bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes
Authors: Mongin Alexander A. , Kimelberg Harold K.
Published: May 2013

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.
PKCζ participates in activation of inflammatory response induced by enteropathogenic E. coli
Authors: Savkovic Suzana D. , Koutsouris Athanasia , Hecht Gail
Published: May 2013

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-κB and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-κB activation but that other signaling molecules such as PKCζ may be involved. The aim of this study was to determine whether PKCζ is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKCζ from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKCζ by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKCζ pseudosubstrate (MYR-PKCζ-PS), or transient expression of a nonfunctional PKCζ significantly suppressed EPEC-induced IκBα phosphorylation. Although PKCζ can activate ERK, MYR-PKCζ-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKCζ can regulate NF-κB activation by interacting with and activating IκB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKCζ and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKCζ-IKK complexes from infected intestinal epithelial cells and recombinant IκBα as a substrate showed a 2.5-fold increase in IκBα phosphorylation. PKCζ can also regulate NF-κB by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKCζ-PS, suggesting that other signaling events may be involved in this particular arm of NF-κB regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKCζ and ERK that lead to NF-κB activation, thus ensuring the proinflammatory response.
Structural and functional relationships between Ca2+ puffs and mitochondria in Xenopusoocytes
Authors: Marchant Jonathan S. , Ramos Viviana , Parker Ian
Published: May 2013

Ca2+ uptake and release from endoplasmic reticulum (ER) and mitochondrial Ca2+ stores play important physiological and pathological roles, and these processes are shaped by interactions that depend on the structural intimacy between these organelles. Here we investigate the morphological and functional relationships between mitochondria, ER, and the sites of intracellular Ca2+ release in Xenopus laevis oocytes by combining confocal imaging of local Ca2+ release events (“Ca2+ puffs”) with mitochondrial localization visualized using vital dyes and subcellularly targeted fluorescent proteins. Mitochondria and ER are localized in cortical bands ∼6–8 μm wide, with the mitochondria arranged as densely packed “islands” interconnected by discrete strands. The ER is concentrated more superficially than mitochondria, and the mean separation between Ca2+ puff sites and mitochondria is ∼2.3 μm. However, a subpopulation of Ca2+ puff sites is intimately associated with mitochondria (∼28% within <600 nm), a greater number than expected if Ca2+ puff sites were randomly distributed. Ca2+release sites close to mitochondria exhibit lower Ca2+ puff activity than Ca2+ puff sites in regions with lower mitochondrial density. Furthermore, Ca2+ puff sites in close association with mitochondria rarely serve as the sites for Ca2+ wave initiation. We conclude that mitochondria play important roles in regulating local ER excitability, Ca2+wave initiation, and, thereby, spatial patterning of global Ca2+ signals.
Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes
Authors: Sathiyaa Ramesh , Vijayan Mathilakath M.
Published: May 2013

We used primary cultures of trout hepatocytes and a physiological dose of cortisol (100 ng/ml), mimicking stressed levels in salmonid fish, to address the impact of glucocorticoid stimulation on glucocorticoid receptor (GR) mRNA abundance and protein content. Cortisol significantly elevated GR mRNA content over a 24-h period; this increase was abolished by actinomycin D, suggesting transcriptional control of GR. However, cortisol significantly decreased GR protein content, leading us to hypothesize that lower GR protein content may be regulating GR mRNA abundance. Indeed, treatment of hepatocytes with MG-132, a proteasomal inhibitor shown to prevent GR degradation by cortisol, abolished cortisol-mediated GR mRNA upregulation. Also, geldanamycin, a heat shock protein 90-specific inhibitor, abolished the GR mRNA increase evident with cortisol but did not modify cortisol-induced increases in abundance of mRNA for phosphoenolpyruvate carboxykinase, a glucocorticoid-responsive gene, or hepatocyte glucose release. Together, our results suggest a negative feedback loop for GR gene regulation by cortisol in trout hepatocytes. The autoregulation of GR may be a crucial step in the physiological stress response process, especially in modulating energy-dependent processes that are glucocorticoid dependent, including gluconeogenesis.
Regulation of the human vitamin C transporters expressed in COS-1 cells by protein kinase C
Authors: Liang Wei-Jun , Johnson Daniel , Ma Li-Sha , Jarvis Simon M.
Published: May 2013

Protein kinase C (PKC) regulation ofl-ascorbic acid transport mediated by the Na+/ascorbic acid transporters, hSVCT1 and hSVCT2, expressed in COS-1 cells was studied using recombinant carboxyl-terminal V5 epitope-tagged forms of the transporters. The PKC activator phorbol 12-myristate 13-acetate (PMA) caused a time-dependent and concentration-dependent decrease (40–60%) in ascorbic acid transport activity. Effects of PMA were not observed with the inactive phorbol ester 4α-phorbol and were reversed by treatment of the cells with the PKC-specific inhibitor Ro-31–8220. Kinetically, the reduction in hSVCT1 and hSVCT2 activity arose from a decrease in maximal velocity with no change in the apparent affinity. Western blot and confocal microscopy analyses indicated that the total pool of hSVCT1 or hSVCT2 proteins expressed in the transfected COS-1 cells remained unaffected by PMA treatment. For hSVCT1 the decrease inl-ascorbic acid correlated with a redistribution of the transporter from the cell surface to intracellular membranes. However, for hSVCT2 there was no apparent change in transporter distribution, suggesting that the PKC-dependent modulation of l-ascorbic acid transport mediated by hSVCT2 was the result of reduced catalytic transport efficiency.
Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells
Authors: Schramek Herbert , Feifel Elisabeth , Marschitz Ingrid , Golochtchapova Nadejda , Gstraunthaler Gerhard , Montesano Roberto
Published: May 2013

Constitutive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form cystlike structures in collagen gels and induces an invasive, myofibroblastlike phenotype. However, the reversibility of these cellular events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been investigated. We now report that loss of constitutively active MEK1 (caMEK1) and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with increased MEK2 protein expression, reexpression of ERK1 protein, and epithelial redifferentiation of these cells. The morphological changes toward an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7) are reflected by the upregulation of epithelial marker proteins, such as E-cadherin, β-catenin, and cytokeratin, by the loss of α-smooth muscle actin expression, and by the ability of these epithelial revertants to form well-organized spherical cysts when grown in three-dimensional collagen gels. Further evidence for a role of the MEK1-ERK1/2 module in epithelial-mesenchymal transition was obtained from the analysis of two novel, spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells). In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein expression, and loss of ERK1 protein expression is associated with phenotypic alterations similar to those observed in transdifferentiated C7caMEK1 cells. C7e1 cells at least partially regained some of their epithelial characteristics at higher passages. In contrast, C7e2 cells maintained a transdifferentiated phenotype at high passage, were unable to generate cystlike epithelial structures, and retained invasive properties when grown on a three-dimensional collagen matrix. We conclude that in renal epithelial MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated with loss of ERK1 protein expression, reduced MEK2 protein expression, and increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2 results in increased MEK2 protein expression and reexpression of ERK1 protein, concomitant with the restoration of epithelial phenotype and the ability to form cystic structures.
Nicotinic acid adenine dinucleotide phosphate: a new intracellular second messenger?
Authors: Chini Eduardo N. , De Toledo Frederico G. S.
Published: May 2013

Nicotinic acid adenine dinucleotide phosphate (NAADP) is one of the most potent stimulators of intracellular Ca2+ release known to date. The role of the NAADP system in physiological processes is being extensively investigated at the present time. Exciting new discoveries in the last 5 years suggest that the NAADP-regulated system may have a significant role in intracellular Ca2+ signaling. The NAADP receptor and its associated Ca2+ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion. However, whether NAADP is a new second messenger or a tool for the discovery of a new Ca2+ channel is still an unanswered question.
αvβ3-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells
Authors: Sajid M. , Zhao R. , Pathak A. , Smyth S. S. , Stouffer G. A.
Published: May 2013

αvβ3-Integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because α-thrombin contributes to neointimal formation, we examined the hypothesis that αvβ3-integrins influence α-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed αvβ3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to α-thrombin were partially inhibited by anti-β3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that α-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by αvβ3 antagonists. β3-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. α-Thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). αvβ3-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from β3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, αvβ3-integrins play an important role in α-thrombin-induced proliferation and focal adhesion formation in RASMC.
Influence of myosin isoforms on contractile properties of intact muscle fibers from Rana pipiens
Authors: Lutz Gordon J. , Sirsi Shashank R. , Shapard-Palmer Sarah A. , Bremner Shannon N. , Lieber Richard L.
Published: May 2013

The myosin heavy chain (MHC) and myosin light chain (MLC) isoforms in skeletal muscle of Rana pipiens have been well characterized. We measured the force-velocity (F- V) properties of single intact fast-twitch fibers from R. pipiens that contained MHC types 1 or 2 (MHC1 or MHC2) or coexpressed MHC1 and MHC2 isoforms. Velocities were measured between two surface markers that spanned most of the fiber length. MHC and MLC isoform content was quantified after mechanics analysis by SDS-PAGE. Maximal shortening velocity ( V max) and velocity at half-maximal tension ( V P 50) increased with percentage of MHC1 (%MHC1). Maximal specific tension (Po/CSA, where Po is isometric tension and CSA is fiber cross-sectional area) and maximal mechanical power ( W max) also increased with %MHC1. MHC concentration was not significantly correlated with %MHC1, indicating that the influence of %MHC1 on Po/CSA and W max was due to intrinsic differences between MHC isoforms and not to concentration. The MLC3-to-MLC1 ratio was not significantly correlated with V max, V P 50, Po/CSA, or W max. These data demonstrate the powerful relationship between MHC isoforms and F- V properties of the two most common R. pipiensfiber types.
Regulation of PDGF production and ERK activation by estrogen is associated with TSC2 gene expression
Authors: Finlay G. A. , Hunter D. S. , Walker C. L. , Paulson K. E. , Fanburg B. L.
Published: May 2013

Mechanisms that regulate the growth response to estrogen (17β-estradiol, E2) are poorly understood. Recently, loss of function of the tuberous sclerosis complex 2 ( TSC2) gene has been associated with E2-related conditions that are characterized by benign cellular proliferation. We examined the growth response to E2 in vascular smooth muscle cells (VSMCs) that possess wild-type TSC2 and compared them with ELT-3 smooth muscle cells that do not express TSC2.In TSC2-expressing VSMCs, growth inhibition in response to E2 was associated with downregulation of platelet-derived growth factor (PDGF), PDGF receptor (PDGFR), and limited activation of extracellular signal-regulated kinase (ERK). In contrast, the growth-promoting effect of E2 in TSC2-null ELT-3 cells was associated with induction of PDGF, robust phosphorylation of PDGFR, and sustained activation of ERK. Furthermore, in ELT-3 cells, cellular growth and ERK activation by E2 were inhibited by the PDGFR inhibitor tyrphostin AG 17 and by PDGF-neutralizing antibody. These results demonstrate that autocrine production of PDGF and augmentation of the ERK pathway leads to estrogen-induced cellular proliferation in TSC2-null cells, a pathway that was downregulated in cells that express TSC2. Understanding the mechanisms that regulate the diverse responses to the steroid hormone estrogen could lead to novel approaches to the treatment of estrogen-related diseases that are characterized by aberrant cell proliferation.
Calcineurin differentially regulates maintenance and growth of phenotypically distinct muscles
Authors: Mitchell Patrick O. , Mills Stephen T. , Pavlath Grace K.
Published: May 2013

Adequate muscle mass is critical for human health. The molecular pathways regulating maintenance and growth of adult skeletal muscle are little understood. Calcineurin (CN) is implicated as a key signaling molecule in hypertrophy. Whether CN is involved in all forms of muscle growth or in different muscles is unknown. Here, we examine the role of CN in regulating maintenance of muscle size and growth of atrophied muscle in the soleus (slow) and plantaris (fast). The CN inhibitor cyclosporin A (CsA) differentially affects muscle growth and maintenance depending on muscle phenotype. The plantaris is more severely affected by CsA than the soleus in both growth conditions. One-week vs. 2-wk CsA treatment suggests that both CN-dependent and CN-independent growth occur in the atrophied soleus, whereas plantaris growth appears to be totally CN dependent. Our results suggest that CN regulates multiple types of muscle growth, depending both on muscle phenotype and stage of myofiber growth. Differential expression of components of the CN pathway occurs and may contribute to the differences between muscles.
Apparent elastic modulus and hysteresis of skeletal muscle cells throughout differentiation
Authors: Collinsworth Amy M. , Zhang Sarah , Kraus William E. , Truskey George A.
Published: May 2013

The effect of differentiation on the transverse mechanical properties of mammalian myocytes was determined by using atomic force microscopy. The apparent elastic modulus increased from 11.5 ± 1.3 kPa for undifferentiated myoblasts to 45.3 ± 4.0 kPa after 8 days of differentiation ( P< 0.05). The relative contribution of viscosity, as determined from the normalized hysteresis area, ranged from 0.13 ± 0.02 to 0.21 ± 0.03 and did not change throughout differentiation. Myosin expression correlated with the apparent elastic modulus, but neither myosin nor β-tubulin were associated with hysteresis. Microtubules did not affect mechanical properties because treatment with colchicine did not alter the apparent elastic modulus or hysteresis. Treatment with cytochalasin D or 2,3-butanedione 2-monoxime led to a significant reduction in the apparent elastic modulus but no change in hysteresis. In summary, skeletal muscle cells exhibited viscoelastic behavior that changed during differentiation, yielding an increase in the transverse elastic modulus. Major contributors to changes in the transverse elastic modulus during differentiation were actin and myosin.
Lipopolysaccharide stimulation of ERK1/2 increases TNF-α production via Egr-1
Authors: Shi Liang , Kishore Raj , McMullen Megan R. , Nagy Laura E.
Published: May 2013

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-α (TNF-α) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-α mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-α production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-α mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-α promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-α mRNA expression in macrophages.
Astrocytes from Na+-K+-Cl−cotransporter-null mice exhibit absence of swelling and decrease in EAA release
Authors: Su Gui , Kintner Douglas B. , Flagella Michael , Shull Gary E. , Sun Dandan
Published: May 2013

We reported previously that inhibition of Na+-K+-Cl− cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K+concentration ([K+]o)-induced swelling and intracellular Cl− accumulation in rat cortical astrocytes. In this report, we extended our study by using cortical astrocytes from NKCC1-deficient (NKCC1−/−) mice. NKCC1 protein and activity were absent in NKCC1−/− astrocytes. [K+]o of 75 mM increased NKCC1 activity approximately fourfold in NKCC1+/+ cells ( P< 0.05) but had no effect in NKCC1−/− astrocytes. Intracellular Cl− was increased by 70% in NKCC1+/+ astrocytes under 75 mM [K+]o ( P < 0.05) but remained unchanged in NKCC1−/− astrocytes. Baseline intracellular Na+ concentration ([Na+]i) in NKCC1+/+ astrocytes was 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM [Na+]i in NKCC1−/− astrocytes ( P < 0.05). Relative cell volume of NKCC1+/+ astrocytes increased by 13 ± 2% in 75 mM [K+]o, compared with a value of 1.0 ± 0.5% in NKCC1−/− astrocytes ( P < 0.05). Regulatory volume increase after hypertonic shrinkage was completely impaired in NKCC1−/− astrocytes. High-[K+]o-induced 14C-labeledd-aspartate release was reduced by ∼30% in NKCC1−/− astrocytes. Our study suggests that stimulation of NKCC1 is required for high-[K+]o-induced swelling, which contributes to glutamate release from astrocytes under high [K+]o.
A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release
Authors: Wyman Travis H. , Bjornsen A. Jason , Elzi David J. , Smith C. Wayne , England Kelly M. , Kelher Marguerite , Silliman Christopher C.
Published: May 2013

Lysophosphatidylcholines (lyso-PCs), generated during blood storage, are etiologic in a two-insult, sepsis-based model of transfusion-related acute lung injury (TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do not activate neutrophils (PMNs). We hypothesized that priming of PMNs alters their reactivity such that a second priming agent causes PMN activation and endothelial cell damage. PMNs were primed or not with LPS and then treated with lyso-PCs, and oxidase activation and elastase release were measured. For coculture experiments, activation of human pulmonary microvascular endothelial cells (HMVECs) was assessed by ICAM-1 expression and chemokine release. HMVECs were stimulated or not with LPS, PMNs were added, cells were incubated with lyso-PCs, and the number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 and release of ENA-78, GROα, and IL-8. PMN-mediated HMVEC damage was dependent on LPS activation of HMVECs, chemokine release, PMN adhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates the microbicidal arsenal, and PMN-mediated HMVEC damage was the result of two insults: HMVEC activation and PMN oxidase assembly.
Characteristics of l-lactic acid transport in basal membrane vesicles of human placental syncytiotrophoblast
Authors: Inuyama Masako , Ushigome Fumihiko , Emoto Akiko , Koyabu Noriko , Satoh Shoji , Tsukimori Kiyomi , Nakano Hitoo , Ohtani Hisakazu , Sawada Yasufumi
Published: May 2013

The characteristics of l-lactic acid transport across the trophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake ofl-[14C]lactic acid into basal membrane vesicles was Na+ independent, and an uphill transport was observed in the presence of a pH gradient ([H+]out > [H+]in).l-[14C]lactic acid uptake exhibited saturation kinetics with a K m value of 5.89 ± 0.68 mM in the presence of a pH gradient. p-Chloromercuribenzenesulfonate and α-cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4′-diisothiocyanostilbene-2,2′-disulfonate did not. Mono- and dicarboxylic acids suppressed the initial uptake. In conclusion, l-lactic acid transport in the basal membrane is H+ dependent and Na+ independent, as is also the case for the brush-border membrane transport, and its characteristics resemble those of monocarboxylic acid transporters. However, there were several differences in the effects of inhibitors between basal and brush-border membrane vesicles, suggesting that the transporter(s) involved in l-lactic acid transport in the basal membrane of placental trophoblast may differ from those in the brush-border membrane.
Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture
Authors: Colegio Oscar R. , Itallie Christina Van , Rahner Christoph , Anderson James Melvin
Published: May 2013

Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER ∼300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na+ more than Cl− (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319–1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only ∼20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na+ relative to Cl−. In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only ∼60 and ∼40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.
K+-induced twitch potentiation is not due to longer action potential
Authors: Yensen Craig , Matar Wadih , Renaud Jean-Marc
Published: May 2013

The objective of this study was to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation at 37°C. Twitch contractions were elicited by field stimulation, and action potentials were measured with conventional microelectrodes. For mouse extensor digitorum longus (EDL) muscle, twitch force was greater at 7–13 mM K+ than at 4.7 mM (control). For soleus muscle, twitch force potentiation was observed between 7 and 11 mM K+. Time to peak and half-relaxation time were not affected by the increase in extracellular K+ concentration in EDL muscle, whereas both parameters became significantly longer in soleus muscle. Decrease in overshoot and prolongation of the action potential duration observed at 9 and 11 mM K+ were mimicked when muscles were respectively exposed to 25 and 50 nM tetrodotoxin (TTX; used to partially block Na+ channels). Despite similar action potentials, twitch force was not potentiated by TTX. It is therefore suggested that the K+-induced potentiation of the twitch in EDL muscle is not due to a prolongation of the action potential and contraction time, whereas a longer contraction, especially the relaxation phase, may contribute to the potentiation in soleus muscle.
Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloidlike manner
Authors: Lim Ivor Jiun , Phan Toan-Thang , Bay Boon-Huat , Qi Robert , Huynh Hung , Tan Walter Tiang-Lee , Lee Seng-Teik , Longaker Michael Thornton
Published: May 2013

Keloid scars represent a pathological response to cutaneous injury, reflecting a new set point between synthesis and degradation biased toward extracellular matrix (ECM) collagen accumulation. Using a serum-free two-chamber coculture model, we recently demonstrated a significant increase in normal fibroblast proliferation when cocultured with keloid-derived keratinocytes. We hypothesized that similar keratinocyte-fibroblast interactions might influence fibroblast collagen production and examined conditioned media and cell lysate from coculture for collagen I and III production by Western blot, allied with Northern analysis for procollagen I and III mRNA. Normal fibroblasts cocultured with keloid keratinocytes produced increased soluble collagen I and III with a corresponding increase in procollagen I and III mRNA transcript levels. This was associated with decreased insoluble collagen from cell lysate. When keloid fibroblasts were cocultured with keloid keratinocytes, both soluble and insoluble collagen were increased with associated procollagen III mRNA upregulation. Transmission electron microscopy of normal fibroblasts cocultured with keloid keratinocytes showed an ECM appearance similar to in vivo keloid tissue, an appearance not seen when normal fibroblasts were cocultured with normal keratinocytes.
Adenylyl cyclase expression and modulation of cAMP in rat taste cells
Authors: Abaffy Tatjana , Trubey Kristina R. , Chaudhari Nirupa
Published: May 2013

cAMP is a second messenger implicated in sensory transduction for taste. The identity of adenylyl cyclase (AC) in taste cells has not been explored. We have employed RT-PCR to identify the AC isoforms present in taste cells and found that AC 4, 6, and 8 are expressed as mRNAs in taste tissue. These proteins are also expressed in a subset of taste cells as revealed by immunohistochemistry. Alterations of cAMP concentrations are associated with transduction of taste stimuli of several classes. The involvement of particular ACs in this modulation has not been investigated. We demonstrate that glutamate, which is a potent stimulus eliciting a taste quality termed umami, causes a decrease in cAMP in forskolin-treated taste cells. The potentiation of this response by inosine monophosphate, the lack of response to d-glutamate, and the lack of response to umami stimuli in nonsensory lingual epithelium all suggest that the cAMP modulation represents umami taste transduction. Because cAMP downregulation via ACs can be mediated through Gαi proteins, we examined the colocalization of the detected ACs with Gαi proteins and found that 66% of AC8 immunopositive taste cells are also positive for gustducin, a taste-specific Gαi protein. Whether AC8 is directly involved in signal transduction of umami taste remains to be established.
Impaired osmotic sensation in mice lacking TRPV4
Authors: Mizuno Atsuko , Matsumoto Naoko , Imai Masashi , Suzuki Makoto
Published: May 2013

The Ca2+-permeable cation channel TRPV4, which is part of the Trp family located in the circumventricular organs, is activated by cell swelling. To investigate the role of TRPV4 in osmotic sensation, we disrupted the TRPV4 gene in mice and examined the effect on osmotic metabolism. Disruption of the mouse TRPV4 gene did not influence either water intake behavior or serum osmolality. Short-term salt ingestion, however, seemed to impair the transient free water clearance. The level of serum arginine vasopressin (AVP) of TRPV4–/– mice was not significantly changed under normal conditions but was significantly increased under stimulated conditions. Incubation of brain slices with graded hyperosmolality suggested an exaggerated response of AVP secretion in TRPV4–/– mice. Thus TRPV4 in the brain may transmit a negative signal to AVP secretion similar to an inhibitory pass through the baroregulatory system. Thus, in the regulation of serum osmolality, TRPV4 is a swell-activated channel that appears to play a role in reversion toward hyposmolality.
Decreased 5α-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage
Authors: Zheng Rui , Samy T. S. Anantha , Schneider Christian P. , Rue Loring W. , Bland Kirby I. , Chaudry Irshad H.
Published: May 2013

Trauma-hemorrhage produces profound immunosuppression in males but not in proestrus females. Prior castration or flutamide treatment of males following trauma-hemorrhage prevents immunosuppression, implicating 5α-dihydrotestosterone for the immunosuppressive effects. 5α-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests both synthesis and catabolism of 5α-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5α-dihydrotestosterone activity in T lymphocytes of males following trauma-hemorrhage is due to decreased catabolism. Accordingly, catabolism of 5α-dihydrotestosterone was assessed in splenic T lymphocytes by examining the activity and expression of enzymes involved. Analysis showed increased synthesis and decreased catabolism of 5α-dihydrotestosterone in intact male T lymphocytes following trauma-hemorrhage. In contrast, reduced 5α-reductase activity and increased expression of 17β-hydroxysteroid dehydrogenase oxidative isomers suggest inactivation of 5α-dihydrotestosterone in precastrated males. Thus our study suggests increased synthesis and decreased catabolism of 5α-dihydrotestosterone as a reason for loss of T lymphocyte functions in intact males following trauma-hemorrhage, as evidenced by decreased release of interleukin-2 and -6.
Potassium depletion increases potassium clearance capacity in skeletal muscles in vivo during acute repletion
Authors: Bundgaard Henning , Kjeldsen Keld
Published: May 2013

Muscular K uptake depends on skeletal muscle Na-K-ATPase concentration and activity. Reduced K uptake is observed in vitro in K-depleted rats. We evaluated skeletal muscle K clearance capacity in vivo in rats K depleted for 14 days. [3H]ouabain binding, α1 and α2 Na-K-ATPase isoform abundance, and K, Na, and Mg content were measured in skeletal muscles. Skeletal muscle K, Na, and Mg and plasma K were measured in relation to intravenous KCl infusion that continued until animals died, i.e., maximum KCl dose was administered. In soleus, extensor digitorum longus (EDL), and gastrocnemius muscles K depletion significantly reduced K content by 18%, 15%, and 19%, [3H]ouabain binding by 36%, 41%, and 68%, and α2 isoform abundance by 34%, 44%, and 70%, respectively. No significant change was observed in α1 isoform abundance. In EDL and gastrocnemius muscles K depletion significantly increased Na (48% and 59%) and Mg (10% and 17%) content, but only tendencies to increase were observed in soleus muscle. K-depleted rats tolerated up to a fourfold higher KCl dose. This was associated with a reduced rate of increase in plasma K and increases in soleus, EDL, and gastrocnemius muscle K of 56%, 42%, and 41%, respectively, but only tendencies to increase in controls. However, whereas K uptake was highest in K-depleted animals, the K uptake rate was highest in controls. In vivo K depletion is associated with markedly increased K tolerance and K clearance despite significantly reduced skeletal muscle Na-K-ATPase concentration. The concern of an increased risk for K intoxication during K repletion seems unwarranted.
Potential role for Id myogenic repressors in apoptosis and attenuation of hypertrophy in muscles of aged rats
Authors: Alway Stephen E. , Degens Hans , Krishnamurthy Gururaj , Smith Cheryl A.
Published: May 2013

Aging attenuates the overload-induced increase in myogenic regulatory transcription factor (MRF) expression and the extent of muscle enlargement. To identify whether mRNA levels of repressors of the MRFs are greater in overloaded muscles from aged animals, overload was achieved in plantaris muscle of aged (33 mo; n = 14) and adult (9 mo; n = 17) rats. After 14 days, plantaris muscles in the overloaded limb were ∼25% and 6% larger in adult and aged rats, respectively, compared with the contralateral limb. Hypertrophied muscles of adult rats had significantly greater levels of mRNA and protein levels for myogenin and MyoD compared with control muscles, but neither MRF increased with overload in muscles of aged rats. Muscles of aged rats had greater Id mRNA (150–700%) and protein repressor (200–6,000%) levels compared with adult rats. BAX and caspase 9 protein levels were 9,500% and 300% greater, respectively, in both control and hypertrophied muscles of aged rats compared with young adult rats. These data are consistent with the hypothesis that aging increases Id transcripts that activate apoptotic pathways involving BAX. This may contribute to sarcopenia by attenuating MRF protein levels in muscles of old animals.
LPP, a LIM protein highly expressed in smooth muscle
Authors: Gorenne Isabelle , Nakamoto Robert K. , Phelps Clayton P. , Beckerle Mary C. , Somlyo Avril V. , Somlyo Andrew P.
Published: May 2013

An 80-kDa protein, prominently expressed in smooth muscle, was microsequenced and identified as LPP, the product of the lipoma-preferred partner gene (Petit MMR, Mols R, Schoenmakers EFPM, Mandahl N, and Van de Ven WJM. Genomics 36: 118–129, 1996). Using a specific anti-LPP antibody, we showed, in Western blots and with immunofluorescence microscopy, the selective expression of LPP in vascular and visceral smooth muscles (∼0.5–1 ng/μg total protein). In other mature (noncultured) tissues, including heart and skeletal muscle, the protein is present only in trace amounts and is closely correlated with the levels of the smooth muscle marker α-actin. In freshly isolated guinea pig bladder smooth muscle cells, immunofluorescence images showed LPP as linear arrays of punctate, longitudinally oriented staining superimposed with vinculin staining on the plasma membrane surface. A corresponding pattern of periodic labeling at the membrane in transverse sections of bladder smooth muscle suggested an association of LPP with peripheral dense bodies. In cultured rat aortic smooth muscle cells, LPP colocalized with vinculin at focal adhesions but not with p120 catenin or α-actinin. Overexpression of the protein increased EGF-stimulated migration of vascular smooth muscle cells in Transwell assays, suggesting the participation of LPP in cell motility. The Rho-kinase inhibitor Y-27632 dissociated focal adhesions and LPP staining at the cell periphery and enhanced the nuclear accumulation of LPP induced by leptomycin B, indicating that LPP has a potential for relocating to the nucleus through a shuttling mechanism that is sensitive to inhibition of Rho-kinase.
Apical Na+/H+ exchange near the base of mouse colonic crypts
Authors: Chu Jingsong , Chu Shaoyou , Montrose Marshall H.
Published: May 2013

Colonic crypts can absorb fluid, but the identity of the absorptive transporters remains speculative. Near the crypt base, the epithelial cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only Cl−secretion. We have applied confocal microscopy in combination with an extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive dye (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to study mouse colonic crypt epithelial cells directly adjacent to the crypt base within an intact mucosal sheet. Measurements of intracellular pH report activation of colonocyte Na+/H+ exchange in response to luminal or serosal Na+. Studies with LY demonstrate the presence of a paracellular fluid flux, but luminal Na+ does not activate Na+/H+ exchange in the nonepithelial cells of the lamina propria, and studies with LY suggest that the fluid bathing colonocyte basolateral membranes is rapidly refreshed by serosal perfusates. The apical Na+/H+ exchange in crypt colonocytes is inhibited equivalently by luminal 20 μM ethylisopropylamiloride and 20 μM HOE-694 but is not inhibited by luminal 20 μM S-1611. Immunostaining reveals the presence of epitopes from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of colonic crypts. Comparison of apical Na+/H+exchange activity in the presence of Cl− with that in the absence of Cl− (substitution by gluconate or nitrate) revealed no evidence of the Cl−-dependent Na+/H+ exchange that had been previously reported as the sole apical Na+/H+ exchange activity in the colonic crypt. Results suggest the presence of an apical Na+/H+ exchanger near the base of crypts with functional attributes similar to those of the cloned NHE2 isoform.
Memory and Self–Neuroscientific Landscapes
Authors: Markowitsch Hans J.
Published: May 2013

Relations between memory and the self are framed from a number of perspectives—developmental aspects, forms of memory, interrelations between memory and the brain, and interactions between the environment and memory. The self is seen as dividable into more rudimentary and more advanced aspects. Special emphasis is laid on memory systems and within them on episodic autobiographical memory which is seen as a pure human form of memory that is dependent on a proper ontogenetic development and shaped by the social environment, including culture. Self and episodic autobiographical memory are seen as interlocked in their development and later manifestation. Aside from content-based aspects of memory, time-based aspects are seen along two lines—the division between short-term and long-term memory and anterograde—future-oriented—and retrograde—past-oriented memory. The state dependency of episodic autobiographical is stressed and implications of it—for example, with respect to the occurrence of false memories and forensic aspects—are outlined. For the brain level, structural networks for encoding, consolidation, storage, and retrieval are discussed both by referring to patient data and to data obtained in normal participants with functional brain imaging methods. It is elaborated why descriptions from patients with functional or dissociative amnesia are particularly apt to demonstrate the facets in which memory, self, and personal temporality are interwoven.
The Number of Candidate Variants in Exome Sequencing for Mendelian Disease under No Genetic Heterogeneity
Authors: Nishino Jo , Mano Shuhei
Published: May 2013

There has been recent success in identifying disease-causing variants in Mendelian disorders by exome sequencing followed by simple filtering techniques. Studies generally assume complete or high penetrance. However, there are likely many failed and unpublished studies due in part to incomplete penetrance or phenocopy. In this study, the expected number of candidate single-nucleotide variants (SNVs) in exome data for autosomal dominant or recessive Mendelian disorders was investigated under the assumption of “no genetic heterogeneity.” All variants were assumed to be under the “null model,” and sample allele frequencies were modeled using a standard population genetics theory. To investigate the properties of pedigree data, full-sibs were considered in addition to unrelated individuals. In both cases, particularly regarding full-sibs, the number of SNVs remained very high without controls. The high efficacy of controls was also confirmed. When controls were used with a relatively large total sample size (e.g.,N=20, 50), filtering incorporating of incomplete penetrance and phenocopy efficiently reduced the number of candidate SNVs. This suggests that filtering is useful when an assumption of no “genetic heterogeneity” is appropriate and could provide general guidelines for sample size determination.
Mathematical Modeling of the High Temperature Treatment of Birch in a Prototype Furnace
Authors: Kocaefe Duygu , Kocaefe Yasar , Younsi Ramdane , Oumarou Noura , Lekounougou S. Thierry
Published: May 2013

In recent years, various wood modification technologies have been commercialized as alternatives to the traditional chemical treatments for wood preservation. The high temperature heat treatment of wood is one of these commercially viable and environmentally friendly alternative wood modification technologies. During this treatment, wood is heated to temperatures above 200°C by contacting it with hot gas. The chemical structure of wood changes leading to increased dimensional stability and resistance to microorganisms. Wood darkens making it aesthetically more attractive. However, it loses some of its elasticity. Therefore, the high temperature heat treatment has to be optimized for each species and each technology. The mathematical modeling is an important tool for optimization. It can also be used as a powerful tool for furnace modification and design. A reliable and predictive model was developed to simulate numerically the heat treatment process. Heat treatment experiments were carried out in the prototype furnace of the University of Quebec at Chicoutimi. The model was validated by comparing the predictions with the experimental data. In this paper, the results of the model applied to birch heat treatment are presented. The model predictions are in good agreement with the data.
Telemedicine in Tinnitus: Feasibility, Advantages, Limitations, and Perspectives
Authors: Guitton Matthieu J.
Published: May 2013

Given the important patient needs for support and treatment, telemedicine—defined by medical approaches supported by the new technologies of information—could provide interesting alternative in tinnitus treatment. By analyzing the published tools and approaches which could be used in the context of telemedicine for tinnitus by health professionals or self-administrated by patients, this review summarizes, presents, and describes the principal telemedicine approaches available presently or in the near future to help assess or treat tinnitus or to offer support to tinnitus sufferers. Several pieces of evidence strongly support the feasibility of telemedicine approaches for tinnitus. Telemedicine can be used to help tinnitus sufferers at several points in the therapeutic process: for early screening, initial evaluation, and diagnosis; for optimizing therapeutic tools, particularly behavioural therapies and virtual reality-enhanced behavioral therapies; for long-term monitoring of patients and provision of online support. Several limitations are, however, discussed in order to optimize the safe development of such approaches. Cost effective and easy to implement, telemedicine is likely to represent an important part of the future of tinnitus therapies and should be progressively integrated by otolaryngologists.
Review and Comment on the Relationship between Primo Vascular System and Meridians
Authors: Cai Ding-Jun , Chen Ji , Zhuang Yi , Liu Mai-Lan , Liang Fan-Rong
Published: May 2013

This paper aims to summarize the recent progress of researches on the primo vascular system (PVS) and to analyze characteristics between PVS and traditional Chinese meridians. With the distribution, position features, identification and origin of PVS, and its function related to meridians elaborated on, we propose that there is still a lack of enough evidence to support the correlation between PVS and traditional Chinese meridians.