bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal Structures of Bifunctional Penicillin-Binding Protein 4 from Listeria monocytogenes
Authors: Jeong Jae-Hee , Kim Yi-Seul , Rojviriya Catleya , Ha Sung-Chul , Kang Beom Sik , Kim Yeon-Gil
Published: May 2013

Penicillin-binding proteins (PBPs), which catalyze the biosynthesis of the peptidoglycan chain of the bacterial cell wall, are the major molecular target of bacterial antibiotics. Here, we present the crystal structures of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase (TP) PBP4 from
Listeria monocytogenes
in the apo-form and covalently linked to two β-lactam antibiotics, ampicillin and carbenicillin. The orientation of the TP domain with respect to the GT domain is distinct from that observed in the previously reported structures of bifunctional PBPs, suggesting interdomain flexibility. In this structure, the active site of the GT domain is occluded by the close apposition of the linker domain, which supports the hypothesis that interdomain flexibility is related to the regulation of GT activity. The acylated structures reveal the mode of action of β-lactam antibiotics toward the class A PBP4 from the human pathogen
L. monocytogenes
. Ampicillin and carbenicillin can access the active site and be acylated without requiring a structural rearrangement. In addition, the active site of the TP domain in the apo-form is occupied by the tartrate molecule via extensive hydrogen bond interactions with the catalytically important residues; thus, derivatives of the tartrate molecule may be useful in the search for new antibiotics to inhibit PBPs.
In Vitro
Efficacies of Clinically Available Drugs against Growth and Viability of an Acanthamoeba castellanii Keratitis Isolate Belonging to the T4 Genotype
Authors: Baig Abdul Mannan , Iqbal Junaid , Khan Naveed Ahmed
Published: May 2013

The effects of clinically available drugs targeting muscarinic cholinergic, adrenergic, dopaminergic, and serotonergic receptors; intracellular calcium levels and/or the function of calcium-dependent biochemical pathways; ion channels; and cellular pumps were tested against a keratitis isolate of
Acanthamoeba castellanii
belonging to the T4 genotype.
In vitro
growth inhibition (amoebistatic) assays were performed by incubating
A. castellanii
with various concentrations of drugs in the growth medium for 48 h at 30°C. To determine amoebicidal effects, amoebae were incubated with drugs in phosphate-buffered saline for 24 h, and viability was determined using trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. Of the eight drugs tested, amlodipine, prochlorperazine, and loperamide showed potent amoebicidal effects, as no viable trophozoites were observed (>95% kill rate), while amiodarone, procyclidine, digoxin, and apomorphine exhibited up to 50% amoebicidal effects. In contrast, haloperidol did not affect viability, but all the drugs tested inhibited
A. castellanii
growth. Importantly, amlodipine, prochlorperazine, and loperamide showed compelling cysticidal effects. The cysticidal effects were irreversible, as cysts treated with the aforementioned drugs did not reemerge as viable amoebae upon inoculation in the growth medium. Except for apomorphine and haloperidol, all the tested drugs blocked trophozoite differentiation into cysts in encystation assays. Given the limited availability of effective drugs to treat amoebal infections, the clinically available drugs tested in this study represent potential agents for managing keratitis and granulomatous amoebic encephalitis caused by
Acanthamoeba
spp. and possibly against other meningoencephalitis-causing amoebae, such as
Balamuthia mandrillaris
and
Naegleria fowleri
.
Genomewide Screen for Modulators of Evolvability under Toxic Antibiotic Exposure
Authors: Méhi Orsolya , Bogos Balázs , Csörgő Bálint , Pál Csaba
Published: May 2013

Antibiotic resistance is generally selected within a window of concentrations high enough to inhibit wild-type growth but low enough for new resistant mutants to emerge. We studied
de novo
evolution of resistance to ciprofloxacin in an
Escherichia coli
knockout library. Five null mutations had little or no effect on intrinsic antibiotic susceptibility but increased the upper antibiotic dosage to which initially sensitive populations could adapt. These mutations affect mismatch repair, translation fidelity, and iron homeostasis.
Congenic Strains for Genetic Analysis of Virulence Traits in Cryptococcus gattii
Authors: Zhu Pinkuan , Zhai Bing , Lin Xiaorong , Idnurm Alexander
Published: May 2013

Cryptococcus gattii
is responsible for a large outbreak of potentially fatal disease that started in the late 1990s on Vancouver Island, Canada. How this fungus and the outbreak isolates in particular cause disease in immunocompetent people is unknown, with differing hypotheses. To explore genetic contributions, a pair of congenic
a
and α mating type strains was generated by a series of 11 backcrosses to introgress the
MAT
locus from a nonoutbreak strain into the background of strain R265, isolated from a Vancouver Island patient. The congenic pair was used to investigate three traits: mitochondrial inheritance, the effect of the
MAT
alleles on virulence, and the impact of a predicted virulence factor on pathogenicity. The two congenic strains show the same virulence in different models of cryptococcosis and equivalent levels of competition in coinfection assays. These results rule out a role of the
MAT
locus and mitochondrial genotype as major virulence factors in the outbreak strains. Disruption of Bwc2, a light-dependent transcription factor, resulted in reduced virulence, consistent with a similar function in the related species
Cryptococcus neoformans
. The
C. gattii
congenic strains represent a new resource for exploring the evolution of virulence in the
C. neoformans-C. gattii
clade.
Congenic Strains of the Filamentous Form of Cryptococcus neoformans for Studies of Fungal Morphogenesis and Virulence
Authors: Zhai Bing , Zhu Pinkuan , Foyle Dylan , Upadhyay Srijana , Idnurm Alexander , Lin Xiaorong
Published: May 2013

Cryptococcus neoformansis an unconventional dimorphic fungus that can grow either as a yeast or in a filamentous form. To facilitate investigation of genetic factors important for its morphogenesis and pathogenicity, congenicaand α strains for a filamentous form were constructed. XL280 (α) was selected as the background strain because of its robust ability to undergo the morphological transition from yeast to the filamentous form. TheMATaallele from a sequenced strain JEC20 was introgressed into the XL280 background to generate the congenicaand α pair strains. The resulting congenic strains were then used to test the impact of mating type on virulence. In both the inhalation and the intravenous infection models of murine cryptococcosis, the congenicaand α strains displayed comparable levels of high virulence. Thea-α coinfections displayed equivalent virulence to the individualaor α infections in both animal models. Further analyses of the mating type distribution ina-α coinfected mice suggested no influence ofa-α interactions on cryptococcal neurotropism, irrespective of the route of inoculation. Furthermore, deletion or overexpression of a known transcription factor, Znf2, in XL280 abolished or enhanced filamentation and biofilm formation, consistent with its established role. Overexpression of Znf2 in XL280 led to attenuation of virulence and a reduced abundance in the brain but not in other organs, suggesting that Znf2 might interfere with cryptococcal neurotropism upon extrapulmonary dissemination. In summary, the congenic strains provide a new resource for the exploration of the relationship inCryptococcusbetween cellular morphology and pathogenesis.
Peripheral cementifying fibroma: a clinical diagnostic dilemma
Authors: Choubey Shikha , Banda Naveen Reddy , Banda Vanaja Reddy , Vyawahare Saket
Published: May 2013

The peripheral ossifying fibroma (POF) is a reactive gingival overgrowth occurring frequently in the anterior maxilla. It originates in the cells of the periodontal ligament and is more common in children and young adults. In the current article a case of gingival over growth, which was thought to be puberty-induced gingivitis was seen in the lower anterior maxillary gingiva. Histology of the excised tissue showed cellular, fibrous connective tissue stroma with calcified osseous calcifications indicative of POF. The definitive diagnosis is established only by histological examination, which revealed the presence of highly cellular connective tissue with focal calcifications. Surgery is the treatment of choice, though the recurrence rate can reach 20% in case of POF. After histological confirmation the recall and clinical evaluation protocol of POF varies due to its increased recurrence rate, which the general dentist should be aware of.
Residential proximity to major roadways and renal function
Authors: Lue Shih-Ho , Wellenius Gregory A , Wilker Elissa H , Mostofsky Elizabeth , Mittleman Murray A
Published: May 2013

Background

Living near major roadways has been associated with increased risk of cardiovascular events, but little is known about its impact on renal function.

Methods

We calculated the estimated glomerular filtration rate (eGFR) for 1103 consecutive Boston-area patients hospitalised with confirmed acute ischaemic stroke between 1999 and 2004. We used linear regression to evaluate the association between eGFR and categories of residential distance to major roadway (0 to ≤50, >50 to ≤100, >100 to ≤200, >200 to ≤400, >400 to ≤1000 and >1000 m) adjusting for age, sex, race, smoking, comorbid conditions, treatment with ACE inhibitor and neighbourhood-level socioeconomic characteristics. In a second analysis, we considered the log of distance to major roadway as a continuous variable.

Results

Patients living closer to a major roadway had lower eGFR than patients living farther away (Ptrend=0.01). Comparing patients living 50 m versus 1000 m from a major roadway was associated with a 3.9 ml/min/1.73 m2lower eGFR (95% CI 1.0 to 6.7; p=0.007): a difference comparable in magnitude to the reduction in eGFR observed for a 4-year increase in age in population-based studies. The magnitude of this association did not differ significantly across categories of age, sex, race, history of hypertension, diabetes or socioeconomic status.

Conclusions

Living near a major roadway is associated with lower eGFR in a cohort of patients presenting with acute ischaemic stroke. If causal, these results imply that exposures associated with living near a major roadway contribute to reduced renal function, an important risk factor for cardiovascular events.
An ontology-driven framework for enhancing reusability of distributed simulation modeling of industrial construction processes
Authors: Saba Farzaneh , Mohamed Yasser
Published: May 2013

This paper describes an ontology-driven framework for developing distributed simulation modeling of construction processes. The research described in the paper is motivated by the necessity of knowledge sharing between distributed simulation modeling collaborators, and reuse and portability challenges in construction simulation models. Our approach addresses these challenges through ontological modeling and linking of construction simulation modeling components including (i) ontology of construction process, (ii) ontology of simulation world view, and (iii) ontology of distributed simulation modeling application tool. Within the paper, ontology driven approach and mapping of ontologies for information transference between simulation components has been described. Another discussed application of ontologies is structuring of simulation modeling development through use of reusable elements. A large-scale distributed simulation model of industrial construction processes has been outlined to illustrate the application of the approach.
Proteins differentially expressed in conidia and mycelia of the entomopathogenic fungusMetarhizium anisopliaesensu stricto
Authors: Su Yubin , Guo Qingfeng , Tu Jie , Li Xiaoxia , Meng Lixue , Cao Liping , Dong Dong , Qiu Junzhi , Guan Xiong
Published: May 2013

Metarhizium anisopliae is a well-characterized entomopathogenic fungus that attacks a variety of insects. Its conidia are involved in its propagation and also in its infection of host insects. To investigate the protein expression profiles and to identify the proteins related to development and pathogenesis, we performed a comparative proteomic analysis of the conidia and mycelia of an M. anisopliae strain (Ma1291). The analysis used 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We detected 898 ± 37 protein spots in conidia and 1072 ± 24 in mycelia of strain Ma1291. A comparison of the 2 protein-expression profiles indicated that only 28% of protein spots were common to both developmental stages. Finally, we identified 30 proteins (19 from conidia and 11 from mycelia). The identified proteins exclusive to conidia were those involved in protective processes, appressorium formation, and degradation of the host cuticle (protease PR1H). The identified proteins exclusive to mycelia included major proteins participating in biosynthetic and energy metabolism, such as UTP-glucose-1-phosphate uridylyltransferase and heat shock protein 70. This research provides the first proteomic analysis of different developmental stages of M. anisopliae, and the results should facilitate clarification of the molecular basis of these epigenetic variations.
TIGHT ANALYSIS OF SHORTEST PATH CONVERGECAST IN WIRELESS SENSOR NETWORKS
Authors: AUGUSTINE JOHN , HAN QI , LODEN PHILIP , LODHA SACHIN , ROY SASANKA
Published: May 2013

We consider the convergecast problem in wireless sensor networks where each sensor has a reading that must reach a designated sink. Since a sensor reading can usually be encoded in a few bytes, more than one reading can readily fit into a standard transmission packet. We assume that each packet hop consumes one unit of energy. Our objective is to minimize the total energy consumed to send all readings to the sink. We show that this problem is NP-hard even when all readings are of fixed size. We then study a class SPEP of distributed algorithms that is completely defined by two properties. Firstly, the packets hop along some shortest path to the sink. Secondly, the nodes use an elementary packing algorithm to pack readings into packets.

Our main technical contribution is a lower bound. We show that no algorithm for UCCP that either follows the shortest path or packs in an elementary manner is a (2 − ϵ)-approximation, for any fixed ϵ > 0. To complement this, we show that SPEP algorithms are [Formula: see text]-approximation for UCCP and 3-approximation for CCP, where k ≥ 2 is the number of readings that can fit within a packet. We conclude with some special cases and experimental observations.
(1,2)-HAMILTONIAN COMPLETION ON A MATCHING
Authors: BIENKOWSKI MARCIN , ZALEWSKI PAWEŁ
Published: May 2013

We consider the problem of computing a minimum-weight Hamiltonian cycle on an undirected graph with edges' weights from set {0, 1, 2}, where 0-weight edges create a perfect matching of the graph. We provide a (4/3)-approximation algorithm and show that the problem is APX-complete.
A NUMERICAL STUDY OF UNIVERSALITY AND SELF-SIMILARITY IN SOME FAMILIES OF FORCED LOGISTIC MAPS
Authors: RABASSA PAU , JORBA ÀNGEL , TATJER JOAN CARLES
Published: May 2013

We explore different two-parametric families of quasi-periodically Forced Logistic Maps looking for universality and self-similarity properties. In the bifurcation diagram of the one-dimensional Logistic Map, it is well known that there exist parameter values sn where the 2n-periodic orbit is superattracting. Moreover, these parameter values lay between the parameters corresponding to two consecutive period doublings. In the quasi-periodically Forced Logistic Maps, these points are replaced by invariant curves, that undergo a (finite) sequence of period doublings.

In this work, we study numerically the presence of self-similarities in the bifurcation diagram of the invariant curves of these quasi-periodically Forced Logistic Maps. Our computations show a remarkable self-similarity for some of these families. We also show that this self-similarity cannot be extended to any quasi-periodic perturbation of the Logistic map.
Contrasting metamorphic records and their implications for tectonic process in the central Sør Rondane Mountains, eastern Dronning Maud Land, East Antarctica
Authors: Adachi Tatsuro , Hokada Tomokazu , Osanai Yasuhito , Nakano Nobuhiko , Baba Sotaro , Toyoshima Tsuyoshi
Published: May 2013

Metamorphic rocks in the central part of Sør Rondane Mountains, eastern Dronning Maud Land, East Antarctica, are classified into three types based on petrological characteristics. (i) The Austkampane area preserves
c.
800 °C and 0.5–0.6 GPa peak metamorphic conditions followed by decompression and subsequent isobaric cooling and later hydration (A-type). (ii) The Brattnipene and eastern Menipa area preserve peak
P–T
conditions of
c.
800 °C and 0.7–0.8 GPa with subsequent isobaric cooling and later hydration (B-type). (iii) The area including Lunckeryggen, southern Walnumfjella and western Menipa preserves an amphibolite–facies peak metamorphic condition with signatures of prograde metamorphism (L-type), which are typically unaffected by the retrograde hydration event. Peak granulite–facies metamorphism of A- and B-type rocks are contemporaneous at
c.
640–600 Ma, but a difference in the
P–T
paths between these rocks can be explained by thrusting of the A-type rock unit onto the B-type rock unit. By contrast, the timing of the metamorphism of the L-type rocks is significantly younger at
c.
550 Ma, possibly related to the intrusion of pegmatites and granitoids. These metamorphic records in the central part of the Sør Rondane Mountains can be a test ground for the regional tectonic processes proposed for the orogeny related to Gondwana formation.

Supplementary material:

Representative mineral compositions are listed at
www.geolsoc.org.uk/SUP18623
Human perception of haptic-to-video and haptic-to-audio skew in multimedia applications
Authors: Silva Juan M. , Orozco Mauricio , Cha Jongeun , Saddik Abdulmotaleb El , Petriu Emil M.
Published: May 2013

The purpose of this research is to assess the sensitivity of humans to perceive asynchrony among media signals coming from a computer application. Particularly we examine haptic-to-video and haptic-to-audio skew. For this purpose we have designed an experimental setup, where users are exposed to a basic multimedia presentation resembling a ping-pong game. For every collision between a ball and a racket, the user is able to perceive auditory, visual, and haptic cues about the collision event. We artificially introduce negative and positive delay to the auditory and visual cues with respect to the haptic stream. We subjectively evaluate the perception of inter-stream asynchrony perceived by the users using two types of haptic devices. The statistical results of our evaluation show perception rates of around 100 ms regardless of modality and type of device.
Modeling the effect of user interactions on mesh-based P2P VoD streaming systems
Authors: Zhao Zhen Wei , Samarth Sameer , Ooi Wei Tsang
Published: May 2013

User interactions such as seeks and pauses are widely supported by existing Peer-to-Peer Video-on-Demand (P2P VoD) streaming systems. Their effect on the streaming system, however, has not been well studied. Seeks cause peers to skip part of the video, making them stay in the system for shorter time, and thus contribute less. On the other hand, only part of the video is downloaded due to seeks, reducing peers' demand from the system. It is unclear which factor dominates the effect of seeks on the streaming system. Pauses during playback, on one hand, allow peers to stay longer in the system and upload more content. When interleaved with seeks, however, long pauses may increase peers' demand unnecessarily as peers may download content that will eventually be skipped by subsequent forward seeks. The collective effect of seeks and pauses, together with the known random peer departure, is unintuitive and needs to be addressed properly so as to understand the effect of human factors on the streaming system performance.

In this article, we develop an analytical model to both qualitatively and quantitatively study the effect of seeks and pauses on mesh-based P2P VoD streaming systems, in particular, the effect on the server cost. Our model can help in understanding how human factors such as seeks and pauses affect the streaming system performance, tuning a P2P VoD system towards better system performance and stability, and providing a framework for capacity planning.
Modeling BitTorrent-like systems with many classes of users
Authors: Liao Wei-Cherng , Papadopoulos Fragkiskos , Psounis Konstantinos , Psomas Constantinos
Published: May 2013

BitTorrent is one of the most successful peer-to-peer systems. Researchers have studied a number of aspects of the system, including its scalability, performance, efficiency and fairness. However, the complexity of the system has forced most prior analytical work to make a number of simplifying assumptions, for example, user homogeneity, or even ignore some central aspects of the protocol altogether, for example, the rate-based Tit-for-Tat (TFT) unchoking scheme, in order to keep the analysis tractable.

Motivated by this, in this article we propose two analytical models that accurately predict the performance of the system while considering the central details of the BitTorrent protocol. Our first model is a steady-state one, in the sense that it is valid during periods of time where the number of users remains fixed. Freed by the complications of user time-dynamics, we account for many of the central details of the BitTorrent protocol and accurately predict a number of performance metrics. Our second model combines prior work on fluid models with our first model to capture the transient behavior as new users join or old users leave, while modelling many major aspects of BitTorrent. To the best of our knowledge, this is the first model that attempts to capture the transient behavior of many classes of heterogeneous users. Finally, we use our analytical methodology to introduce and study the performance of a flexible token-based scheme for BitTorrent, show how this scheme can be used to block freeriders and tradeoff between higher-bandwidth and lower-bandwidth users performance, and evaluate the scheme's parameters that achieve a target operational point.
Characterizing per-application network traffic using entropy
Authors: Petkov Vladislav , Rajagopal Ram , Obraczka Katia
Published: May 2013

The Internet has been evolving into a more heterogeneous internetwork with diverse new applications imposing more stringent bandwidth and QoS requirements. Already new applications such as YouTube, Hulu, and Netflix are consuming a large fraction of the total bandwidth. We argue that, in order to engineer future internets such that they can adequately cater to their increasingly diverse and complex set of applications while using resources efficiently, it is critical to be able to characterize the load that emerging and future applications place on the underlying network. In this article, we investigate entropy as a metric for characterizing per-flow network traffic complexity. While previous work has analyzed aggregated network traffic, we focus on studying isolated traffic flows. Per-application flow characterization caters to the need of network control functions such as traffic scheduling and admission control at the edges of the network. Such control functions necessitate differentiating network traffic on a per-application basis. The “entropy fingerprints” that we get from our entropy estimator summarize many characteristics of each application's network traffic. Not only can we compare applications on the basis of peak entropy, but we can also categorize them based on a number of other properties of the fingerprints.
Subcellular redistribution of the renal betaine transporter during hypertonic stress
Authors: Kempson Stephen A. , Parikh Vaibhave , Xi Lixuan , Chu Shaoyou , Montrose Marshall H.
Published: May 2013

The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the NH2 terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent Km for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2-3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins.
Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages
Authors: Okugawa Shu , Ota Yasuo , Kitazawa Takatoshi , Nakayama Kuniko , Yanagimoto Shintaro , Tsukada Kunihisa , Kawada Miki , Kimura Satoshi
Published: May 2013

The mechanisms by which lipopolysaccharide (LPS) is recognized, and how such recognition leads to innate immune responses, are poorly understood. Stimulation with LPS induces the activation of a variety of proteins, including mitogen-activated protein kinases (MAPKs) and NF-κB. Activation of protein tyrosine kinases (PTKs) is also necessary for a number of biological responses to LPS. We used a murine macrophage-like cell line, RAW264.7, to demonstrate that Janus kinase (JAK)2 is tyrosine phosphorylated immediately after LPS stimulation. Anti-Toll-like receptor (TLR)4 neutralization antibody inhibits the phosphorylation of JAK2 and the c-Jun NH2-terminal protein kinase (JNK). Both the JAK inhibitor AG490 and the kinase-deficient JAK2 protein reduce the phosphorylation of JNK and phosphatidylinositol 3-kinase (PI3K) via LPS stimulation. Pharmacological inhibition of the kinase activity of PI3K with LY-294002 decreases the phosphorylation of JNK. Finally, we show that JAK2 is involved in the production of IL-1β and IL-6. PI3K and JNK are also important for the production of IL-1β. These results suggest that LPS induces tyrosine phosphorylation of JAK2 via TLR4 and that JAK2 regulates phosphorylation of JNK mainly through activation of PI3K. Phosphorylation of JAK2 via LPS stimulation is important for the production of IL-1β via the PI3K/JNK cascade. Thus JAK2 plays a pivotal role in LPS-induced signaling in macrophages.
More than apical: distribution of SGLT1 in Caco-2 cells
Authors: Kipp Helmut , Khoursandi Saeed , Scharlau Daniel , Kinne Rolf K. H.
Published: May 2013

We investigated the distribution of the endogenous sodium-d-glucose cotransporter (SGLT1) in polarized Caco-2 cells, a model for enterocytes. A cellular organelle fraction was separated by free-flow electrophoresis and subjected to the analysis of endogenous and exogenous marker enzymes for various membrane vesicle components. Furthermore, the presence of SGLT1 was tested by an ELISA assay employing newly developed epitope specific antibodies. Thereby it was found that the major amount of SGLT1 resided in intracellular compartments and only a minor amount in apical plasma membranes. The distribution ratio between intracellular SGLT1 and apical membrane-associated SGLT1 was ∼2:1. Further immunocytochemical investigation of SGLT1 distribution in fixed Caco-2 cells by epifluorescence and confocal microscopy revealed that the intracellular compartments containing SGLT1 were associated with microtubules. Elimination of SGLT1 synthesis by incubation of cells with cycloheximide did not significantly reduce the size of the intracellular SGLT1 pool. Furthermore, the half-life of SGLT1 in Caco-2 cells was determined to be 2.5 days by metabolic labeling followed by immunoprecipitation. Our data suggest that most of the intracellular SGLT1 are not transporters en route from biosynthesis to their cellular destination but represent an intracellular reserve pool. We therefore propose that intracellular compartments containing SGLT1 are involved in the regulation of SGLT1 abundance at the apical cell surface.
NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells
Authors: Zhang Jianliang , Xia Shen-Ling , Block Edward R. , Patel Jawaharlal M.
Published: May 2013

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca2+]i) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively ( P < 0.05). [Ca2+]i in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca2+]i in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca2+]i, suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca2+]i and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca2+]i and capacitive calcium entry in porcine PAEC.
VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration
Authors: van Wetering Sandra , van den Berk Nadia , van Buul Jaap D. , Mul Frederik P. J. , Lommerse Ingrid , Mous Rogier , Klooster Jean-Paul ten , Zwaginga Jaap-Jan , Hordijk Peter L.
Published: May 2013

Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the β1- and β2-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1β-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation.
Redox signaling of cardiac HSF1 DNA binding
Authors: Paroo Zain , Meredith Michael J. , Locke Marius , Haist James V. , Karmazyn Morris , Noble Earl G.
Published: May 2013

Experiments involving chemical induction of the heat shock response in simple biological systems have generated the hypothesis that protein denaturation and consequential binding of heat shock transcription factor 1 (HSF1) to proximal heat shock elements (HSEs) on heat shock protein ( hsp) genes are the result of oxidation and/or depletion of intracellular thiols. The purpose of the present investigation was to determine the role of redox signaling of HSF1 in the intact animal in response to physiological and pharmacological perturbations. Heat shock and exercise induced HSF1-HSE DNA binding in the rat myocardium ( P < 0.001) in the absence of changes in reduced glutathione (GSH), the major nonprotein thiol in the cell. Ischemia-reperfusion, which decreased GSH content ( P < 0.05), resulted in nonsignificant HSF1-HSE formation. This dissociation between physiological induction of HSF1 and changes in GSH was not gender dependent. Pharmacological ablation of GSH withl-buthionine-[ S, R]-sulfoximine (BSO) treatment increased myocardial HSF1-HSE DNA binding in estrogen-naive animals ( P = 0.007). Thus, although physiological induction of HSF1-HSE DNA binding is likely regulated by mediators of protein denaturation other than cellular redox status, the proposed signaling pathway may predominate with pharmacological oxidation and may represent a plausible and accessible strategy in the development of HSP-based therapies.
Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-β-induced nuclear accumulation
Authors: Roelen Bernard A. J. , Cohen Ori S. , Raychowdhury Malay K. , Chadee Deborah N. , Zhang Ying , Kyriakis John M. , Alessandrini Alessandro A. , Lin Herbert Y.
Published: May 2013

Smad4, the common Smad, is central for transforming growth factor (TGF)-β superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-β and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-β-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.
Role of αvβ3-integrin in TNF-α-induced endothelial cell migration
Authors: Gao Baochong , Saba Thomas M. , Tsan Min-Fu
Published: May 2013

Tumor necrosis factor-α (TNF-α), one of the major inflammatory cytokines, is known to influence endothelial cell migration. In this study, we demonstrate that exposure of calf pulmonary artery endothelial cells to TNF-α caused an increase in the formation of membrane protrusions and cell migration. Fluorescence microscopy revealed an increase in αvβ3focal contacts but a decrease in α5β1 focal contacts in TNF-α-treated cells. In addition, both cell-surface and total cellular expression of αvβ3-integrins increased significantly, whereas the expression of α5β1-integrins was unaltered. Only focal contacts containing αvβ3- but not α5β1-integrins were present in membrane protrusions of cells at the migration front. In contrast, robust focal contacts containing α5β1-integrins were present in cells behind the migration front. A blocking antibody to αvβ3, but not a blocking antibody to α5-integrins, significantly inhibited TNF-α-induced cell migration. These results indicate that in response to TNF-α, endothelial cells may increase the activation and ligation of αvβ3 while decreasing the activation and ligation of α5β1-integrins to facilitate cell migration, a process essential for vascular wound healing and angiogenesis.
Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts
Authors: Carton Iris , Hermans Diane , Eggermont Jan
Published: May 2013

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way.
L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis
Authors: Yakubu Momoh A. , Leffler Charles W.
Published: May 2013

We investigated the role of intracellular calcium concentration ([Ca2+]i) in endothelin-1 (ET-1) production, the effects of potential vasospastic agents on [Ca2+]i, and the presence of L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells. Primary cultures of endothelial cells isolated from piglet cerebral microvessels were used. Confluent cells were exposed to either the thromboxane receptor agonist U-46619 (1 μM), 5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 μM) alone or after pretreatment with the Ca2+-chelating agent EDTA (100 mM), the L-type Ca2+ channel blocker verapamil (10 μM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 μM) for 15 min. ET-1 production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT), or 3.9 (LPA) fmol/μg protein, respectively. Such elevated ET-1 biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. To investigate the presence of L-type voltage-dependent Ca2+channels in endothelial cells, the [Ca2+]isignal was determined fluorometrically by using fura 2-AM. Superfusion of confluent endothelial cells with U-46619, 5-HT, or LPA significantly increased [Ca2+]i. Pretreatment of endothelial cells with high K+ (60 mM) or nifedipine (4 μM) diminished increases in [Ca2+]i induced by the vasoactive agents. These results indicate that 1) elevated [Ca2+]i signals are involved in ET-1 biosynthesis induced by specific spasmogenic agents, 2) the increases in [Ca2+]i induced by the vasoactive agents tested involve receptor as well as L-type voltage-dependent Ca2+ channels, and 3) primary cultures of cerebral microvascular endothelial cells express L-type voltage-dependent Ca2+ channels.
Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra
Authors: Sergeant G. P. , Thornbury K. D. , McHale N. G. , Hollywood M. A.
Published: May 2013

Freshly dispersed interstitial cells from the rabbit urethra were studied by using the perforated-patch technique. When cells were voltage clamped at −60 mV and exposed to 10 μM norepinephrine (NE) at 80-s intervals, either large single inward currents or a series of oscillatory inward currents of diminishing amplitude were evoked. These currents were blocked by either phentolamine (1 μM) or prazosin (1 μM), suggesting that the effects of NE were mediated via α1-adrenoceptors. NE-evoked currents were depressed by the blockers of Ca2+-activated Cl− currents, niflumic acid (10 μM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversal potential of the above currents changed in a predictable manner when the Cl− equilibrium potential was altered, again suggesting that they were due to activation of a Cl−conductance. NE-evoked currents were decreased by 10 μM cyclopiazonic acid, suggesting that they were dependent on store-released Ca2+. Inhibition of NE-evoked currents by the phospholipase C inhibitor 2-nitro-4-carboxyphenyl- N, N-diphenylcarbamate (100 μM) suggested that NE releases Ca2+ via an inositol 1,4,5-trisphosphate (IP3)-dependent mechanism. These results support the idea that stimulation of α1-adrenoceptors releases Ca2+ from an IP3-sensitive store, which in turn activates Ca2+-activated Cl− current in freshly dispersed interstitial cells of the rabbit urethra. This elevates slow wave frequency in these cells and may underlie the mechanism responsible for increased urethral tone during nerve stimulation.
Insulin inhibits PDGF-directed VSMC migration via NO/ cGMP increase of MKP-1 and its inactivation of MAPKs
Authors: Jacob Asha , Molkentin Jeffery D. , Smolenski Albert , Lohmann Suzanne M. , Begum Najma
Published: May 2013

In this study, we examined the role of insulin in the control of vascular smooth muscle cell (VSMC) migration in the normal vasculature. Platelet-derived growth factor (PDGF) increased VSMC migration, which was inhibited by pretreatment with insulin in a dose-dependent manner. Insulin also caused a 60% decrease in PDGF-stimulated mitogen-activated protein kinase (MAPK) phosphorylation and activation. Insulin inhibition of MAPK was accompanied by a rapid induction of MAPK phosphatase (MKP-1), which inactivates MAPKs by dephosphorylation. Pretreatment with inhibitors of the nitric oxide (NO)/cGMP pathway, blocked insulin-induced MKP-1 expression and restored PDGF-stimulated MAPK activation and migration. In contrast, adenoviral infection of VSMCs with MKP-1 or cGMP-dependent protein kinase Iα (cGK Iα), the downstream effector of cGMP signaling, blocked the activation of MAPK and prevented PDGF-directed VSMC migration. Expression of antisense MKP-1 RNA prevented insulin's inhibitory effect and restored PDGF-directed VSMC migration and MAPK phosphorylation. We conclude that insulin inhibition of VSMC migration may be mediated in part by NO/cGMP/cGK Iα induction of MKP-1 and consequent inactivation of MAPKs.
Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication
Authors: Dufresne Julie , Finnson Kenneth W. , Gregory Mary , Cyr Daniel G.
Published: May 2013

In the epididymis, Cx43 forms gap junctions between principal and basal cells but not between adjacent principal cells. Cx30.3, 31.1, and 32 were identified in adult rat epididymis by RT-PCR, whereas Cx26 was present in young rats. Postnatal development studies indicate that Cx26 mRNA was detectable only in the caput-corpus region of the epididymis and that levels increased by fivefold during the first 4 wk postnatally, when epithelial cells differentiate, and decrease to nondetectable levels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout the epididymis in young rats and began to increase in the second and third weeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32 were localized to the lateral plasma membranes between adjacent epithelial cells of the epididymis. Colocalization studies indicate that Cx26 and Cx32 exist either independently of one another or can colocalize along the lateral plasma membrane of epithelial cells in young rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.
Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases
Authors: Boo Yong Chool , Jo Hanjoong
Published: May 2013

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases—protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases—are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.