bluuebunny/crossref_metadata_2025 huggingface dataset:
Problems caused by the Three Gorges Dam construction in the Yangtze River basin: a review
Authors: Li Kaifeng , Zhu Cheng , Wu Li , Huang Linyan
Published: May 2013

Dam is an important way of water-resources utilization in large rivers. To date, more than 50 000 dams with various sizes have been constructed in the Yangtze River basin, with many other dams proposed to be constructed by 2020. Dam construction has played significant roles in flood control, irrigation, navigation, and energy supply; however, the enormous negative effects, such as landslides, ecological problems, and water quality decline, could surpass positive gains. Although a long and complicated evaluation process had been carried out and the countermeasures for numerous foreseen negative impacts of the Three Gorges Dam (TGD) had been implemented, many uncertainties and debating opinions on the benefits and costs of this project still exist. In this review, we synthesize the negative impacts that have occurred as a result of the TGD, including reservoir-triggered seismicity, landslides, water quality control, ecological problems, siltation, and sediment discharge decline to assure an environmentally friendly operation of the TGD and regional sustainable development in the Yangtze River basin, especially in the Three Gorges Reservoir region.
ENERGY-EFFICIENT THRESHOLD CIRCUITS COMPUTING MOD FUNCTIONS
Authors: SUZUKI AKIRA , UCHIZAWA KEI , ZHOU XIAO
Published: May 2013

We prove that the modulus function MODm of n variables can be computed by a threshold circuit C of energy e and size s = O(e(n/m)1/(e − 1)) for any integer e ≥ 2, where the energy e is defined to be the maximum number of gates outputting "1" over all inputs to C, and the size s to be the number of gates in C. Our upper bound on the size s almost matches the known lower bound s = Ω(e(n/m)1/e). We also consider an extreme case where threshold circuits have energy 1, and prove that such circuits need at least 2(n − m)/2 gates to compute MODm of n variables.
MINIMUM MOSAIC INFERENCE OF A SET OF RECOMBINANTS
Authors: BLIN GUILLAUME , RIZZI ROMEO , SIKORA FLORIAN , VIALETTE STEPHANE
Published: May 2013

In this paper, we investigate the central problem of finding recombination events. It is commonly assumed that a present population is a descendent of a small number of specific sequences called founders. The recombination process consists in given two equal length sequences, generates a third sequence of the same length by concatenating the prefix of one sequence with the suffix of the other sequence. Due to recombination, a present sequence (called a recombinant) is thus composed of blocks from the founders. A major question related to founder sequences is the so-called Minimum Mosaic problem: using the natural parsimony criterion for the number of recombinations, find the "best" founders. In this article, we prove that the Minimum Mosaic problem given haplotype recombinants with no missing values is NP-hard when the number of founders is given as part of the input and propose some exact exponential-time algorithms for the problem, which can be considered polynomial provided some extra information. Notice that Rastas and Ukkonen proved that the Minimum Mosaic problem is NP-hard using a somewhat unrealistic mutation cost function. The aim of this paper is to provide a better complexity insight of the problem.
ON THE NUMBER AND DISTRIBUTIONS OF LIMIT CYCLES OF A PLANAR QUARTIC VECTOR FIELD
Authors: WU YUHAI , HAN MAOAN
Published: May 2013

In this paper, the number and distributions of limit cycles of a planar quartic vector field are considered. Through perturbation technique and qualitative analysis of differential equation, it is shown that the quartic vector field has 21 limit cycles as parameters satisfy proper conditions. The distributions of limit cycles in the above perturbed planar quartic vector field are also presented.
SIMPLE MEMRISTIVE TIME-DELAY CHAOTIC SYSTEMS
Authors: PHAM VIET-THANH , BUSCARINO ARTURO , FORTUNA LUIGI , FRASCA MATTIA
Published: May 2013

Memristive systems have appeared in various application fields from nonvolatile memory devices and biological structures to chaotic circuits. In this paper, we propose two nonlinear circuits based on memristive systems in the presence of delay, i.e. memristive systems in which the state of the memristor depends on the time-delay. Both systems can exhibit chaotic behavior and, notably, in the second model, only a capacitor and a memristor are required to obtain chaos.
A reward-and-punishment-based approach for concept detection using adaptive ontology rules
Authors: Bhatt Chidansh A. , Atrey Pradeep K. , Kankanhalli Mohan S.
Published: May 2013

Despite the fact that performance improvements have been reported in the last years, semantic concept detection in video remains a challenging problem. Existing concept detection techniques, with ontology rules, exploit the static correlations among primitive concepts but not the dynamic spatiotemporal correlations. The proposed method rewards (or punishes) detected primitive concepts using dynamic spatiotemporal correlations of the given ontology rules and updates these ontology rules based on the accuracy of detection. Adaptively learned ontology rules significantly help in improving the overall accuracy of concept detection as shown in the experimental result.
Multifeature analysis and semantic context learning for image classification
Authors: Zhang Qianni , Izquierdo Ebroul
Published: May 2013

This article introduces an image classification approach in which the semantic context of images and multiple low-level visual features are jointly exploited. The context consists of a set of semantic terms defining the classes to be associated to unclassified images. Initially, a multiobjective optimization technique is used to define a multifeature fusion model for each semantic class. Then, a Bayesian learning procedure is applied to derive a context model representing relationships among semantic classes. Finally, this context model is used to infer object classes within images. Selected results from a comprehensive experimental evaluation are reported to show the effectiveness of the proposed approaches.
Effective transfer tagging from image to video
Authors: Yang Yang , Yang Yi , Shen Heng Tao
Published: May 2013

Recent years have witnessed a great explosion of user-generated videos on the Web. In order to achieve an effective and efficient video search, it is critical for modern video search engines to associate videos with semantic keywords automatically. Most of the existing video tagging methods can hardly achieve reliable performance due to deficiency of training data. It is noticed that abundant well-tagged data are available in other relevant types of media (e.g., images). In this article, we propose a novel video tagging framework, termed as
Cross-Media Tag Transfer
(CMTT), which utilizes the abundance of well-tagged images to facilitate video tagging. Specifically, we build a “cross-media tunnel” to transfer knowledge from images to videos. To this end, an optimal kernel space, in which distribution distance between images and video is minimized, is found to tackle the domain-shift problem. A novel cross-media video tagging model is proposed to infer tags by exploring the intrinsic local structures of both labeled and unlabeled data, and learn reliable video classifiers. An efficient algorithm is designed to optimize the proposed model in an iterative and alternative way. Extensive experiments illustrate the superiority of our proposal compared to the state-of-the-art algorithms.
APRICOD
Authors: Zhao Zhen Wei , Ooi Wei Tsang
Published: May 2013

Content discovery is a major source of latency in peer-to-peer (P2P) media streaming systems, especially in the presence of noncontinuous user access, such as random seek in Video-on-Demand (VoD) streaming and teleportation in a Networked Virtual Environment (NVE). After the aforementioned user interactions, streaming systems often need to initiate the content discovery process to identify where to retrieve the requested media objects. Short content lookup latency is demanded to ensure smooth user experience. Existing content discovery systems based on either a Distributed Hash Table (DHT) or gossip mechanism cannot cope with noncontinuous access efficiently due to their long lookup latency.

In this work, we propose an access-pattern-driven distributed caching middleware named APRICOD, which caters for fast and scalable content discovery in peer-to-peer media streaming systems, especially when user interactions are present. APRICOD exploits correlations among media objects accessed by users, and adapts to shift in the user access pattern automatically. We first present a general APRICOD design that can be used with any existing content discovery system. We then present an implementation of APRICOD on top of Pastry, which we use to evaluate APRICOD. Our evaluation in a 1024-node system, using a Second Life trace with 5,735 users and a VoD trace with 54 users, shows that APRICOD can effectively resolve all continuous access queries with a single hop deterministically with node failure as an exception, and resolve noncontinuous access queries with a single hop with high probability.
Reversible simulations of elastic collisions
Authors: Perumalla Kalyan S. , Protopopescu Vladimir A.
Published: May 2013

Consider a system of
N
identical hard spherical particles moving in a
d
-dimensional box and undergoing elastic, possibly multiparticle, collisions. We develop a new algorithm that recovers the precollision state from the post-collision state of the system, across a series of consecutive collisions, with essentially no memory overhead. The challenge in achieving reversibility for an
n
-particle collision (where, in general,
n ≪ N
) arises from the presence of
nd - d - 1
degrees of freedom (arbitrary angles) during each collision, as well as from the complex geometrical constraints placed on the colliding particles. To reverse the collisions in a traditional simulation setting, all of the particular realizations of these degrees of freedom (angles) during the forward simulation must be tracked. This requires memory proportional to the number of collisions, which grows very fast with
N
and
d
, thereby severely limiting the de facto applicability of the scheme. This limitation is addressed here by first performing a pseudorandomization of angles, which ensures determinism in the reverse path for any values of
n
and
d
. To address the more difficult problem of geometrical and dynamic constraints, a new approach is developed which correctly samples the constrained phase space. Upon combining the pseudorandomization with correct phase space sampling, perfect reversibility of collisions is achieved, as illustrated for
n
≤ 3,
d
= 2, and
n
= 2,
d
= 3. This result enables, for the first time, reversible simulations of elastic collisions with essentially zero memory accumulation. In principle, the approach presented here could be generalized to larger values of
n
. The reverse computation methodology presented here uncovers important issues of irreversibility in conventional models, and the difficulties encountered in arriving at a reversible model for one of the most basic and widely used physical system processes, namely, elastic collisions for hard spheres. Insights and solution methodologies, with regard to accurate phase space coverage with reversible random sampling proposed in this context, can help serve as models and/or starting points for other reversible simulations.
pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway
Authors: Machen Terry E. , Leigh Mary Jae , Taylor Carmen , Kimura Tohru , Asano Shinji , Moore Hsiao-Ping H.
Published: May 2013

The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the α- and β-subunits of human H+/K+-ATPase (H+/K+-α,β cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-α,β cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-α,β cells (pHTGN 6.36) and vector-transfected (“mock”) cells (pHTGN 6.34); pHRE was also similar in H+/K+-α,β (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.0–7.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8,000) active in the TGN to balance a large, passive H+ leak (with PH ∼10–3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be ∼4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.
Isolated mouse pancreatic β-cells show cell-specific temporal response pattern
Authors: Larsson-Nyrén Gerd , Pakhtusova Natalia , Sehlin Janove
Published: May 2013

The length of the silent lag time before elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) differs between individual pancreatic β-cells. One important question is whether these differences reflect a random phenomenon or whether the length of lag time is inherent in the individual β-cell. We compared the lag times, initial dips, and initial peak heights for [Ca2+]i from two consecutive glucose stimulations (with either 10 or 20 mM glucose) in individual ob/ob mouse β-cells with the fura 2 technique in a microfluorimetric system. There was a strong correlation between the lengths of the lag times in each β-cell (10 mM glucose: r = 0.94, P < 0.001; 20 mM glucose: r = 0.96, P < 0.001) as well as between the initial dips in [Ca2+]i (10 mM glucose: r = 0.93, P < 0.001; 20 mM glucose: r = 0.79, P < 0.001) and between the initial peak heights (10 mM glucose: r = 0.51, P < 0.01; 20 mM glucose: r = 0.77, P < 0.001). These data provide evidence that the response pattern, including both the length of the lag time and the dynamics of the subsequent [Ca2+]i, is specific for the individual β-cell.
Oxidative stress-induced cell death of human oral neutrophils
Authors: Sato Eisuke F. , Higashino Masahiro , Ikeda Kazuo , Wake Ryotaro , Matsuo Mitsuyoshi , Utsumi Kozo , Inoue Masayasu
Published: May 2013

Polymorphonuclear leukocytes (PMN) play crucial roles in protecting hosts against invading microbes and in the pathogenesis of inflammatory tissue injury. Although PMN migrate into mucosal layers of digestive and respiratory tracts, only limited information is available of their fate and function in situ. We previously reported that, unlike circulating PMN (CPMN), PMN in the oral cavity spontaneously generate superoxide radical and nitric oxide (NO) in the absence of any stimuli. When cultured for 12 h under physiological conditions, oral PMN (OPMN) showed morphological changes that are characteristic of those of apoptosis. Upon agarose gel electrophoresis, nuclear DNA samples isolated from OPMN revealed ladder-like profiles characteristic of nucleosomal fragmentation.l-cysteine, reduced glutathione (GSH), and herbimycin A, a protein tyrosine kinase inhibitor, suppressed the activation of caspase-3 and apoptosis of OPMN. Neither thiourea, superoxide dismutase (SOD), nor catalase inhibited the activation of caspase-3 and apoptosis. Moreover, N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibitor for caspase-3, inhibited the fragmentation of DNA. These results suggested that oxidative stress and/or tyrosine-kinase-dependent pathway(s) activated caspase-3 in OPMN, thereby inducing their apoptosis.
Ca2+-dependent inhibition of NHE3 requires PKCα which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes
Authors: Lee-Kwon Whaseon , Kim Jae Ho , Choi Jung Woong , Kawano Kazuya , Cha Boyoung , Dartt Darlene A. , Zoukhri Driss , Donowitz Mark
Published: May 2013

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKCα pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCα from cytosol to membrane. PKCα bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCα and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCα is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.
Osmotic stress-induced remodeling of the cortical cytoskeleton
Authors: Di Ciano Caterina , Nie Zilin , Szászi Katalin , Lewis Alison , Uruno Takehito , Zhan Xi , Rotstein Ori D. , Mak Alan , Kapus András
Published: May 2013

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.
Placental ceruloplasmin homolog is regulated by iron and copper and is implicated in iron metabolism
Authors: Danzeisen Ruth , Fosset Cedric , Chariana Zehane , Page Kenneth , David Samuel , McArdle Harry J.
Published: May 2013

We previously reported an endogenous, membrane-bound Cu oxidase with homology to ceruloplasmin in BeWo cells, a placental choriocarcinoma cell line. In this previous study, ceruloplasmin immunoreactivity was localized to the perinuclear region and non-brush-border membranes. Here, we show that azide-sensitive oxidase activity is enriched in the same fractions, correlating subcellular localization of enzyme activity with ceruloplasmin immunoreactivity. Expression of the placental Cu oxidase is inversely proportional to Fe status and directly proportional to Cu status at enzyme and protein levels. To identify a role for the Cu oxidase, cells were exposed to 59Fe-transferrin for 18 h in an environment of 20% O2 or 5% O2. At 5% O2, Cu-deficient cells retain significantly more59Fe than control cells. This excess in 59Fe accumulation is caused by a significant decrease in 59Fe release. These results indicate that downregulation of the placental Cu oxidase in BeWo cells impairs Fe release. This effect is only apparent in an environment of limited O2.
PGE2 reduces arachidonic acid release in murine podocytes: evidence for an autocrine feedback loop
Authors: Lemieux Lyne I. , Rahal Sherine S. , Kennedy Chris R. J.
Published: May 2013

Increased glomerular prostaglandin E2 (PGE2) production is associated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. We investigated the signaling pathways that regulate the synthesis and actions of PGE2 in glomerular podocytes. To study its actions, we assessed the ability of PGE2 to regulate the production of its own precursor, arachidonic acid (AA), in a mouse podocyte cell line. PGE2 dose-dependently reduced phorbol ester (PMA)-mediated AA release. Inhibition of PMA-stimulated AA release by PGE2 was found to be cAMP/PKA-dependent, because PGE2 significantly increased levels of this second messenger, whereas the inhibitory actions of PGE2 were reversed by PKA inhibition and reproduced by the cAMP-elevating agents forskolin and IBMX. PGE2 synthesis in this podocyte cell line increased fourfold at 60 min in response to PMA, coinciding with upregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However, PGE2 synthesis was significantly reduced by COX-1-selective inhibition, yet to a lesser extent by COX-2-selective inhibition. Our findings suggest that PMA-stimulated PGE2 synthesis in mouse podocytes requires both basal COX-1 activity and induced COX-2 expression, and that PGE2 reduces PMA-stimulated AA release in a cAMP/PKA-dependent manner. Such an autocrine regulatory loop might have important consequences for podocyte and glomerular function in the context of renal diseases involving PGE2 synthesis.
No decrease in myonuclear number after long-term denervation in mature mice
Authors: Wada K.-I. , Takahashi H. , Katsuta S. , Soya H.
Published: May 2013

Age-related but not artificially induced muscle fiber atrophy has been shown to occur without any decrease in myonuclear number, although these results remain controversial. The present study was carried out to clarify whether age difference affects the degree of decrease in myonuclear number occurring with denervation-induced fiber atrophy. After denervation of 3-wk-old (young) and 4-mo-old (mature) mice, single myofibers were isolated from the plantaris muscles by alkali maceration, and their fiber cross-sectional area (CSA), myonuclear number, and cytoplasm-to-myonucleus (C/N) ratios were analyzed. Fiber CSA in both young and mature mice decreased with denervation. Myonuclear number decreased in young mice 5 and 10 days after denervation but was unchanged in mature mice 10 and 120 days after denervation. C/N ratio decreased in mature mice but was unchanged in denervated young mice. These results suggest that age differences affect the degree of decrease of myonuclear number with denervation and that fiber cytoplasmic atrophy may occur without decrease in myonuclear number.
Functional reconstitution of an eicosanoid-modulated Cl− channel from bovine tracheal smooth muscle
Authors: Salvail Dany , Cloutier Martin , Rousseau Eric
Published: May 2013

We describe the biochemical properties of an eicosanoid-modulated Cl−channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability ( P o). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl− channel is a 55-kDa glycosylated monomer with voltage- and Ca2+concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 μM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P o of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 μM but failed to alter the P o of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET− anions with the channel pore, whereas the alteration of P o requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.
Skeletal muscle sarcoplasmic reticulum contains a NADH-dependent oxidase that generates superoxide
Authors: Xia Ruohong , Webb Jason A. , Gnall Lisa L. M. , Cutler Kerry , Abramson Jonathan J.
Published: May 2013

Skeletal muscle sarcoplasmic reticulum (SR) is shown to contain an NADH-dependent oxidase (NOX) that reduces molecular oxygen to generate superoxide. Its activity is coupled to an activation of the Ca2+ release mechanism, as evident by stimulation in the rate of high-affinity ryanodine binding. NOX activity, coupled to the production of superoxide, is not derived from the mitochondria but is SR in origin. The SR preparation also contains a significant NADH oxidase activity, which is not coupled to the production of superoxide and appears to be mitochondrial in origin. This mitochondrial component is preferentially associated with the terminal cisternae region of the SR. Its activity is inhibited by diphenylene iodonium (10 μM), antimycin A (200 nM), and rotenone (40 nM) but is not coupled to the generation of superoxide or the stimulation of the ryanodine receptor. The rate of superoxide production per milligram of protein is larger in SR than in mitochondria. This NOX may be a major source of oxidative stress in muscle.
RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells
Authors: Carton Iris , Trouet Dominique , Hermans Diane , Barth Holger , Aktories Klaus , Droogmans Guy , Jorgensen Nanna K. , Hoffmann Else K. , Nilius Bernd , Eggermont Jan
Published: May 2013

Cell swelling triggers in most cell types an outwardly rectifying anion current, I Cl,swell, via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I Cl,swell. However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of Gα (a Rho activating heterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5′- O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway.
Claudins create charge-selective channels in the paracellular pathway between epithelial cells
Authors: Colegio Oscar R. , Van Itallie Christina M. , McCrea Heather J. , Rahner Christoph , Anderson James Melvin
Published: May 2013

Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl−. These results support a model where claudins create charge-selective channels in the paracellular space.
A critical role for PKCζ in endothelin-1-induced uterine contractions at the end of pregnancy
Authors: Di Liberto G. , Dallot E. , Eude-Le Parco I. , Cabrol D. , Ferré F. , Breuiller-Fouché M.
Published: May 2013

We have previously shown that protein kinase C (PKC) ζ and/or PKCδ are necessary for endothelin-1 (ET-1)-induced human myometrial contraction at the end of pregnancy (Eude I, Paris P, Cabrol D, Ferré F, and Breuiller-Fouché M. Biol Reprod 63: 1567–1573, 2000). Here, we report that the selective inhibitor of PKCδ isoform, Rottlerin, does not prevent ET-1-induced contractions, whereas LY-294002, a phosphatidylinositol (PI) 3-kinase inhibitor, affects the contractile response. This study characterized the in vitro contractile response of cultured human pregnant myometrial cells to ET-1 known to induce in vitro contractions of intact uterine smooth muscle strips. Cultured myometrial cells incorporated into collagen lattices have the capacity to reduce the size of these lattices, referred to as lattice contraction. Neither the selective conventional PKC isoform inhibitor, Gö-6976, or rottlerin affected myometrial cell-mediated gel contraction by ET-1, whereas this effect was blocked by LY-294002. We found that treatment of myometrial cell lattices with an inhibitory peptide specific for PKCζ or with an antisense against PKCζ resulted in a significant loss of ET-1-induced contraction. Evidence is also presented by using confocal microscopy that ET-1 induced translocation of PKCζ to a structure coincident with the actin-rich microfilaments of the cytoskeleton. We have shown that PKCζ has a role in the actin organization in ET-1-stimulated cells. Accordingly, our results suggest that PKCζ plays a role in myometrial contraction in pregnant women.
Glu496Ala polymorphism of human P2X7receptor does not affect its electrophysiological phenotype
Authors: Boldt Wolfgang , Klapperstück Manuela , Büttner Cora , Sadtler Sven , Schmalzing Günther , Markwardt Fritz
Published: May 2013

A glutamate to alanine exchange at amino acid position 496 of the human P2X7 receptor was recently shown to be associated with a loss of function in human B lymphocytes in terms of ATP-induced ethidium+ uptake, Ba2+ influx, and induction of apoptosis (Gu BJ, Zhang WY, Worthington RA, Sluyter R, Dao-Ung P, Petrou S, Barden JA, and Wiley JS. J Biol Chem 276: 11135–11142, 2001). Here we analyzed the effect of the Glu496 to Ala exchange on the channel properties of the human P2X7 receptor expressed in Xenopus oocytes with the two-microelectrode voltage-clamp technique. The amplitudes of ATP-induced whole cell currents characteristic of functional expression, kinetic properties including ATP concentration dependence, and permeation behavior were not altered by this amino acid exchange. Also in HEK293 cells, the Ala496 mutant mediated typical P2X7 receptor-dependent currents like the parent Glu496 hP2X7 receptor. Because the function of the P2X7 receptor as an ATP-gated channel for small cations including Ba2+ remained unaffected by this mutation, we conclude that Glu496 plays a critical role in pore formation but does not determine the ion channel properties of the human P2X7 receptor.
Specific iNOS-targeted antisense knockdown in endothelial cells
Authors: Hemmrich Karsten , Suschek Christoph V. , Lerzynski Guido , Schnorr Oliver , Kolb-Bachofen Victoria
Published: May 2013

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.
l-Carnitine: a potential treatment for blocking apoptosis and preventing skeletal muscle myopathy in heart failure
Authors: Vescovo Giorgio , Ravara Barbara , Gobbo Valerio , Sandri Marco , Angelini Annalisa , Della Barbera Mila , Dona Massimo , Peluso Gianfranco , Calvani Menotti , Mosconi Luigi , Dalla Libera Luciano
Published: May 2013

Skeletal muscle in congestive heart failure is responsible for increased fatigability and decreased exercise capacity. A specific myopathy with increased expression of fast-type myosins, myocyte atrophy, secondary to myocyte apoptosis triggered by high levels of circulating tumor necrosis factor-α (TNF-α) has been described. In an animal model of heart failure, the monocrotaline-treated rat, we have observed an increase of apoptotic skeletal muscle nuclei. Proapoptotic agents, caspase-3 and -9, were increased, as well as serum levels of TNF-α and its second messenger sphingosine. Treatment of rats withl-carnitine, known for its protective effect on muscle metabolism injuries, was found to inhibit caspases and to decrease the levels of TNF-α and sphingosine, as well as the number of apoptotic myonuclei. Staurosporine was used in in vitro experiments to induce apoptosis in skeletal muscle cells in culture. Whenl-carnitine was applied to skeletal muscle cells, before staurosporine treatment, we observed a reduction in apoptosis. These findings show that l-carnitine can prevent apoptosis of skeletal muscles cells and has a role in the treatment of congestive heart failure-associated myopathy.
GSK-3β negatively regulates skeletal myotube hypertrophy
Authors: Vyas Dharmesh R. , Spangenburg Espen E. , Abraha Tsghe W. , Childs Thomas E. , Booth Frank W.
Published: May 2013

To determine whether changes in glycogen synthase kinase-3β (GSK-3β) phosphorylation contribute to muscle hypertrophy, we delineated the effects of GSK-3β activity on C2C12 myotube size. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible gene activity and possible modulation of NFAT activation by GSK-3β. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., both inhibit GSK-3β activity) increased the area of C2C12 myotubes by 80 and 85%, respectively. The application of IGF-I (250 ng/ml) elevated GSK-3β phosphorylation and reduced GSK-3β kinase activity by ∼800% and ∼25%, respectively. LY-294002 (100 μM) and wortmannin (150 μM), specific inhibitors of phosphatidylinositol 3′-kinase, attenuated IGF-I-induced GSK-3β phosphorylation by 67 and 92%, respectively. IGF-I suppressed the kinase activity of GSK-3β. IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporter activity. Cotransfection of a constitutively active GSK-3β (cGSK-3β) inhibited the induction by IGF-I of NFAT-inducible reporter activity. LiCl, which inhibits GSK-3β, removed the block by cGSK-3β on IGF-I-inducible NFAT-responsive reporter gene activity. These data suggest that the IGF-I-induced increase in skeletal myotube size is signaled, in part, through the inhibition of GSK-3β.
Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite
Authors: Mori N. , Suzuki R. , Furuno T. , McKay D. M. , Wada M. , Teshima R. , Bienenstock J. , Nakanishi M.
Published: May 2013

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 ± 0.04 vs. 0.05 ± 0.01 μm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.
Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells
Authors: Thi Mia M. , Kojima Takashi , Cowin Stephen C. , Weinbaum Sheldon , Spray David C.
Published: May 2013

We tested the hypothesis that fluid shear stress (τ) modifies the expression, function, and distribution of junctional proteins [connexin (Cx)43, Cx45, and zona occludens (ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1 cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to τ-values of 5 or 20 dyn/cm2 for 1–3 h. Immunostaining indicated that at 5 dyn/cm2, the distribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cell membranes; at 20 dyn/cm2, disruption was more severe. Intercellular coupling was significantly decreased at both shear stress levels. Western blots showed the downregulation of membrane-bound Cx43 and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at different levels of shear stress. Similarly, Northern blots revealed that expression of Cx43, Cx45, and ZO-1 was selectively up- and downregulated in response to different shear stress levels. These results indicate that in cultured bone cells, fluid shear stress disrupts junctional communication, rearranges junctional proteins, and determines de novo synthesis of specific connexins to an extent that depends on the magnitude of the shear stress. Such disconnection from the bone cell network may provide part of the signal whereby the disconnected cells or the remaining network initiate focal bone remodeling.
Confocal ratiometric voltage imaging of cultured human keratinocytes reveals layer-specific responses to ATP
Authors: Burgstahler Ralf , Koegel Heidi , Rucker Franz , Tracey David , Grafe Peter , Alzheimer Christian
Published: May 2013

Recent evidence suggests that changes in membrane potential influence the proliferation and differentiation of keratinocytes. To further elucidate the role of changes in membrane potential for their biological fate, the electrical behavior of keratinocytes needs to be studied under complex conditions such as multilayered cultures. However, electrophysiological recordings from cells in the various layers of a complex culture would be extremely difficult. Given the high spatial resolution of confocal imaging and the availability of novel voltage-sensitive dyes, we combined these methods in an attempt to develop a viable alternative for recording membrane potentials in more complex tissue systems. As a first step, we used confocal ratiometric imaging of fluorescence resonance energy transfer (FRET)-based voltage-sensitive dyes. We then validated this approach by comparing the optically recorded voltage signals in HaCaT keratinocytes with the electrophysiological signals obtained by whole cell recordings of the same preparation. We demonstrate 1) that optical recordings allow precise multisite measurements of voltage changes evoked by the extracellular signaling molecules ATP and bradykinin and 2) that responsiveness to ATP differs in various layers of cultured keratinocytes.
Polyamines regulate β-catenin tyrosine phosphorylation via Ca2+ during intestinal epithelial cell migration
Authors: Guo Xin , Rao Jaladanki N. , Liu Lan , Rizvi Mort , Turner Douglas J. , Wang Jian-Ying
Published: May 2013

Polyamines are essential for early mucosal restitution that occurs by epithelial cell migration to reseal superficial wounds after injury. Normal intestinal epithelial cells are tightly bound in sheets, but they need to be rapidly disassembled during restitution. β-Catenin is involved in cell-cell adhesion, and its tyrosine phosphorylation causes disassembly of adhesion junctions, enhancing the spreading of cells. The current study determined whether polyamines are required for the stimulation of epithelial cell migration by altering β-catenin tyrosine phosphorylation. Migration of intestinal epithelial cells (IEC-6 line) after wounding was associated with an increase in β-catenin tyrosine phosphorylation, which decreased the binding activity of β-catenin to α-catenin. Polyamine depletion by α-difluoromethylornithine reduced cytoplasmic free Ca2+concentration ([Ca2+]cyt), prevented induction of β-catenin phosphorylation, and decreased cell migration. Elevation of [Ca2+]cyt induced by the Ca2+ ionophore ionomycin restored β-catenin phosphorylation and promoted migration in polyamine-deficient cells. Decreased β-catenin phosphorylation through the tyrosine kinase inhibitor herbimycin-A or genistein blocked cell migration, which was accompanied by reorganization of cytoskeletal proteins. These results indicate that β-catenin tyrosine phosphorylation plays a critical role in polyamine-dependent cell migration and that polyamines induce β-catenin tyrosine phosphorylation at least partially through [Ca2+]cyt.
Functions of the orexinergic/hypocretinergic system
Authors: Kukkonen Jyrki P. , Holmqvist Tomas , Ammoun Sylwia , Åkerman Karl E. O.
Published: May 2013

Orexin A and orexin B are hypothalamic peptides that act on their targets via two G protein-coupled receptors (OX1 and OX2 receptors). In the central nervous system, the cell bodies producing orexins are localized in a narrow region within the lateral hypothalamus and project mainly to regions involved in feeding, sleep, and autonomic functions. Via putative pre- and postsynaptic effects, orexins increase synaptic activity in these regions. In isolated neurons and cells expressing recombinant receptors orexins cause Ca2+ elevation, which is mainly dependent on influx. The activity of orexinergic cells appears to be controlled by feeding- and sleep-related signals via a variety of neurotransmitters/hormones from the brain and other tissues. Orexins and orexin receptors are also found outside the central nervous system, particularly in organs involved in feeding and energy metabolism, e.g., gastrointestinal tract, pancreas, and adrenal gland. In the present review we focus on the physiological properties of the cells that secrete or respond to orexins.
p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle
Authors: Hawke T. J. , Meeson A. P. , Jiang N. , Graham S. , Hutcheson K. , DiMaio J. M. , Garry D. J.
Published: May 2013

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired.