bluuebunny/crossref_metadata_2025 huggingface dataset:
Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells
Authors: Aulestia Francisco J. , Redondo Pedro C. , Rodríguez-García Arancha , Rosado Juan A. , Salido Ginés M. , Alonso Maria Teresa , García-Sancho Javier
Published: February 2011

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.
Autotransporter passenger domain secretion requires a hydrophobic cavity at the extracellular entrance of the β-domain pore
Authors: Zhai Yujia , Zhang Kai , Huo Yanwu , Zhu Yanshi , Zhou Qiangjun , Lu Jiuwei , Black Isobel , Pang Xiaoyun , Roszak Aleksander W. , Zhang Xujia , Isaacs Neil W. , Sun Fei
Published: February 2011

Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence. In the present study, we have solved the crystal structure of the BrkA β-domain at 3 Å (1 Å=0.1 nm) resolution. Special features are a hairpin-like structure formed by the external loop L4, which is observed fortuitously sitting inside the pore of the crystallographic adjacent β-domain, and a previously undiscovered hydrophobic cavity formed by patches on loop L4 and β-strands S5 and S6. This adopts a ubiquitous structure characteristic of all AT β-domains. Mutagenesis studies have demonstrated that the hairpin-like structure and hydrophobic cavity are crucial for BrkA passenger domain (virulence effector) translocation. This structure helps in understanding the molecular mechanism of AT assembly and secretion and provides a potential target for anti-pertussis drug design.
Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells
Authors: Patel Nitin , Tahara Stanley M. , Malik Punam , Kalra Vijay K.
Published: February 2011

PAI-1 (plasminogen activator inhibitor-1) is a key physiological inhibitor of fibrinolysis. Previously, we have reported PlGF (placental growth factor)-mediated transcriptional up-regulation of PAI-1 (SERPINE1) mRNA expression via activation of HIF-1α (hypoxia-inducible factor-1α) and AP-1 (activator protein-1) in HPMVECs (human pulmonary microvascular endothelial cells), which resulted in elevated PAI-1 in humans with SCA (sickle cell anaemia). In the present study, we have identified the role of post-transcriptional mechanism(s) of PlGF-mediated accumulation of PAI-1 mRNA in HPMVECs by examining the role of microRNAs (miRNAs/miRs) in PlGF-induced PAI-1 mRNA stability. Our results show reduced expression of miR-30c and miR-301a, but not of miR-99a, in response to PlGF, which have evolutionarily conserved binding sites in the 3′-UTR (3′-untranslated region) of PAI-1 mRNA. Transfection of anti-miR-30c or anti-miR-301a oligonucleotides resulted in increased PAI-1 mRNA levels, which were increased further with PlGF stimulation. Conversely, overexpression of pre-miR-30c or pre-miR-301a resulted in an attenuation of PlGF-induced PAI-1 mRNA and protein levels. Luciferase reporter assays using wild-type and mutant 3′-UTR constructs confirmed that the PAI-1 3′-UTR is indeed a direct target of miR-30c and miR-301a. Finally, plasma levels of miR-30c and miR-301a were significantly down-regulated in patients with SCA compared with normal controls. These results provide a post-transcriptional regulatory mechanism of PlGF-induced PAI-1 elevation.
Trans-interaction of nephrin and Neph1/Neph3 induces cell adhesion that associates with decreased tyrosine phosphorylation of nephrin
Authors: Heikkilä Eija , Ristola Mervi , Havana Marika , Jones Nina , Holthöfer Harry , Lehtonen Sanna
Published: February 2011

Slit diaphragms are specialized junctions between glomerular epithelial cells (podocytes) that are crucial for glomerular ultrafiltration. The Ig superfamily members nephrin and Neph1 are essential components of the slit diaphragm, whereas the role of Neph1 homologue Neph3 in the slit diaphragm is unknown. In the present paper we show that Neph3 homodimerizes and heterodimerizes with nephrin and Neph1. We further investigated whether these interactions play a role in cell adhesion by using mouse L fibroblasts that lack endogenous cell-adhesion activity and found that Neph1 and Neph3 are able to induce cell adhesion alone, whereas nephrin needs to trans-interact with Neph1 or Neph3 in order to promote formation of cell–cell contacts. Tyrosine phosphorylation of nephrin was down-regulated after nephrin trans-interacted with either Neph1 or Neph3 leading to formation of cell–cell contacts. We further found that the expression of Neph3 was increased in nephrin-deficient mouse podocytes. The findings of the present paper show that nephrin and Neph1 or Neph3 trans-interactions promote cell-contact formation, suggesting that they may also function together in slit diaphragm assembly.
A new principle of oligomerization of plant DEG7 protease based on interactions of degenerated protease domains
Authors: Schuhmann Holger , Mogg Ulrike , Adamska Iwona
Published: February 2011

Deg/HtrA proteases are a large group of ATP-independent serine endoproteases found in almost every organism. Their usual domain arrangement comprises a trypsin-type protease domain and one or more PDZ domains. All Deg/HtrA proteases form homo-oligomers with trimers as the basic unit, where the active protease domain mediates the interaction between individual monomers. Among the members of the Deg/HtrA protease family, the plant protease DEG7 is unique since it contains two protease domains (one active and one degenerated) and four PDZ domains. In the present study, we investigated the oligomerization behaviour of this unusual protease using yeast two-hybrid analysis in vivo and with recombinant protein in vitro. We show that DEG7 forms trimeric complexes, but in contrast with other known Deg/HtrA proteases, it shows a new principle of oligomerization, where trimerization is based on the interactions between degenerated protease domains. We propose that, during evolution, a duplicated active protease domain degenerated and specialized in protein–protein interaction and complex formation.
The fission yeast Schizosaccharomyces pombe has two distinct tRNase ZLs encoded by two different genes and differentially targeted to the nucleus and mitochondria
Authors: Gan Xuhua , Yang Jing , Li Jun , Yu Haiyan , Dai Hongmei , Liu Jinyu , Huang Ying
Published: February 2011

tRNase Z is the endonuclease that is involved in tRNA 3′-end maturation by removal of the 3′-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3′-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.
Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach
Authors: Zhang Weiqiang , Penmatsa Himabindu , Ren Aixia , Punchihewa Chandanamali , Lemoff Andrew , Yan Bing , Fujii Naoaki , Naren Anjaparavanda P.
Published: February 2011

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein–protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)–NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR–NHERF2–LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2–NHERF2 interaction. We show that this compound can disrupt the LPA2–NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl− channel function can be finely tuned by modulating PDZ domain-based protein–protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl− channels.
Comparing the potential renal protective activity of desferrioxamine B and the novel chelator desferrioxamine B-N-(3-hydroxyadamant-1-yl)carboxamide in a cell model of myoglobinuria
Authors: Groebler Ludwig K. , Liu Joe , Shanu Anu , Codd Rachel , Witting Paul K.
Published: February 2011

Accumulating Mb (myoglobin) in the kidney following severe burns promotes oxidative damage and inflammation, which leads to acute renal failure. The potential for haem–iron to induce oxidative damage has prompted testing of iron chelators [e.g. DFOB (desferrioxamine B)] as renal protective agents. We compared the ability of DFOB and a DFOB-derivative {DFOB-AdAOH [DFOB-N-(3-hydroxyadamant-1-yl)carboxamide]} to protect renal epithelial cells from Mb insult. Loading kidney-tubule epithelial cells with dihydrorhodamine-123 before exposure to 100 μM Mb increased rhodamine-123 fluorescence relative to controls (absence of Mb), indicating increased oxidative stress. Extracellular Mb elicited a reorganization of the transferrin receptor as assessed by monitoring labelled transferrin uptake with flow cytometry and inverted fluorescence microscopy. Mb stimulated HO-1 (haem oxygenase-1), TNFα (tumour necrosis factor α), and both ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) gene expression and inhibited epithelial monolayer permeability. Pre-treatment with DFOB or DFOB-AdAOH decreased Mb-mediated rhodamine-123 fluorescence, HO-1, ICAM and TNFα gene expression and restored monolayer permeability. MCP-1 (monocyte chemotactic protein 1) secretion increased in cells exposed to Mb-insult and this was abrogated by DFOB or DFOB-AdAOH. Cells treated with DFOB or DFOB-AdAOH alone showed no change in permeability, MCP-1 secretion or HO-1, TNFα, ICAM or VCAM gene expression. Similarly to DFOB, incubation of DFOB-AdAOH with Mb plus H2O2 yielded nitroxide radicals as detected by EPR spectroscopy, indicating a potential antioxidant activity in addition to metal chelation; Fe(III)-loaded DFOB-AdAOH showed no nitroxide radical formation. Overall, the chelators inhibited Mb-induced oxidative stress and inflammation and improved epithelial cell function. DFOB-AdAOH showed similar activity to DFOB, indicating that this novel low-toxicity chelator may protect the kidney after severe burns.
Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants
Authors: Bal Naresh C. , Jena Nivedita , Sopariwala Danesh , Balaraju Tuniki , Shaikh Sana , Bal Chandralata , Sharon Ashoke , Gyorke Sandor , Periasamy Muthu
Published: February 2011

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.
When a module is not a domain: the case of the REJ module and the redefinition of the architecture of polycystin-1
Authors: Schröder Samantha , Fraternali Franca , Quan Xueping , Scott David , Qian Feng , Pfuhl Mark
Published: February 2011

The extracellular region of a group of cell-surface receptors known as the polycystic kidney disease 1 family, containing, among others, polycystin-1, has been controversially described as containing four FNIII (fibronectin type III) domains or one REJ (receptor of egg jelly protein) module in the same portion of polypeptide. Stimulated by recent atomic force microscopy work, we re-examined the similarity of these four domains with a FNIII sequence profile showing the evolutionary relationship. Two of the predicted domains could be expressed in bacteria and refolded to give a protein suitable for biophysical study, and one of these expressed solubly. CD spectroscopy showed that both domains contain a significant amount of β-sheet, in good agreement with theoretical predictions. Confirmation of independent folding as a domain is obtained from highly co-operative thermal and urea unfolding curves. Excellent dispersion of peaks in the high-field region of one-dimensional NMR spectra confirms the presence of a hydrophobic core. Analytical ultracentrifugation and analytical gel filtration agree very well with the narrow linewidths in the NMR spectra that at least one of the domains is monomeric. On the basis of this combined theoretical and experimental analysis, we show that the extracellular portion of polycystin-1 does indeed contain β-sheet domains, probably FNIII, and that, consequently, the REJ module is not a single domain.
ER stress modulates cellular metabolism
Authors: Wang Xiaoli , Eno Colins O. , Altman Brian J. , Zhu Yanglong , Zhao Guoping , Olberding Kristen E. , Rathmell Jeffrey C. , Li Chi
Published: February 2011

Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak−/−Bax−/− haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak−/−Bax−/− cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.
Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1
Authors: Sheerin David J. , Buchanan Jeremy , Kirk Chris , Harvey Dawn , Sun Xiaolin , Spagnuolo Julian , Li Sheng , Liu Tong , Woods Virgil A. , Foster Toshi , Jones William T. , Rakonjac Jasna
Published: February 2011

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A–RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.
Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans
Authors: Kosel Mandy , Wild Waltraud , Bell Alexandra , Rothe Michael , Lindschau Carsten , Steinberg Christian E. W. , Schunck Wolf-Hagen , Menzel Ralph
Published: February 2011

Caenorhabditis elegans harbours several CYP (cytochrome P450) genes that are homologous with mammalian CYP isoforms important to the production of physiologically active AA (arachidonic acid) metabolites. We tested the hypothesis that mammals and C. elegans may share similar basic mechanisms of CYP-dependent eicosanoid formation and action. We focused on CYP33E2, an isoform related to the human AA-epoxygenases CYP2C8 and CYP2J2. Co-expression of CYP33E2 with the human NADPH–CYP reductase in insect cells resulted in the reconstitution of an active microsomal mono-oxygenase system that metabolized EPA (eicosapentaenoic acid) and, with lower activity, also AA to specific sets of regioisomeric epoxy- and hydroxy-derivatives. The main products included 17,18-epoxyeicosatetraenoic acid from EPA and 19-hydroxyeicosatetraenoic acid from AA. Using nematode worms carrying a pCYP33E2::GFP reporter construct, we found that CYP33E2 is exclusively expressed in the pharynx, where it is predominantly localized in the marginal cells. RNAi (RNA interference)-mediated CYP33E2 expression silencing as well as treatments with inhibitors of mammalian AA-metabolizing CYP enzymes, significantly reduced the pharyngeal pumping frequency of adult C. elegans. These results demonstrate that EPA and AA are efficient CYP33E2 substrates and suggest that CYP–eicosanoids, influencing in mammals the contractility of cardiomyocytes and vascular smooth muscle cells, may function in C. elegans as regulators of the pharyngeal pumping activity.
Endoplasmic reticulum stress induced by hepatitis B virus X protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4
Authors: Cho Hyun Kook , Cheong Kyu Jin , Kim Hye Young , Cheong JaeHun
Published: February 2011

Chronic hepatitis B is a disease of the liver that can progress to cirrhosis and liver cancer. The HBx (hepatitis B virus X) protein of hepatitis B virus is a multifunctional regulator that induces ER (endoplasmic reticulum) stress by previously unknown mechanisms. ER stress plays a critical role in inflammatory induction and COX2 (cyclo-oxygenase 2) is an important mediator of this inflammation. In the present study, we demonstrate the molecular mechanisms of HBx on induction of ER stress and COX2 expression. In addition, HBx reduced expression of enzymes which are involved in mitochondrial β-oxidation of fatty acids and the mitochondrial inner membrane potential. The reduction in intracellular ATP levels by HBx induced the unfolded protein response and COX2 expression through the eIF2α (eukaryotic initiation factor 2α)/ATF4 (activating transcription factor 4) pathway. We confirmed that ATF4 binding to the COX2 promoter plays a critical role in HBx-mediated COX2 induction. The results of the present study suggest that HBV infection contributes to induction of hepatic inflammation through dysfunction of cellular organelles including the ER and mitochondria.
The Cdc42-associated kinase ACK1 is not autoinhibited but requires Src for activation
Authors: Chan Wing , Sit Soon-Tuck , Manser Ed
Published: February 2011

The non-RTK (receptor tyrosine kinase) ACK1 [activated Cdc42 (cell division cycle 42)-associated kinase 1] binds a number of RTKs and is associated with their endocytosis and turnover. Its mode of activation is not well established, but models have suggested that this is an autoinhibited kinase. Point mutations in its SH3 (Src homology 3)- or EGF (epidermal growth factor)-binding domains have been reported to activate ACK1, but we find neither of the corresponding W424K or F820A mutations do so. Indeed, deletion of the various ACK1 domains C-terminal to the catalytic domain are not associated with increased activity. A previous report identified only one major tyrosine phosphorylated protein of 60 kDa co-purified with ACK1. In a screen for new SH3 partners for ACK1 we found multiple Src family kinases; of these c-Src itself binds best. The SH2 and SH3 domains of Src interact with ACK1 Tyr518 and residues 623–652 respectively. Src targets the ACK1 activation loop Tyr284, a poor autophosphorylation site. We propose that ACK1 fails to undergo significant autophosphorylation on Tyr284in vivo because it is basophilic (whereas Src is acidophilic). Subsequent ACK1 activation downstream of receptors such as EGFR (EGF receptor) (and Src) promotes turnover of ACK1 in vivo, which is blocked by Src inhibitors, and is compromised in the Src-deficient SYF cell line. The results of the present study can explain why ACK1 is responsive to so many external stimuli including RTKs and integrin ligation, since Src kinases are commonly recruited by multiple receptor systems.
The C2 domain of Tollip, a Toll-like receptor signalling regulator, exhibits broad preference for phosphoinositides
Authors: Ankem Gayatri , Mitra Sharmistha , Sun Furong , Moreno Anna C. , Chutvirasakul Boonta , Azurmendi Hugo F. , Li Liwu , Capelluto Daniel G. S.
Published: February 2011

TLRs (Toll-like receptors) provide a mechanism for host defence immune responses. Activated TLRs lead to the recruitment of adaptor proteins to their cytosolic tails, which in turn promote the activation of IRAKs (interleukin-1 receptor-associated kinases). IRAKs act upon their transcription factor targets to influence the expression of genes involved in the immune response. Tollip (Toll-interacting protein) modulates IRAK function in the TLR signalling pathway. Tollip is multimodular, with a conserved C2 domain of unknown function. We found that the Tollip C2 domain preferentially interacts with phosphoinositides, most notably with PtdIns3P (phosphatidylinositol 3-phosphate) and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), in a Ca2+-independent manner. However, NMR analysis demonstrates that the Tollip C2 domain binds Ca2+, which may be required to target the membrane interface. NMR and lipid–protein overlay analyses suggest that PtdIns3P and PtdIns(4,5)P2 share interacting residues in the protein. Kinetic studies reveal that the C2 domain reversibly binds PtdIns3P and PtdIns(4,5)P2, with affinity values in the low micromolar range. Mutational analysis identifies key PtdIns3P- and PtdIns(4,5)P2-binding conserved basic residues in the protein. Our findings suggest that basic residues of the C2 domain mediate membrane targeting of Tollip by interaction with phosphoinositides, which contribute to the observed partition of the protein in different subcellular compartments.
HID-1 is a novel player in the regulation of neuropeptide sorting
Authors: Yu Yong , Wang Lifen , Jiu Yaming , Zhan Yi , Liu Lin , Xia Zhiping , Song Eli , Xu Pingyong , Xu Tao
Published: February 2011

Peptide hormones and neuropeptides are packaged and stored in a specialized intracellular organelle called the dense core vesicle. It remains elusive how peptide cargoes are correctly sorted. In the present study, we show that a highly conserved Golgi-localized protein named HID-1 acts to prevent mis-sorting of peptide cargoes to lysosomes for degradation via a PtdIns3P-dependent trafficking pathway. Epistasis analysis suggests that rab-2 is epistatic to hid-1.
Identification and characterization of the DNA replication origin recognition complex gene family in the silkworm Bombyx mori
Authors: Yang Hui-Peng , Luo Su-Juan , Li Yi-Nü , Zhang Yao-Zhou , Zhang Zhi-Fang
Published: February 2011

The ORC (origin recognition complex) binds to the DNA replication origin and recruits other replication factors to form the pre-replication complex. The cDNA and genomic sequences of all six subunits of ORC in Bombyx mori (BmORC1–6) were determined by RACE (rapid amplification of cDNA ends) and bioinformatic analysis. The conserved domains were identified in BmOrc1p–6p and the C-terminal of BmOrc6p features a short sequence that may be specific for Lepidoptera. As in other organisms, each of the six BmORC subunits had evolved individually from ancestral genes in early eukaryotes. During embryo development, the six genes were co-regulated, but different ratios of the abundance of mRNAs were observed in 13 tissues of the fifth instar day-6 larvae. Infection by BmNPV (B. mori nucleopolyhedrovirus) initially decreased and then increased the abundance of BmORC. We suggest that some of the BmOrc proteins may have additional functions and that BmOrc proteins participate in the replication of BmNPV.
Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats
Authors: Benes Jan , Kazdova Ludmila , Drahota Zdenek , Houstek Josef , Medrikova Dasa , Kopecky Jan , Kovarova Nikola , Vrbacky Marek , Sedmera David , Strnad Hynek , Kolar Michal , Petrak Jiri , Benada Oldrich , Skaroupkova Petra , Cervenka Ludek , Melenovsky Vojtech
Published: February 2011

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg−1 of body weight·day−1, 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6–12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10–90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified ‘free’ fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial 14C-glucose and 14C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.
Circulating levels of non-phosphorylated undercarboxylated matrix Gla protein are associated with disease severity in patients with chronic heart failure
Authors: Ueland Thor , Dahl Christen P. , Gullestad Lars , Aakhus Svend , Broch Kaspar , Skåardal Rita , Vermeer Cees , Aukrust Pål , Schurgers Leon J.
Published: February 2011

We recently demonstrated that circulating MGP [matrix Gla (γ-carboxylated glutamate) protein] levels were associated with left ventricular dysfunction and increased mortality in patients with symptomatic aortic stenosis. We hypothesized that patients with chronic HF (heart failure) would have dysregulated MGP levels. We examined plasma dp-cMGP (non-phosphorylated carboxylated MGP) and dp-ucMGP (non-phosphorylated undercarboxylated MGP) in 179 patients with chronic HF and matched healthy controls as well as the relationship between MGP and cardiac dysfunction as assessed by echocardiographic measurements, inflammation [CRP (C-reactive protein)] and neurohormonal activation [NT-proBNP (N-terminal proB-type natriuretic peptide)] and the prognostic value of MGP levels in relation to mortality in these patients. We found markedly enhanced plasma dp-cMGP and, in particular, of dp-ucMGP in chronic HF with increasing levels with disease severity. Elevated MGP species were associated with ischaemic aetiology, increased CRP and NT-proBNP levels, as well as systolic and diastolic dysfunction. Finally, dp-ucMGP was associated with long-term heart transplant-free survival (n=48) in univariate, but not in multivariate, analysis. However, plasma dp-ucMGP was markedly higher in patients who died because of progression of HF (n=12) and gave prognostic information also in multivariate analysis. In conclusion, a dysregulated MGP system could be involved in left ventricular dysfunction in patients with chronic HF.
Asymptotics of solutions of a quasilinear system involving curl
Authors: Pan Xing-Bin
Published: February 2011

This paper concerns the asymptotic behavior of the solutions of a singularly perturbed quasilinear system involving a small parameter λ and a quadratic form gM(curl H) = 〈Mcurl H, curl H〉. This system arises in the mathematical theory of nucleation of instability of the Meissner states of anisotropic superconductors, and the location of the maximum points of the quadratic form suggests the location where instability begins to nucleate. The existence and regularity of the solutions of this system for fixed λ have been established by Pan [“A quasilinear system involving the operator curl,” Calculus Var. Partial Differ. Equ. 36, 317 (2009)]. In this paper we find an optimal bound of the boundary data for the solvability for all small λ and find the asymptotic behavior of the solutions as λ goes to zero. In particular it is proved that the maximum points of the quadratic form approach a point on the domain boundary where the tangential component of the boundary datum is the maximal. In the special case where the applied magnetic field has constant direction and constant magnitude, the maximum points of the quadratic form approach a point on the domain boundary where the applied field is tangential to the surface.
An improved single crystal adsorption calorimeter for determining gas adsorption and reaction energies on complex model catalysts
Authors: Fischer-Wolfarth Jan-Henrik , Hartmann Jens , Farmer Jason A. , Flores-Camacho J. Manuel , Campbell Charles T. , Schauermann Swetlana , Freund Hans-Joachim
Published: February 2011

A new ultrahigh vacuum microcalorimeter for measuring heats of adsorption and adsorption-induced surface reactions on complex single crystal-based model surfaces is described. It has been specifically designed to study the interaction of gaseous molecules with well-defined model catalysts consisting of metal nanoparticles supported on single crystal surfaces or epitaxial thin oxide films grown on single crystals. The detection principle is based on the previously described measurement of the temperature rise upon adsorption of gaseous molecules by use of a pyroelectric polymer ribbon, which is brought into mechanical/thermal contact with the back side of the thin single crystal. The instrument includes (i) a preparation chamber providing the required equipment to prepare supported model catalysts involving well-defined nanoparticles on clean single crystal surfaces and to characterize them using surface analysis techniques and in situ reflectivity measurements and (ii) the adsorption/reaction chamber containing a molecular beam, a pyroelectric heat detector, and calibration tools for determining the absolute reactant fluxes and adsorption heats. The molecular beam is produced by a differentially pumped source based on a multichannel array capable of providing variable fluxes of both high and low vapor pressure gaseous molecules in the range of 0.005–1.5 × 1015 molecules cm−2 s−1 and is modulated by means of the computer-controlled chopper with the shortest pulse length of 150 ms. The calorimetric measurements of adsorption and reaction heats can be performed in a broad temperature range from 100 to 300 K. A novel vibrational isolation method for the pyroelectric detector is introduced for the reduction of acoustic noise. The detector shows a pulse-to-pulse standard deviation ≤15 nJ when heat pulses in the range of 190–3600 nJ are applied to the sample surface with a chopped laser. Particularly for CO adsorption on Pt(111), the energy input of 15 nJ (or 120 nJ cm−2) corresponds to the detection limit for adsorption of less than 1.5 × 1012 CO molecules cm−2 or less than 0.1% of the monolayer coverage (with respect to the 1.5 × 1015 surface Pt atoms cm−2). The absolute accuracy in energy is within ∼7%–9%. As a test of the new calorimeter, the adsorption heats of CO on Pt(111) at different temperatures were measured and compared to previously obtained calorimetric data at 300 K.
Infrared spectroscopic study of pulsed laser deposited Fe3O4 thin film on Si (111) substrate across Verwey transition temperature
Authors: Master Ridhi , Tiwari Shailja , Choudhary R. J. , Deshpande U. P. , Shripathi T. , Phase D. M.
Published: February 2011

We present low-temperature infrared measurements of magnetite (Fe3O4) thin films on Si (111) substrate across the Verwey transition temperature (TV). The line parameters of the most intense t1u mode observed in the Fourier transform infra red spectrum of the film is studied as a function of temperature. We observe that mode frequency increases abruptly at 130 K, and full width at half maxima of the mode increases abruptly at 113 K. The observations point out that structural transition may start earlier at 130 K than the actual Verwey transition (121 K) and that complete at 113 K. These results are consistent with the higher transition temperature as observed in resistivity and magnetization measurements.
Excitation of surface plasma wave at TiN/air interface in the Kretschmann geometry
Authors: Chen N. C. , Lien W. C. , Liu C. R. , Huang Y. L. , Lin Y. R. , Chou C. , Chang S. Y. , Ho C. W.
Published: February 2011

Since titanium nitride appears golden and has a high conductivity, the possibility that it can be used in surface plasma wave applications in a manner similar to gold but with very strong scratch-resistance, is of interest. This work considers this possibility using the Kretschmann configuration, measuring the angle-dependent reflectivity as well as the wavelength-dependent reflectivity. Both sets of results demonstrate the excitation of a surface plasma wave at the TiN/air interface by an incident p-wave. The thickness of TiN that most efficiently couples the incident p-wave to the surface plasma wave is around 35 nm. All of the experiments are accompanied by corresponding numerical simulations.
Dielectric decrement as a source of ion-specific effects
Authors: Ben-Yaakov Dan , Andelman David , Podgornik Rudi
Published: February 2011

Many theoretical studies were devoted in the past to ion-specific effects trying to interpret a large body of experimental evidence, such as surface tension at air/water interfaces and force measurements between charged objects. Although several mechanisms were suggested to explain the results, such as dispersion forces and specific surface-ion interactions, we would like to suggest another source of ion-specificity originating from the local variations of the dielectric constant due to the presence of ions in the solution. We present a mean-field model to account for the heterogeneity of the dielectric constant caused by the ions. In particular, for ions that decrease the dielectric constant we find a depletion of ions from the vicinity of charged surfaces. For a two-plate system, the same effect leads to an increase of the pressure in between two surfaces. Our results suggest that the effect of ions on the local dielectric constant should be taken into account when interpreting experiments that address ion-specific effects.
Wave propagation and noncollisional heating in neutral loop and helicon discharges
Authors: Celik Y. , Crintea D. L. , Luggenhölscher D. , Czarnetzki U. , Ishijima T. , Sugai H.
Published: February 2011

Heating mechanisms in two types of magnetized low pressure rf (13.56 MHz) discharges are investigated: a helicon discharge and a neutral loop discharge. Radial B-dot probe measurements demonstrate that the neutral loop discharge is sustained by helicon waves as well. Axial B-dot probe measurements reveal standing wave and beat patterns depending on the dc magnetic field strength and plasma density. In modes showing a strong wave damping, the plasma refractive index attains values around 100, leading to electron-wave interactions. In strongly damped modes, the radial plasma density profiles are mainly determined by power absorption of the propagating helicon wave, whereas in weakly damped modes, inductive coupling dominates. Furthermore, an azimuthal diamagnetic drift is identified. Measurements of the helicon wave phase demonstrate that initial plane wave fronts are bent during their axial propagation due to the inhomogeneous density profile. A developed analytical standing wave model including Landau damping reproduces very well the damping of the axial helicon wave field. This comparison underlines the theory whereupon Landau damping of electrons traveling along the field lines at speeds close to the helicon phase velocity is the main damping mechanism in both discharges.
Investigation of critical behavior in Pr0.55Sr0.45MnO3 by using the field dependence of magnetic entropy change
Authors: Fan Jiyu , Pi Li , Zhang Lei , Tong Wei , Ling Langsheng , Hong Bo , Shi Yangguang , Zhang Weichun , Lu Di , Zhang Yuheng
Published: February 2011

One method of calculating critical exponents based on the field dependence on magnetic entropy change was applied to study critical behavior in Pr0.55Sr0.45MnO3. By using the obtained critical exponents, the modified Arrott plot [A. Arrott and J. E. Noakes, Phys. Rev. Lett. 19, 786 (1967)] is consistent with that by using conventional method. The calculated critical exponents not only obey the scaling theory, but also inosculate the deduced results from the Kouvel–Fisher method [J. S. Kouvel and M. E. Fisher, Phys. Rev. 136, A1626 (1964)]. It eliminates the drawback due to utilization of multistep nonlinear fitting in a conventional manner. Therefore, this means can be applied to investigate critical behavior.
Wave scattering by many small bodies and applications
Authors: Ramm A. G. , Rona A.
Published: February 2011

Wave scattering problem by many bodies is studied in the case when the bodies are small, ka ≪ 1, where a is the characteristic size of a body. The limiting case when a → 0 and the total number of the small bodies is M = O(a−(2 − κ)), where κ ∈ (0, 1) is a number, are studied.
Numerical simulations of space charge waves in InP films and microwave frequency conversion under negative differential conductivity
Authors: Garcia-Barrientos Abel , Palankovski Vassil
Published: February 2011

Numerical simulations of amplification and propagation of space charge waves in InP films is investigated theoretically. A microwave frequency conversion using the negative differential conductivity phenomenon is carried out when the harmonics of the input signal are generated. An increment in the amplification is observed in n-InP films at essentially higher frequencies f<70 GHz, when compared with n-GaAs films f<44 GHz. This work provides a way to achieve a frequency conversion and amplification of micrometer and millimeter waves.
Stochastic resonance of a domain wall in a stripe with two pinning sites
Authors: Martinez Eduardo , Finocchio Giovanni , Carpentieri Mario
Published: February 2011

We report on the observation of stochastic resonance phenomenon of a single domain wall in a ferromagnetic stripe with two pinning sites. Under a weak oscillating field, the wall performs irregular transitions between both constrictions in the presence of thermal fluctuations. Ours results indicate that synchronized wall transitions with the driving field can be achieved at the optimal level of noise. The stochastic resonance is quantified by computing the output signal power as a function of temperature. The analysis points out that this system could be used to design well-controlled amplification devices, which could find application as nanodetectors.
Refractive Index of Water and Its Dependence on Wavelength, Temperature, and Density
Authors: Thormählen I. , Straub J. , Grigull U.
Published: February 2011

A survey of the available experimental data and the existing equations for the refractive index of water is given. The dependence of the molar refraction on wavelength, temperature, and density is shown over an extended range. Based upon the electromagnetic theory of light an equation for the refractive index of water with wavelength, temperature, and density as independent variables is constructed. Its coefficients are directly deduced from all available experimental data by least-squares fit. The range of validity of wavelength is restricted by the theory for normal dispersion to 182 nm≤λ≤2770 nm. The range of temperature and density is given by the available experimental data. Interpolations between the single measured points are possible and the following range of validity can be recommended: for temperature −10 °C≤T≤500 °C and for density 0.0028 kg/m3 ≤ρ≤1045 kg/m3. Good agreement exists between the new relation, the available experimental data, and several existing equations.