stabley John N. , Dominguez James M. , Davis Robert T. , McCullough Danielle J. , Muller-Delp Judy M. , Delp Michael D.
Published: October 2012

With old age, blood flow to the high-oxidative red skeletal muscle is reduced and blood flow to the low-oxidative white muscle is elevated during exercise. Changes in the number of feed arteries perforating the muscle are thought to contribute to this altered hyperemic response during exercise. We tested the hypothesis that exercise training would ameliorate age-related differences in blood flow during exercise and feed artery structure in skeletal muscle. Young (6–7 mo old, n = 36) and old (24 mo old, n = 25) male Fischer 344 rats were divided into young sedentary (Sed), old Sed, young exercise-trained (ET), and old ET groups, where training consisted of 10–12 wk of treadmill exercise. In Sed and ET rats, blood flow to the red and white portions of the gastrocnemius muscle (GastRed and GastWhite) and the number and luminal cross-sectional area (CSA) of all feed arteries perforating the muscle were measured at rest and during exercise. In the old ET group, blood flow was greater to GastRed (264 ± 13 and 195 ± 9 ml·min−1·100 g−1 in old ET and old Sed, respectively) and lower to GastWhite (78 ± 5 and 120 ± 6 ml·min−1·100 g−1 in old ET and old Sed, respectively) than in the old Sed group. There was no difference in the number of feed arteries between the old ET and old Sed group, although the CSA of feed arteries from old ET rats was larger. In young ET rats, there was an increase in the number of feed arteries perforating the muscle. Exercise training mitigated old age-associated differences in blood flow during exercise within gastrocnemius muscle. However, training-induced adaptations in resistance artery morphology differed between young (increase in feed artery number) and old (increase in artery CSA) animals. The altered blood flow pattern induced by exercise training with old age would improve the local matching of O2 delivery to consumption within the skeletal muscle.
Transfusion of hemoglobin-based oxygen carriers in the carboxy state is beneficial during transient focal cerebral ischemia
Authors: Zhang Jian , Cao Suyi , Kwansa Herman , Crafa Daina , Kibler Kathleen K. , Koehler Raymond C.
Published: October 2012

Exchange transfusion of large volumes of hemoglobin (Hb)-based oxygen carriers can protect the brain from middle cerebral artery occlusion (MCAO). Hb in the carboxy state (COHb) may provide protection at relatively low volumes by enhancing vasodilation. We determined whether transfusion of rats with 10 ml/kg PEGylated COHb [polyethylene glycol (PEG)-COHb] at 20 min of 2-h MCAO was more effective in reducing infarct volume compared with non-carbon monoxide (CO) PEG-Hb. After PEG-COHb transfusion, whole blood and plasma COHb was <3%, indicating rapid release of CO. PEG-COHb transfusion significantly reduced infarct volume (15 ± 5% of hemisphere; mean ± SE) compared with that in the control group (35 ± 6%), but non-CO PEG-Hb did not (24 ± 5%). Chemically dissimilar COHb polymers were also effective. Induction of MCAO initially produced 34 ± 2% dilation of pial arterioles in the border region that subsided to 10 ± 1% at 2 h. Transfusion of PEG-COHb at 20 min of MCAO maintained pial arterioles in a dilated state (40 ± 5%) at 2 h, whereas transfusion of non-CO PEG-Hb had an intermediate effect (22 ± 3%). When transfusion of PEG-COHb was delayed by 90 min, laser-Doppler flow in the border region increased from 57 ± 9 to 82 ± 13% of preischemic baseline. These data demonstrate that PEG-COHb is more effective than non-CO PEG-Hb at reducing infarct volume, sustaining cerebral vasodilation, and improving collateral perfusion in a model of transient focal cerebral ischemia when given at a relatively low dose (plasma Hb concentration < 1 g/dl). Use of acellular Hb as a CO donor that is rapidly converted to an oxygen carrier in vivo may permit potent protection at low transfusion volumes.
Spatial gradient in TTX sensitivity of axons at the crayfish opener neuromuscular junction
Authors: Lin Jen-Wei
Published: October 2012

At the crayfish opener neuromuscular junction, axons branch repeatedly before synapsing onto muscle fibers as varicosities. Excitability of these axons was examined with two-electrode current clamp before and after partial block of Na+ channels with 1 nM tetrodotoxin. 4-Aminopyridine (200 μM) was added to homogenize nonuniformity in K+ channel density. The impact of tetrodotoxin was evaluated in terms of action potential (AP) amplitude, rate of rise, and threshold. All three parameters were more severely affected at the secondary than the primary branching point (BP). Both BPs fired continuously during 1-s current steps before tetrodotoxin. After tetrodotoxin, the secondary BP fired only in brief bursts, whereas the primary BP still fired continuously. Despite this diminished excitability at the secondary BP, no failure in orthodromic AP conduction was observed. AP waveform at terminals (APf) was examined with voltage indicators. For orthodromic APs, reduction in AP amplitude and prolongation of AP rise time in tetrodotoxin were more pronounced in terminals than at the secondary BP. For APs initiated at the secondary BP, APf sometimes failed to show a spikelike waveform in tetrodotoxin. This degraded APf was not due to averaging variable AP invasion into terminals, because the variance of APf traces did not increase in tetrodotoxin. Tetrodotoxin applied in the absence of 4-aminopyridine showed an impact on the distal axon similar but less distinct than that recorded with 4-aminopyridine. In conclusion, the distal axon is more sensitive to tetrodotoxin than the proximal axon, such that AP waveform degrades as it propagates toward terminals in tetrodotoxin.
Nucleic-Acid-Binding Chromophores as Efficient Indicators of Aptamer-Target Interactions
Authors: Sarpong Kwabena , Datta Bhaskar
Published: October 2012

The binding affinity and specificity of nucleic acid aptamers have made them valuable candidates for use as sensors in diagnostic applications. In particular, chromophore-functionalized aptamers offer a relatively simple format for detection and quantification of target molecules. We describe the use of nucleic-acid-staining reagents as an effective tool for detecting and signaling aptamer-target interactions. Aptamers varying in size and structure and targeting a range of molecules have been used in conjunction with commercially available chromophores to indicate and quantify the presence of cognate targets with high sensitivity and selectivity. Our assay precludes the covalent modification of nucleic acids and relies on the differential fluorescence signal of chromophores when complexed with aptamers with or without their cognate target. We also evaluate factors that are critical for the stability of the complex between the aptamer and chromophore in presence or absence of target molecules. Our results indicate the possibility of controlling those factors to enhance the sensitivity of target detection by the aptamers used in such assays.
Changing Academic Medicine: Strategies Used by Academic Leaders of Integrative Medicine—A Qualitative Study
Authors: Witt Claudia M. , Holmberg Christine
Published: October 2012

In Western countries, complementary and alternative medicine (CAM) is more and more provided by practitioners and family doctors. To base this reality of health care provision on an evidence-base, academic medicine needs to be included in the development. In the study we aimed to gain information on a structured approach to include CAM in academic health centers. We conducted a semistructured interview study with leading experts of integrative medicine to analyze strategies of existing academic institutions of integrative medicine. The study sample consisted of a purposive sample of ten leaders that have successfully integrated CAM into medical schools in the USA, Great Britain, and Germany and the Director of the National Center for Alternative and Complementary Medicine. Analysis was based on content analysis. The prerequisite to foster change in academic medicine was a strong educational and professional background in academic medicine and research methodologies. With such a skill set, the interviewees identified a series of strategies to align themselves with colleagues from conventional medicine, such as creating common goals, networking, and establishing well-functioning research teams. In addition, there must be a vision of what should be needed to be at the center of all efforts in order to implement successful change.
An Odontoid Fracture Causing Apnea, Cardiac Instability, and Quadriplegia
Authors: Bowers Christian A. , Jost Gregory F. , Dailey Andrew T.
Published: October 2012

Odontoid fractures are typically associated with low rates of acute neurologic deficit and morbidity/mortality in nonelderly patients. In the patient in this case, traumatic injury triggered by a syncopal event led to a combined C1-C2 fracture and a fatal spinal cord injury with apnea, quadriplegia, and cardiovascular instability. We briefly review the anatomical basis for the pathophysiology of cardiac dysfunction following high-cervical spine injury and present an example of a worst-case scenario.
Combined Kidney-Liver, Heart-Liver, and Kidney-Pancreas Transplantations from a Single Deceased Donor
Authors: Ravaioli Matteo , Serenari Matteo , Cescon Matteo , Martin Suarez Sofia , Cucchetti Alessandro , Ercolani Giorgio , Del Gaudio Massimo , Catena Fausto , Morelli Cristina , Arpesella Giorgio , Pinna Antonio Daniele
Published: October 2012

Splitting the liver for two adults to increase the donor pool is still a debated issue, especially for combined organ transplantation. We described a case of liver-splitting procedure for two adults, which was successful even in the presence of combined organ transplantation. Three adult combined organ transplantations from one deceased donor were performed, with, use of split liver grafts in two patients: a combined heart-right split liver, a left kidney-left split liver, and a right kidney-pancreas transplantation. Despite a not perfect match between the graft type and recipient, the prevention of small-for-size syndrome by ligature of the splenic artery, and/or hemiportocaval shunt in the patient receiving the left split liver, and the maximal reduction of ischemia time were the main factors contributing to the success of the procedure. This is the first report of combined heart and split liver in two adults which may suggest new strategies for organ transplantations.
Cyclin D1 Activity Regulates Autophagy and Senescence in the Mammary Epithelium
Authors: Brown Nelson E. , Jeselsohn Rinath , Bihani Teeru , Hu Miaofen G. , Foltopoulou Parthena , Kuperwasser Charlotte , Hinds Philip W.
Published: October 2012

Overexpression of cyclin D1 is believed to endow mammary epithelial cells (MEC) with a proliferative advantage by virtue of its contribution to pRB inactivation. Accordingly, abrogation of the kinase-dependent function of cyclin D1 is sufficient to render mice resistant to breast cancer initiated by ErbB2. Here, we report that mouse cyclin D1KE/KE MECs (deficient in cyclin D1 activity) upregulate an autophagy-like process but fail to implement ErbB2-induced senescence in vivo. In addition, immortalized cyclin D1KE/KE MECs retain high rates of autophagy and reduced ErbB2-mediated transformation in vitro. However, highlighting its dual role during tumorigenesis, downregulation of autophagy led to an increase in senescence in cyclin D1KE/KE MECs. Autophagy upregulation was also confirmed in human mammary epithelial cells (HMEC) subjected to genetic and pharmacologic inhibition of cyclin D1 activity and, similar to our murine system, simultaneous inhibition of Cdk4/6 and autophagy in HMECs enhanced the senescence response. Collectively, our findings suggest a previously unrecognized function of cyclin D1 in suppressing autophagy in the mammary epithelium. Cancer Res; 72(24); 6477–89. ©2012 AACR.
Oxidative Stress-Regulated Lentiviral TK/GCV Gene Therapy for Lung Cancer Treatment
Authors: Leinonen Hanna M. , Ruotsalainen Anna-Kaisa , Määttä Ann-Marie , Laitinen Heidi M. , Kuosmanen Suvi M. , Kansanen Emilia , Pikkarainen Jere T. , Lappalainen Jari P. , Samaranayake Haritha , Lesch Hanna P. , Kaikkonen Minna U. , Ylä-Herttuala Seppo , Levonen Anna-Liisa
Published: October 2012

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells. Cancer Res; 72(23); 6227–35. ©2012 AACR.
Regression of Human Prostate Cancer Xenografts in Mice by AMG 212/BAY2010112, a Novel PSMA/CD3-Bispecific BiTE Antibody Cross-Reactive with Non-Human Primate Antigens
Authors: Friedrich Matthias , Raum Tobias , Lutterbuese Ralf , Voelkel Markus , Deegen Petra , Rau Doris , Kischel Roman , Hoffmann Patrick , Brandl Christian , Schuhmacher Joachim , Mueller Peter , Finnern Ricarda , Fuergut Melanie , Zopf Dieter , Slootstra Jerry W. , Baeuerle Patrick A. , Rattel Benno , Kufer Peter
Published: October 2012

For treatment of patients with prostate cancer (PCa), we developed a novel T cell-engaging (BiTE) antibody designated AMG 212 or BAY2010112 that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. AMG 212/BAY2010112 induced target cell-dependent activation and cytokine release of T cells, and efficiently redirected T cells for lysis of target cells. In addition to Chinese hamster ovary cells stably expressing human or cynomolgus monkey PSMA, T cells redirected by AMG 212/BAY2010112 also lysed human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC-3-huPSMA, and LnCaP at half maximal BiTE concentrations between 0.1 and 4 ng/mL (1.8–72 pmol/L). No lysis of PSMA-negative human PCa cell lines PC-3 and DU145 was observed. The subcutaneous (s.c.) formation of tumors from PC-3-huPSMA cells in NOD/SCID mice was significantly prevented by once daily intravenous (i.v.) injection of AMG 212/BAY2010112 at a dose level as low as 0.005 mg/kg/d. Rapid tumor shrinkage with complete remissions were observed in NOD/SCID mice bearing established s.c. 22Rv1 xenografts after repeated daily treatment with AMG 212/BAY2010112 by either the i.v. or s.c. route. Of note, 22Rv1 tumors were grown in the absence of human T cells followed by intraperitoneal injection of T cells 3 days before BiTE treatment. No effects on tumor growth were observed in the absence of human T cells or AMG 212/BAY2010112. On the basis of these preclinical results, AMG 212/BAY2010112 appears as a promising new BiTE antibody for the treatment of patients with PSMA-expressing PCa. Mol Cancer Ther; 11(12); 2664–73. ©2012 AACR.
Sorafenib-Mediated Targeting of the AAA+ ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death
Authors: Yi Ping , Higa Arisa , Taouji Said , Bexiga Mariana G. , Marza Esther , Arma Daniela , Castain Claire , Le Bail Brigitte , Simpson Jeremy C. , Rosenbaum Jean , Balabaud Charles , Bioulac-Sage Paulette , Blanc Jean-Frédéric , Chevet Eric
Published: October 2012

The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer–derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal–regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA+ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients. Mol Cancer Ther; 11(12); 2610–20. ©2012 AACR.
The Role of Bcl-xL in Synergistic Induction of Apoptosis by Mapatumumab and Oxaliplatin in Combination with Hyperthermia on Human Colon Cancer
Authors: Song Xinxin , Kim Seog-Young , Lee Yong J.
Published: October 2012

Colorectal cancer is the third leading cause of cancer-related mortality in the world. The main cause of death because of colorectal cancer is hepatic metastases, which can be treated using isolated hepatic perfusion (IHP), allowing treatment of colorectal metastasis with various methods. In this study, we present a novel potent multimodality strategy comprising humanized death receptor 4 (DR4) antibody mapatumumab in combination with oxaliplatin and hyperthermia to treat human colon cancer cells. Oxaliplatin and hyperthermia sensitized colon cancer cells to mapatumumab in the mitochondrial-dependent apoptotic pathway and increased reactive oxygen species (ROS) production, leading to Bcl-xL phosphorylation at serine 62 in a c-jun-NH2-kinase (JNK)-dependent manner. Overexpression of Bcl-xL reduced the efficacy of the multimodality treatment, whereas phosphorylation of Bcl-xL decreased its antiapoptotic activity. The multimodality treatment dissociated Bcl-xL from Bax, allowing Bax oligomerization to induce cytochrome c release from mitochondria. In addition, the multimodality treatment significantly inhibited colorectal cancer xenografts' tumor growth. The successful outcome of this study will support the application of multimodality strategy to colorectal hepatic metastases. Mol Cancer Res; 10(12); 1567–79. ©2012 AACR.
Measuring Oncogenic Signaling Pathways in Cancer with PET: An Emerging Paradigm from Studies in Castration-Resistant Prostate Cancer
Authors: Evans Michael J.
Published: October 2012

As parallel advances in cancer biology and drug development continue to elevate the role of targeted therapies in oncology, the need for imaging biomarkers that systematically measure the biology associated with therapeutic intervention has become more urgent. Although the molecular imaging community has a commitment to develop technologies to this end, few investigational radiotracers directly measure the biology of common oncogenic signaling pathways often addressed by targeted therapies. Visible progress has been achieved with a handful of radiotracers rationally designed to intercalate the pathobiology of prostate cancer, a molecularly heterogeneous disease nevertheless broadly defined by a fairly small repertoire of recurrent oncogenic lesions.

Significance: That variable treatment responses or emergent resistance phenotypes are often documented in humans argues strongly for diagnostic technologies that can be realistically applied posttherapy to capture the dynamic patterns of disease response. The purpose of this review is to describe a collection of radiotracers developed to measure the pathobiology of prostate cancer for improved treatment monitoring, placing particular emphasis on the biologic rationale for their preparation. A chronologic description of radiotracer development programs is outlined, primarily to stress how an ongoing dialectic between earlier and more contemporary imaging technologies has accelerated discovery. Cancer Discov; 2(11); 985–94. ©2012 AACR.
Fatty Acids Regulate Endothelial Lipase and Inflammatory Markers in Macrophages and in Mouse Aorta
Authors: Jung Un Ju , Torrejon Claudia , Chang Chuchun L. , Hamai Hiroko , Worgall Tilla S. , Deckelbaum Richard J.
Published: October 2012

Objective—

Macrophage endothelial lipase (EL) is associated with increased atherosclerosis and inflammation. Because of their anti-inflammatory properties we hypothesized that n-3 fatty acids, in contrast to saturated fatty acids, would lower macrophages and arterial EL and inflammatory markers.

Methods and Results—

Murine J774 and peritoneal macrophages were incubated with eicosapentaenoic acid or palmitic acid in the presence or absence of lipopolysaccaride (LPS). LPS increased EL mRNA and protein. Palmitic acid alone or with LPS dose-dependently increased EL mRNA and protein. In contrast, eicosapentaenoic acid dose-dependently abrogated effects of LPS or palmitic acid on increasing EL expression. EL expression closely linked to peroxisome proliferator activated receptor (PPAR)γ expression. Eicosapentaenoic acid blocked rosiglitazone (a PPARγ agonist)-mediated EL activation and GW9662 (a PPARγ antagonist)-blocked palmitic acid-mediated EL stimulation. Eicosapentaenoic acid alone or with LPS blunted LPS-mediated stimulation of macrophage proinflammatory interleukin-6, interleukin-12p40, and toll-like receptor-4 mRNA and increased anti-inflammatory interleukin-10 and mannose receptor mRNA. In vivo studies in low density lipoprotein receptor knockout mice showed that high saturated fat rich diets, but not n-3 diets, increased arterial EL, PPARγ, and proinflammatory cytokine mRNA.

Conclusion—

n-3 fatty acids, in contrast to saturated fatty acids, decrease EL in parallel with modulating pro- and anti-inflammatory markers, and these effects on EL link to PPARγ.