bluuebunny/crossref_metadata_2025 huggingface dataset:
MECHANISM AND CONTROL OF V(D)J RECOMBINATION AT THE IMMUNOGLOBULIN HEAVY CHAIN LOCUS
Authors: Jung David , Giallourakis Cosmas , Mostoslavsky Raul , Alt Frederick W.
Published: January 2006

V(D)J recombination assembles antigen receptor variable region genes from component germline variable (V), diversity (D), and joining (J) gene segments. For B cells, such rearrangements lead to the production of immunoglobulin (Ig) proteins composed of heavy and light chains. V(D)J is tightly controlled at the Ig heavy chain locus (IgH) at several different levels, including cell-type specificity, intra- and interlocus ordering, and allelic exclusion. Such controls are mediated at the level of gene segment accessibility to V(D)J recombinase activity. Although much has been learned, many long-standing questions regarding the regulation of IgH locus rearrangements remain to be elucidated. In this review, we summarize advances that have been made in understanding how V(D)J recombination at the IgH locus is controlled and discuss important areas for future investigation.
INTESTINAL AND PULMONARY MUCOSAL T CELLS: Local Heroes Fight to Maintain the Status Quo
Authors: Lefrançois Leo , Puddington Lynn
Published: January 2006

Mucosal immunity in the lung and intestine is controlled by complex multifaceted systems. While mucosal T cells are essential for protection against invading pathogens owing to their proximity to the outside world, powerful systems must also be in place to harness ongoing inflammatory processes. In each site, distinct anatomical structures play key roles in mounting and executing both protective and deleterious mucosal T cell responses. Although analogies can be drawn regarding the immune systems of these two organs, there are substantial dissimilarities necessitated by unique physiologic constraints. Here, we discuss how T cell activation and effector function are generated in the mucosae.
MULTIPLEXED PROTEIN ARRAY PLATFORMS FOR ANALYSIS OF AUTOIMMUNE DISEASES
Authors: Balboni Imelda , Chan Steven M. , Kattah Michael , Tenenbaum Jessica D. , Butte Atul J. , Utz Paul J.
Published: January 2006

Several proteomics platforms have emerged in the past decade that show great promise for filling in the many gaps that remain from earlier studies of the genome and from the sequencing of the human genome itself. This review describes applications of proteomics technologies to the study of autoimmune diseases. We focus largely on biased technology platforms that are capable of analyzing a large panel of known analytes, as opposed to techniques such as two-dimensional gel electrophoresis (2DIGE) or mass spectroscopy that represent unbiased approaches (as reviewed in 1 ). At present, the main analytes that can be systematically studied in autoimmunity include autoantibodies, cytokines and chemokines, components of signaling pathways, and cell-surface receptors. We review the most commonly used platforms for such studies, citing important discoveries and limitations that exist. We conclude by reviewing advances in biomedical informatics that will eventually allow the human proteome to be deciphered.
COMPARTMENTALIZED RAS/MAPK SIGNALING
Authors: Mor Adam , Philips Mark R.
Published: January 2006

Signal transduction down the Ras/MAPK pathway, including that critical to T cell activation, proliferation, and differentiation, has been generally considered to occur at the plasma membrane. It is now clear that the plasma membrane does not represent the only platform for Ras/MAPK signaling. Moreover, the plasma membrane itself is no longer considered a uniform structure but rather a patchwork of microdomains that can compartmentalize signaling. Signaling on internal membranes was first recognized on endosomes. Genetically encoded fluorescent probes for signaling events such as GTP/GDP exchange on Ras have revealed signaling on a variety of intracellular membranes, including the Golgi apparatus. In fibroblasts, Ras is activated on the plasma membrane and Golgi with distinct kinetics. The pathway by which Golgi-associated Ras becomes activated involves PLCγ and RasGRP1 and may also require retrograde trafficking of Ras from the plasma membrane to the Golgi as a consequence of depalmitoylation. Thus, the Ras/MAPK pathway represents a clear example of compartmentalized signaling.
DETERMINANTS OF LYMPHOID-MYELOID LINEAGE DIVERSIFICATION
Authors: Laiosa Catherine V. , Stadtfeld Matthias , Graf Thomas
Published: January 2006

In recent years, investigators have made great progress in delineating developmental pathways of several lymphoid and myeloid lineages and in identifying transcription factors that establish and maintain their fate. However, the developmental branching points between these two large cell compartments are still controversial, and little is known about how their diversification is induced. Here, we give an overview of determinants that play a role at lymphoid-myeloid junctures, in particular transcription factors and cytokine receptors. Experiments showing that myeloid lineages can be reversibly reprogrammed into one another by transcription factor network perturbations are used to highlight key principles of lineage commitment. We also discuss experiments showing that lymphoid-to-myeloid but not myeloid-to-lymphoid conversions can be induced by the enforced expression of a single transcription factor. We close by proposing that this asymmetry is related to a higher complexity of transcription factor networks in lymphoid cells compared with myeloid cells, and we suggest that this feature must be considered when searching for mechanisms by which hematopoietic stem cells become committed to lymphoid lineages.
REFLECTIONS ON PHYSICAL CHEMISTRY: Science and Scientists
Authors: Jortner Joshua
Published: January 2006

▪ Abstract  This is the story of a young person who grew up in Tel-Aviv during the period of the establishment of the State of Israel and was inspired to become a physical chemist by the cultural environment, by the excellent high-school education, and by having been trained by some outstanding scientists at the Hebrew University of Jerusalem and, subsequently, by the intellectual environment and high-quality scientific endeavor at the University of Chicago. Since serving as the first chairman of the Chemistry Department of the newly formed Tel-Aviv University he has been immersed in research, in the training of young scientists, and in intensive and extensive international scientific collaboration. Together with the members of his “scientific family” he has explored the phenomena of energy acquisition, storage and disposal and structure-dynamics-function relations in large molecules, condensed phase, clusters and biomolecules, and is looking forward to many future adventures in physical chemistry.

“What to leave out and what to put in? That's the problem.” Hugh Lofting, Doctor Dolittle's Zoo, 1925
COHERENT EXCITATION OF VIBRATIONAL MODES IN METALLIC NANOPARTICLES
Authors: Hartland Gregory V.
Published: January 2006

▪ Abstract  Excitation of metal nanoparticles with subpicosecond laser pulses causes a rapid increase in the lattice temperature, which can impulsively excite the phonon modes of the particle that correlate with the expansion coordinates. The vibrational periods depend on the size, shape, and elastic constants of the particles. Thus, time-resolved spectroscopy can be used to examine the material properties of nanometer-sized objects. This review provides a brief overview of the steady-state and time-resolved electronic spectroscopy of metal particles, which is important for understanding why vibrational motion appears in transient absorption traces. I also describe how the vibrational modes observed in the experiments are assigned, and what information can be obtained from the measurements. Our work has been mainly concerned with noble metal particles (gold and silver) in aqueous solution. The different shapes that have been examined to date include spheres, rods, and triangles, all with different sizes.
DYNAMICAL STUDIES OF THE OZONE ISOTOPE EFFECT: A Status Report
Authors: Schinke R. , Grebenshchikov S. Yu. , Ivanov M.V. , Fleurat-Lessard P.
Published: January 2006

▪ Abstract  Dynamical studies of the recombination of O and O2 to form ozone are reviewed. The focus is the intriguing isotope dependence of the recombination rate coefficient as observed by Mauersberger and coworkers in the last decade. The key quantity for understanding of this dependence appears to be the difference of zero-point energies of the two fragmentation channels to which excited ozone can dissociate, i.e., X + YZ ← XYZ* → XY + Z, where X, Y, and Z stand for the three isotopes of oxygen. Besides the isotope dependence, the variation of the recombination rate coefficient with pressure and temperature is also addressed. Despite the numerous approaches of recent years, the recombination of ozone is far from being satisfactorily explained; there are still several essential questions to be solved by detailed theoretical analysis. We mainly discuss—and critically assess—the results of our own investigations of the ozone kinetics. The work of other research groups is also evaluated.
SCANNING TUNNELING MICROSCOPY MANIPULATION OF COMPLEX ORGANIC MOLECULES ON SOLID SURFACES
Authors: Otero Roberto , Rosei Federico , Besenbacher Flemming
Published: January 2006

▪ Abstract  Organic molecules adsorbed on solid surfaces display a fascinating variety of new physical and chemical phenomena ranging from self-assembly and molecular recognition to nonlinear optical properties and current rectification. Both the fundamental interest in these systems and the promise of technological applications have motivated a strong research effort in understanding and controlling these properties. Scanning tunneling microscopy (STM) and, in particular, its ability to manipulate individual adsorbed molecules, has become a powerful tool for studying the adsorption geometry and the conformation and dynamics of single molecules and molecular aggregates. Here we review selected case studies demonstrating the enormous capabilities of STM manipulations to explore basic physiochemical properties of adsorbed molecules. In particular, we emphasize the role of STM manipulations in studying the coupling between the multiple degrees of freedom of adsorbed molecules, the phenomenon of molecular molding, and the possibility of creating and breaking individual chemical bonds in a controlled manner, i.e., the concept of single-molecule chemistry.
SINGLE-MOLECULE ELECTRICAL JUNCTIONS
Authors: Selzer Yoram , Allara David L.
Published: January 2006

▪ Abstract  The objective of this review is to describe current experimental research of single-molecule electrical junctions in the context of various theoretical frameworks, with emphasis on the application of single-electron transistor theory to molecular junctions. Molecule quantum dots are at least an order of magnitude smaller than semiconductor quantum dots, which allows the study of many of the same mesoscopic and many-body effects at far higher temperatures. We discuss processes such as cotunneling, sequential tunneling, and incoherent tunneling, as well as the Kondo effect, Zeeman splitting, and the Coulomb diamond. Goals for future experimental work are outlined.
Measurement of myocardial free radical production during exercise using EPR spectroscopy
Authors: Traverse Jay H. , Nesmelov Yuri E. , Crampton Melanie , Lindstrom Paul , Thomas David D. , Bache Robert J.
Published: January 2006

Exercise is associated with an increase in oxygen flux through the mitochondrial electron transport chain that has recently been demonstrated to increase the production of reactive oxygen species (ROS) in skeletal muscle. This study examined whether exercise also causes free radical production in the heart. We measured ROS production in seven chronically instrumented dogs during rest and treadmill exercise (6.4 km/h at 10° grade; and heart rate, 204 ± 3 beats/min) using electron paramagnetic resonance spectroscopy in conjunction with the spin trap α-phenyl- tert-butylnitrone (PBN) (0.14 mol/l) in blood collected from the aorta and coronary sinus (CS). To improve signal detection, the free radical adducts were deoxygenated over a nitrogen stream for 15 min and extracted with toluene. The hyperfine splitting constants of the radicals were αN = 13.7 G and αH = 1.0 G, consistent with an alkoxyl or carbon-centered radical. Resting aortic and CS PBN adduct concentrations were 6.7 and 6.3 × 108 arbitrary units ( P = not significant). Both aortic and CS adduct concentrations increased during exercise, but there was no significant difference between the aortic and CS concentrations. Thus, in contrast to skeletal muscle, submaximal treadmill exercise did not result in detectable free radical production by the heart.
Hypoxia promotes relaxation of bovine coronary arteries through lowering cytosolic NADPH
Authors: Gupte Sachin A. , Wolin Michael S.
Published: January 2006

Hypoxia relaxes endothelium-denuded bovine coronary arteries (BCA) through mechanisms that do not appear to involve reactive oxygen species, prostaglandins, or nitric oxide. Because of similarities in the relaxation of BCA to hypoxia (Po2= 8–10 Torr) and inhibitors of the pentose phosphate pathway (PPP) including 6-aminonicotinamide and epiandrosterone, we measured NADPH and NADP and found that hypoxia caused NADPH oxidation (decreased NADPH/NADP). The relaxation to hypoxia was similar to previously reported properties of relaxation to PPP inhibitors in that both responses were associated with glutathione oxidation and depressed intracellular calcium release and calcium influx-mediated contractile responses. Inhibitors of potassium channels had minimal effects on these relaxation responses. Relaxation to hypoxia and PPP inhibitors were attenuated by a thiol reductant (3 mM dithiothreitol) and by eliciting contraction with an activator of protein kinase C (phorbol 12,13-dibutyrate). In the presence of contraction to U-46619, relaxation to hypoxia and PPP inhibitors were attenuated by the sarco(endo)plasmic reticulum Ca2+-ATPase pump inhibitor 200 μM cyclopiazonic acid and by 10 mM pyruvate. Hypoxia decreased BCA levels of glucose-6-phosphate but not ATP. Pyruvate prevented the hypoxia-elicited decrease in glucose-6-phosphate and glutathione oxidation, and it increased NADPH levels under hypoxia to levels observed under normoxia. Thus hypoxia causes a metabolic stress on the PPP that promotes BCA relaxation through processes controlled by lowering the levels of cytosolic NADPH.
Heterogeneous response of microvascular endothelial cells to shear stress
Authors: Hong D. , Jaron D. , Buerk D. G. , Barbee K. A.
Published: January 2006

We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.
Alterations in dihydropyridine receptors in dystrophin-deficient cardiac muscle
Authors: Woolf Peter J. , Lu Sai , Cornford-Nairn Renee , Watson Michael , Xiao Xiao-Hui , Holroyd Sean M. , Brown Lindsay , Hoey Andrew J.
Published: January 2006

The deficiency of dystrophin, a critical membrane stabilizing protein, in the mdx mouse causes an elevation in intracellular calcium in myocytes. One mechanism that could elicit increases in intracellular calcium is enhanced influx via the L-type calcium channels. This study investigated the effects of the dihydropyridines BAY K 8644 and nifedipine and alterations in dihydropyridine receptors in dystrophin-deficient mdx hearts. A lower force of contraction and a reduced potency of extracellular calcium ( P < 0.05) were evident in mdx left atria. The dihydropyridine agonist BAY K 8644 and antagonist nifedipine had 2.7- and 1.9-fold lower potencies in contracting left atria ( P < 0.05). This corresponded with a 2.0-fold reduction in dihydropyridine receptor affinity evident from radioligand binding studies of mdx ventricular homogenates ( P < 0.05). Increased ventricular dihydropyridine receptor protein was evident from both radioligand binding studies and Western blot analysis and was accompanied by increased mRNA levels ( P < 0.05). Patch-clamp studies in isolated ventricular myocytes showed no change in L-type calcium current density but revealed delayed channel inactivation ( P < 0.05). This study indicates that a deficiency of dystrophin leads to changes in dihydropyridine receptors and L-type calcium channel properties that may contribute to enhanced calcium influx. Increased influx is a potential mechanism for the calcium overload observed in dystrophin-deficient cardiac muscle.
Pressure-calcium relationships in perfused mouse hearts
Authors: MacGowan Guy A. , Kirk Jonathan A. , Evans Caroline , Shroff Sanjeev G.
Published: January 2006

We explored the relationship between left ventricular (LV) pressure and intracellular free calcium concentration ([Ca]i) in the isolated perfused mouse heart. [Ca]i(rhod-2) and LV pressure were recorded simultaneously. In response to increases in LV volume (Frank-Starling, FS, protocol), there were increases in developed pressure (up to 250%), with no changes in pressure morphology (rise or relaxation time) or [Ca]i(magnitude and morphology) for up to 10 min. During transient increases in the stimulus interval at a fixed LV volume (mechanical restitution, MR, protocol), developed pressure increased significantly (31.3 ± 1.2%), with relatively small changes in peak systolic [Ca]i(7.4 ± 1.4%). The relaxation of [Ca]i, however, was prolonged (30.0 ± 5.5%), resulting in prolonged pressure relaxation (21.2 ± 1.9%) and increased area under the calcium transient that paralleled the increase in developed pressure (1:1 ratio). A model-based analysis showed that changes in LV pressure during the MR protocol could be completely explained by altered [Ca]i; it was not necessary to invoke any changes in model parameters (i.e., dynamic processes that link calcium to pressure). For the FS data, the model predicted only a change in the gain parameter; however, this change alone cannot reproduce well-established length-dependent changes in the steady-state force-pCa relationship. In summary, the mouse myocardium appears to be unique in that significant changes in peak developed pressure can occur with little or no change in the peak [Ca]i. Additionally, unlike other mammalian species, load-dependent prolongation of pressure relaxation is absent in the mouse heart, and pressure relaxation is primarily governed by intracellular free calcium relaxation.
Role of adiponectin receptors in endothelin-induced cellular hypertrophy in cultured cardiomyocytes and their expression in infarcted heart
Authors: Fujioka Daisuke , Kawabata Ken-ichi , Saito Yukio , Kobayashi Tsuyoshi , Nakamura Takamitsu , Kodama Yasushi , Takano Hajime , Obata Jyun-ei , Kitta Yoshinobu , Umetani Ken , Kugiyama Kiyotaka
Published: January 2006

Adiponectin, an adipocyte-derived protein, has cardioprotective actions. We elucidated the role of the adiponectin receptors AdipoR1 and AdipoR2 in the effects of adiponectin on endothelin-1 (ET-1)-induced hypertrophy in cultured cardiomyocytes, and we examined the expression of adiponectin receptors in normal and infarcted mouse hearts. Recombinant full-length adiponectin suppressed the ET-1-induced increase in cell surface area and [3H]leucine incorporation into cultured cardiomyocytes compared with cells treated with ET-1 alone. Transfection of small interfering RNA (siRNA) specific for AdipoR1 or AdipoR2 reversed the suppressive effects of adiponectin on ET-1-induced cellular hypertrophy in cultured cardiomyocytes. Adiponectin induced phosphorylation of AMP-activated protein kinase (AMPK) and inhibited ET-1-induced ERK1/2 phosphorylation, which were also reversible by transfection of siRNA for AdipoR1 or AdipoR2 in cultured cardiomyocytes. Transfection of siRNA for α2-catalytic subunits of AMPK reduced the inhibitory effects of adiponectin on ET-1-induced cellular hypertrophy and ERK1/2 phosphorylation. Effects of globular adiponectin were similar to those of full-length adiponectin, and siRNA for AdipoR1 reversed the actions of globular adiponectin. Compared with normal left ventricle, expression levels of AdipoR1 mRNA and protein were decreased in the remote, as well as the infarcted, area after myocardial infarction in mouse hearts. In conclusion, AdipoR1 and AdipoR2 mediate the suppressive effects of full-length and globular adiponectin on ET-1-induced hypertrophy in cultured cardiomyocytes, and AMPK is involved in signal transduction through these receptors. AdipoR1 and AdipoR2 might play a role in the pathogenesis of ET-1-related cardiomyocyte hypertrophy after myocardial infarction.
Multimodal functional cardiac MRI in creatine kinase-deficient mice reveals subtle abnormalities in myocardial perfusion and mechanics
Authors: Nahrendorf Matthias , Streif Jörg U. , Hiller Karl-Heinz , Hu Kai , Nordbeck Peter , Ritter Oliver , Sosnovik David , Bauer Lisa , Neubauer Stefan , Jakob Peter M. , Ertl Georg , Spindler Matthias , Bauer Wolfgang R.
Published: January 2006

A decrease in the supply of ATP from the creatine kinase (CK) system is thought to contribute to the evolution of heart failure. However, previous studies on mice with a combined knockout of the mitochondrial and cytosolic CK (CK−/−) have not revealed overt left ventricular dysfunction. The aim of this study was to employ novel MRI techniques to measure maximal myocardial velocity ( Vmax) and myocardial perfusion and thus determine whether abnormalities in the myocardial phenotype existed in CK−/− mice, both at baseline and 4 wk after myocardial infarction (MI). As a result, myocardial hypertrophy was seen in all CK−/− mice, but ejection fraction (EF) remained normal. Vmax, however, was significantly reduced in the CK−/− mice [wild-type, 2.32 ± 0.09 vs. CK−/−, 1.43 ± 0.16 cm/s, P < 0.05; and wild-type MI, 1.53 ± 0.11 vs. CK−/− MI, 1.26 ± 0.11 cm/s, P = not significant (NS), P < 0.05 vs. baseline]. Myocardial perfusion was also lower in the CK−/− mice (wild-type, 6.68 ± 0.27 vs. CK−/−, 4.12 ± 0.63 ml/g·min, P < 0.05; and wild-type MI, 3.97 ± 0.65 vs. CK−/− MI, 3.71 ± 0.57 ml/g·min, P = NS, P < 0.05 vs. baseline), paralleled by a significantly reduced capillary density (histology). In conclusion, myocardial function in transgenic mice may appear normal when only gross indexes of performance such as EF are assessed. However, the use of a combination of novel MRI techniques to measure myocardial perfusion and mechanics allowed the abnormalities in the CK−/− phenotype to be detected. The myocardium in CK-deficient mice is characterized by reduced perfusion and reduced maximal contraction velocity, suggesting that the myocardial hypertrophy seen in these mice cannot fully compensate for the absence of the CK system.
Regulation of antisense RNA expression during cardiac MHC gene switching in response to pressure overload
Authors: Haddad F. , Qin A. X. , Bodell P. W. , Zhang L. Y. , Guo H. , Giger J. M. , Baldwin K. M.
Published: January 2006

Hypertension has been shown to cause cardiac hypertrophy and a shift in myosin heavy chain (MHC) gene expression from the faster α- to slower β-MHC isoform. The expression of the β- and α-MHC pre-mRNAs, mRNAs, as well as the newly discovered antisense β-RNA were analyzed in three regions of the normal control (NC) and 12-day pressure-overloaded (AbCon) hearts: the left ventricle apex, left ventricle base, and the septum. The RNA analyses in the AbCon heart targeted both the 5′ and the 3′ ends of each RNA molecule. β-MHC mRNA expression significantly increased relative to control in all three regions, regardless of the target site (5′ or 3′ end). In contrast, β-MHC pre-mRNA expression in the AbCon heart depended on the site of the measurement (5′ vs. 3′ end). For example, whereas the pre-mRNA did not change when targeted at the 3′ end (last intron), it increased significantly in the AbCon heart when measurement targeted the 5′ end (2nd intron) of the 25-kb molecule. Analyses of the antisense β-RNA revealed that its expression in the AbCon heart was significantly decreased relative to control regardless of its measurement site. A negative correlation was observed between the β-mRNA expression and the antisense β-RNA ( P < 0.05), suggesting an inhibitory role of antisense RNA on the sense β-MHC gene expression. In contrast, a positive correlation was observed between the antisense β-RNA and the α-MHC pre-mRNA ( P < 0.05). This latter observation along with the α-MHC gene position relative to that of the β-antisense suggest that the α-MHC sense and β-antisense transcription are coregulated likely via common intergenic regulatory sequences. Our results suggest that the increased β-MHC expression in the AbCon heart not only is the result of increased β-MHC transcription but also involves an antisense β-RNA regulation scheme. Although the exact mechanism concerning antisense regulation is not clear, it could involve modulation of both transcriptional activity of the β-MHC gene and posttranscriptional processing.
Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction
Authors: Hassan Ghada S. , Williams Terence M. , Frank Philippe G. , Lisanti Michael P.
Published: January 2006

We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient (Cav-1−/−) mice and characterized these cells with respect to their proliferation, migration, and Ca2+response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2′-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in Cav-1−/−compared with wild-type (Cav-1+/+) AoSMCs. Cav-1−/−AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor p27Kip1. The Ca2+response was examined in the presence of ET-1 and assessed by confocal microscopy with the Ca2+-sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-1−/−AoSMCs exhibited a faster and larger increase in free intracellular Ca2+than Cav-1+/+cells. The ET-1-induced response in Cav-1−/−cells was mediated by the ETBreceptor, as shown using the ETBreceptor antagonist BQ-788 and the ETAreceptor antagonist BQ-123. In Cav-1−/−cells, ETAreceptor expression was reduced and ETBreceptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced Ca2+response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and Ca2+-mediated signaling.
Estrogen receptor-α mediates acute myocardial protection in females
Authors: Wang Meijing , Crisostomo Paul , Wairiuko George M. , Meldrum Daniel R.
Published: January 2006

Sex differences in myocardial recovery have been reported after acute ischemia and reperfusion injury. Estrogen and the estrogen receptor are critical determinants of cardiovascular sex differences. However, the mechanistic pathways responsible for these differences remain unknown. We hypothesized that estrogen receptor-α is an important modulator of 1) myocardial functional recovery after ischemia and 2) inflammatory signaling via MAPK. To study this, adult male and female wild-type (WT) and estrogen receptor-α knockout (ER1KO) mouse hearts were isolated, perfused via Langendorff model, and subjected to 20 min of ischemia and 60 min of reperfusion. Myocardial contractile function (left ventricular developed pressure and positive and negative first derivative of pressure) was continuously recorded. After ischemia-reperfusion, hearts were assessed for expression of inflammatory cytokines (ELISA) and activation of MAPK and caspase-3 (Western blot analysis). Data were analyzed with two-way ANOVA or Student's t-test, and P < 0.05 was statistically significant. ER1KO females exhibited significantly less functional recovery than WT females and were similar to WT males. Activated ERK was increased in female WT hearts compared with female ER1KO. Activated JNK was decreased in female WT hearts compared with female ER1KO. No significant differences were found between male WT, female WT, male ER1KO, and female ER1KO in activated p38 MAPK, proinflammatory cytokine expression, and proapoptotic signaling. Estrogen receptor-α plays a role in the protection observed in the female heart. Differential activation of MAPK may mediate this protection. Further studies are necessary to delineate these mechanistic pathways.
Transforming growth factor-β1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair?
Authors: Lechapt-Zalcman E. , Prulière-Escabasse V. , Advenier D. , Galiacy S. , Charrière-Bertrand C. , Coste A. , Harf A. , d'Ortho M.-P. , Escudier E.
Published: January 2006

In vivo, transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMPs) present at the site of airway injury are thought to contribute to epithelial wound repair. As TGF-β1 can modulate MMP expression and MMPs play an important role in wound repair, we hypothesized that TGF-β1 may enhance airway epithelial repair via MMPs secreted by epithelial cells. We evaluated the in vitro influence of TGF-β1 on wound repair in human airway epithelial cells cultured under conditions allowing differentiation. The results showed that TGF-β1 accelerated in vitro airway wound repair, whereas MMP inhibitors prevented this acceleration. In parallel, we examined the effect of TGF-β1 on the expression of MMP-2 and MMP-9. TGF-β1 induced a dramatic increase of MMP-2 expression with an increased steady-state level of MMP-2 mRNA, contrasting with a slight increase in MMP-9 expression. To confirm the role of MMP-2, we subsequently evaluated the effect of MMP-2 on in vitro airway wound repair and demonstrated that the addition of MMP-2 reproduced the acceleration of wound repair induced by TGF-β1. These results strongly suggest that TGF-β1 increases in vitro airway wound repair via MMP-2 upregulation. It also raises the issue of a different in vivo biological role of MMP-2 and MMP-9 depending on the cytokine microenvironment.
Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells
Authors: Bogatcheva Natalia V. , Wang Peiyi , Birukova Anna A. , Verin Alexander D. , Garcia Joe G. N.
Published: January 2006

In this study, we demonstrate that challenge of endothelial cells (EC) with NaF, a recognized G protein activator and protein phosphatase inhibitor, leads to a significant Erk activation, with increased phosphorylation of the well-known Erk substrate caldesmon. Inhibition of the Erk MAPK, MEK, by U0126 produces a marked decrease in NaF-induced caldesmon phosphorylation. NaF transiently increases the activity of the MEK kinase known as Raf-1 (∼3- to 4-fold increase over basal level), followed by a sustained Raf-1 inhibition (∼3- to 4-fold decrease). Selective Raf-1 inhibitors (ZM-336372 and Raf-1 inhibitor 1) significantly attenuate NaF-induced Erk and caldesmon phosphorylation. Because we have previously shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) participates in Erk activation in thrombin-challenged cells, we next explored if CaMKII is involved in NaF-induced EC responses. We found that in NaF-treated EC, CaMKII activity increases in a time-dependent manner with maximal activity at 10 min (∼4-fold increase over a basal level). Pretreatment with KN93, a specific CaMKII inhibitor, attenuates NaF-induced barrier dysfunction and Erk phosphorylation. The Rho inhibitor C3 exotoxin completely abolishes NaF-induced CaMKII activation. Collectively, these data suggest that sequential activation of Raf-1, MEK, and Erk is modulated by Rho-dependent CaMKII activation and represents important NaF-induced signaling response. Caldesmon phosphorylation occurring by an Erk-dependent mechanism in NaF-treated pulmonary EC may represent a link between NaF stimulation and contractile responses of endothelium.
Effects of macrophage inducible nitric oxide synthase in murine septic lung injury
Authors: Farley K. S. , Wang L. F. , Razavi H. M. , Law C. , Rohan M. , McCormack D. G. , Mehta S.
Published: January 2006

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS−/− AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 ± 0.1 vs. 1.4 ± 0.1 μg EB·g lung−1·min−1; P < 0.05) and MPO activity (37 ± 4 vs. 67 ± 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 ± 0.3 μg EB·g lung−1·min−1) but not with iNOS−/− donor AM. In iNOS−/− mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS−/− mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.
Viscoelastic and dynamic nonlinear properties of airway smooth muscle tissue: roles of mechanical force and the cytoskeleton
Authors: Ito Satoru , Majumdar Arnab , Kume Hiroaki , Shimokata Kaoru , Naruse Keiji , Lutchen Kenneth R. , Stamenović Dimitrije , Suki Béla
Published: January 2006

The viscoelastic and dynamic nonlinear properties of guinea pig tracheal smooth muscle tissues were investigated by measuring the storage (G′) and loss (G") moduli using pseudorandom small-amplitude length oscillations between 0.12 and 3.5 Hz superimposed on static strains of either 10 or 20% of initial length. The G" and G′ spectra were interpreted using a linear viscoelastic model incorporating damping (G) and stiffness (H), respectively. Both G and H were elevated following an increase in strain from 10 to 20%. There was no change in harmonic distortion ( Kd), an index of dynamic nonlinearity, between 10 and 20% strains. Application of methacholine at 10% strain significantly increased G and H while it decreased Kd. Cytochalasin D, isoproterenol, and HA-1077, a Rho-kinase inhibitor, significantly decreased both G and H but increased Kd. Following cytochalasin D, G, H, and Kd were all elevated when mean strain increased from 10 to 20%. There were no changes in hysteresivity, G/H, under any condition. We conclude that not all aspects of the viscoelastic properties of tracheal smooth muscle strips are similar to those previously observed in cultured cells. We attribute these differences to the contribution of the extracellular matrix. Additionally, using a network model, we show that the dynamic nonlinear behavior, which has not been observed in cell culture, is associated with the state of the contractile stress and may derive from active polymerization within the cytoskeleton.
Inhaled marijuana smoke disrupts mitochondrial energetics in pulmonary epithelial cells in vivo
Authors: Sarafian Theodore A. , Habib Nancy , Oldham Michael , Seeram Navindra , Lee Ru-Po , Lin Laura , Tashkin Donald P. , Roth Michael D.
Published: January 2006

Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8–10 times higher than those measured in blood (75 ± 38 ng/g wet wt tissue vs. 9.2 ± 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.
Streptococcus pneumoniaeinduced p38 MAPK- and NF-κB-dependent COX-2 expression in human lung epithelium
Authors: N’Guessan Philippe Dje , Hippenstiel Stefan , Etouem Mirabelle O. , Zahlten Janine , Beermann Wiebke , Lindner David , Opitz Bastian , Witzenrath Martin , Rosseau Simone , Suttorp Norbert , Schmeck Bernd
Published: January 2006

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE2, are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE2production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE2. Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IκBα and DNA binding of NF-κB. A specific peptide inhibitor of the IκBα kinase complex blocked pneumococci-induced PGE2release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE2release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-κB activation: dominant-negative mutants of p38 MAPK isoforms α, β2, γ, and δ blocked S. pneumoniae-induced NF-κB activation. In addition, recruitment of NF-κB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-κB-controlled COX-2 expression and subsequent PGE2release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.
Discordant regulatory changes in monocrotaline-induced megalocytosis of lung arterial endothelial and alveolar epithelial cells
Authors: Mukhopadhyay Somshuvra , Sehgal Pravin B.
Published: January 2006

Monocrotaline (MCT) causes pulmonary hypertension in the rat by a mechanism characterized by megalocytosis (enlarged cells with enlarged endoplasmic reticulum and Golgi and a cell cycle arrest) of pulmonary arterial endothelial (PAEC), arterial smooth muscle, and type II alveolar epithelial cells. In cell culture, although megalocytosis is associated with a block in entry into mitosis in both lung endothelial and epithelial cells, DNA synthesis is stimulated in endothelial but inhibited in epithelial cells. The molecular mechanism(s) for this dichotomy are unclear. While MCTP-treated PAEC and lung epithelial (A549) cells both showed an increase in the “promitogenic” transcription factor STAT3 levels and in the IL-6-induced nuclear pool of PY-STAT3, this was transcriptionally inactive in A549 but not in PAEC cells. This lack of transcriptional activity of STAT3 in A549 cells correlated with the cytoplasmic sequestration of the STAT3 coactivators CBP/p300 and SRC1/NcoA in A549 cells but not in PAEC. Both cell types displayed a Golgi trafficking block, loss of caveolin-1 rafts, and increased nuclear Ire1α, but an incomplete unfolded protein response (UPR) with little change in levels of UPR-induced chaperones including GRP78/BiP. There were discordant alterations in cell cycle regulatory proteins in the two cell types such as increase in levels of both cyclin D1 and p21 simultaneously, but with a decrease in cdc2/cdk1, a kinase required for entry into mitosis. While both cell types showed increased cytoplasmic geminin, the DNA synthesis-initiating protein Cdt1 was predominantly nuclear in PAEC but remained cytoplasmic in A549 cells, consistent with the stimulation of DNA synthesis in the former but an inhibition in the latter cell type. Thus differences in cell type-specific alterations in subcellular trafficking of critical regulatory molecules (such as CBP/p300, SRC1/NcoA, Cdt1) likely account for the dichotomy of the effects of MCTP on DNA synthesis in endothelial and epithelial cells.
Levosimendan protects against experimental endotoxemic acute renal failure
Authors: Zager Richard A. , Johnson Ali C. , Lund Steve , Hanson Sherry Y. , Abrass Christine K.
Published: January 2006

Endotoxemia induces a hemodynamic form of acute renal failure (ARF; renal vasoconstriction ± reduced glomerular ultrafiltration coefficient, Kf; minimal/no histological damage). We tested whether levosimendan (LS), an ATP-sensitive K+ (KATP) channel opener with cardiac ionotropic and possible anti-inflammatory properties, might have utility in combating this form of ARF. CD-1 mice were injected with LPS ± LS. LS effects on LPS-induced systemic inflammation (plasma TNF-α/MCP-1; cardiorenal mRNAs), plasma NO levels, and azotemia were assessed. Because KATP channel opening has been reported to mediate hypoxic tubular injury, possible adverse LS effects on ischemic ARF and ATP depletion injury were sought. Effects of diazoxide (another KATP channel agonist) and glibenclamide (a channel antagonist) on hypoxic tubular injury also were assessed. Finally, the ability of LS to alter rat mesangial cell (MC) contraction in response to ANG II (elevated in sepsis) was tested. LS conferred almost complete protection against LPS-induced ARF, without any apparent reduction in the LPS-induced inflammatory response. Neither LS nor diazoxide altered ATP depletion-mediated tubule injury (in vivo or in vitro). Conversely, glibenclamide induced a marked and direct cytotoxic effect. LS completely blocked ANG II-induced MC contraction, an action likely to increase Kf. We concluded that 1) LS can confer marked protection against LPS-induced ARF; 2) this likely stems from vasoactive properties, rather than reductions in LPS-induced inflammation; and 3) KATP channel agonists (but not antagonists) appear to be devoid of toxic proximal tubular cell effects. This suggests that LS, and other KATP channel agonists, have a margin of safety if employed in situations (sepsis syndrome, heart failure) in which severe renal vasoconstriction might lead to ischemic ARF.
Distinct pattern of lung gene expression in theCftr-KO mice developing spontaneous lung disease compared with their littermate controls
Authors: Guilbault Claudine , Novak Jaroslav P. , Martin Patricia , Boghdady Marie-Linda , Saeed Zienab , Guiot Marie-Christine , Hudson Thomas J. , Radzioch Danuta
Published: January 2006

Cystic fibrosis (CF) is caused by a defect in the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel. Dysfunction of the CFTR protein results in salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility, and severe pulmonary disease. Most of the morbidity and mortality of CF patients results from pulmonary complications. Differences in susceptibility to bacterial infection and variable degree of CF lung disease among CF patients remain unexplained. Many phenotypic expressions of the disease do not directly correlate with the type of mutation in the Cftr gene. Using a unique CF mouse model that mimics aspects of human CF lung disease, we analyzed the differential gene expression pattern between the normal lungs of wild-type mice (WT) and the affected lungs of CFTR knockout mice (KO). Using microarray analysis followed by quantitation of candidate gene mRNA and protein expression, we identified many interesting genes involved in the development of CF lung disease in mice. These findings point to distinct mechanisms of gene expression regulation between mice with CF and control mice.