bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative contribution of PDX-1, MafA and E47/β2 to the regulation of the human insulin promoter
Authors: Docherty Hilary M. , Hay Colin W. , Ferguson Laura A. , Barrow John , Durward Elaine , Docherty Kevin
Published: July 2005

The insulin promoter binds a number of tissue-specific and ubiquitous transcription factors. Of these, the homoeodomain protein PDX-1 (pancreatic duodenal homeobox factor-1), the basic leucine zipper protein MafA and the basic helix–loop–helix heterodimer E47/BETA2 (β-cell E box transactivator 2; referred to here as β2) bind to important regulatory sites. Previous studies have shown that PDX-1 can interact synergistically with E47 and β2 to activate the rat insulin 1 promoter. The aim of the present study was to determine the relative contribution of PDX-1, MafA and E47/β2 in regulating the human insulin promoter, and whether these factors could interact synergistically in the context of the human promoter. Mutagenesis of the PDX-1, MafA and E47/β2 binding sites reduced promoter activity by 60, 74 and 94% respectively, in INS-1 β-cells. In the islet glucagonoma cell line αTC1.6, overexpression of PDX-1 and MafA separately increased promoter activity approx. 2.5–3-fold, and in combination approx. 6-fold, indicating that their overall effect was additive. Overexpression of E47 and β2 had no effect. In HeLa cells, PDX-1 stimulated the basal promoter by approx. 40-fold, whereas MafA, E47 and β2 each increased activity by less than 2-fold. There was no indication of any synergistic effects on the human insulin promoter. On the other hand, the rat insulin 1 promoter and a mutated version of the human insulin promoter, in which the relevant regulatory elements were separated by the same distances as in the rat insulin 1 promoter, did exhibit synergy. PDX-1 was shown further to activate the endogenous insulin 1 gene in αTC1.6 cells, whereas MafA activated the insulin 2 gene. In combination, PDX-1 and MafA activated both insulin genes. Chromatin immunoprecipitation assays confirmed that PDX-1 increased the association of acetylated histones H3 and H4 with the insulin 1 gene and MafA increased the association of acetylated histone H3 with the insulin 2 gene.
Spectral and catalytic properties of aryl-alcohol oxidase, a fungal flavoenzyme acting on polyunsaturated alcohols
Authors: Ferreira Patricia , Medina Milagros , Guillén Francisco , Martínez María Jesús , Van Berkel Willem J. H. , Martínez Ángel T.
Published: July 2005

Spectral and catalytic properties of the flavoenzyme AAO (aryl-alcohol oxidase) from Pleurotus eryngii were investigated using recombinant enzyme. Unlike most flavoprotein oxidases, AAO does not thermodynamically stabilize a flavin semiquinone radical and forms no sulphite adduct. AAO catalyses the oxidative dehydrogenation of a wide range of unsaturated primary alcohols with hydrogen peroxide production. This differentiates the enzyme from VAO (vanillyl-alcohol oxidase), which is specific for phenolic compounds. Moreover, AAO is optimally active in the pH range of 5–6, whereas VAO has an optimum at pH 10. Kinetic studies showed that AAO is most active with p-anisyl alcohol and 2,4-hexadien-1-ol. AAO converts m- and p-chlorinated benzyl alcohols at a similar rate as it does benzyl alcohol, but introduction of a p-methoxy substituent in benzyl alcohol increases the reaction rate approx. 5-fold. AAO also exhibits low activity on aromatic aldehydes. 19F NMR analysis showed that fluorinated benzaldehydes are converted into the corresponding benzoic acids. Inhibition studies revealed that the AAO active site can bind a wide range of aromatic ligands, chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid being the best competitive inhibitors. Uncompetitive inhibition was observed with 4-methoxybenzylamine. The properties described above render AAO a unique oxidase. The possible mechanism of AAO binding and oxidation of substrates is discussed in the light of the results of the inhibition and kinetic studies.
Growth factors induce differential phosphorylation profiles of the Hrs–STAM complex: a common node in signalling networks with signal-specific properties
Authors: Row Paula E. , Clague Michael J. , Urbé Sylvie
Published: July 2005

Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule) form a heterodimeric complex that associates with endosomal membranes and is tyrosine-phosphorylated in response to a variety of growth factors including EGF (epidermal growth factor), HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). Phosphorylation of the Hrs–STAM complex requires receptor endocytosis. We show that an intact UIM (ubiquitin interaction motif) within Hrs is a conserved requirement for Hrs phosphorylation downstream of both EGF and HGF stimulations. Consistent with this, expression of a dominant-negative form of the E3 ubiquitin ligase, c-Cbl, inhibits EGF- and HGF-dependent Hrs phosphorylation. Despite this conservation, kinase inhibitor profiles using PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and SU6656 indicate that distinct non-receptor tyrosine kinases couple EGF, HGF and PDGF stimulation with the tyrosine phosphorylation of the Hrs–STAM complex. Crucially, analysis with phospho-specific antibodies indicates that these kinases generate a signal-specific, combinatorial phosphorylation profile of the Hrs–STAM complex, with the potential of diversifying tyrosine kinase receptor signalling through a common element.
Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells
Authors: Moneer Zahid , Pino Irene , Taylor Emily J. A. , Broad Lisa M. , Liu Yingjie , Tovey Stephen C. , Staali Leila , Taylor Colin W.
Published: July 2005

Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439–448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13–21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237–244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3).
Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT
Authors: Hui Kwok-Min , Orriss George L. , Schirmer Tilman , Magnadóttir Bergljót , Schifferli Jürg A. , Inal Jameel M.
Published: July 2005

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the β-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224–437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 α, β and γ chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4β chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2–CRIT and C2–C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.
The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity
Authors: Woodman Zenda L. , Schwager Sylva L. U. , Redelinghuys Pierre , Carmona Adriana K. , Ehlers Mario R. W. , Sturrock Edward D.
Published: July 2005

sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the kcat for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates.
Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697
Authors: Wilson David P. , Susnjar Marija , Kiss Enikő , Sutherland Cindy , Walsh Michael P.
Published: July 2005

The signal transduction pathway whereby the TxA2 (thromboxane A2) mimetic U-46619 activates vascular smooth muscle contraction was investigated in de-endothelialized rat caudal artery. U-46619-evoked contraction was inhibited by the TP receptor (TxA2 receptor) antagonist SQ-29548, the ROK (Rho-associated kinase) inhibitors Y-27632 and H-1152, the MLCK (myosin light-chain kinase) inhibitors ML-7, ML-9 and wortmannin, the voltagegated Ca2+-channel blocker nicardipine, and removal of extracellular Ca2+; the protein kinase C inhibitor GF109203x had no effect. U-46619 elicited Ca2+ sensitization in α-toxin-permeabilized tissue. U-46619 induced activation of the small GTPase RhoA, consistent with the involvement of ROK. Two downstream targets of ROK were investigated: CPI-17 [protein kinase C-potentiated inhibitory protein for PP1 (protein phosphatase type 1) of 17 kDa], a myosin light-chain phosphatase inhibitor, was not phosphorylated at the functional site (Thr-38); phosphorylation of MYPT1 (myosin-targeting subunit of myosin light-chain phosphatase) was significantly increased at Thr-855, but not Thr-697. U-46619-evoked contraction correlated with phosphorylation of the 20 kDa light chains of myosin. We conclude that: (i) U-46619 induces contraction via activation of the Ca2+/calmodulin/MLCK pathway and of the RhoA/ROK pathway; (ii) Thr-855 of MYPT1 is phosphorylated by ROK at rest and in response to U-46619 stimulation; (iii) Thr-697 of MYPT1 is phosphorylated by a kinase other than ROK under resting conditions, and is not increased in response to U-46619 treatment; and (iv) neither ROK nor protein kinase C phosphorylates CPI-17 in this vascular smooth muscle in response to U-46619.
Radiation-induced cell death: importance of lysosomal destabilization
Authors: Persson H. Lennart , Kurz Tino , Eaton John W. , Brunk Ulf T.
Published: July 2005

The mechanisms involved in radiation-induced cellular injury and death remain incompletely understood. In addition to the direct formation of highly reactive hydroxyl radicals (HO·) by radiolysis of water, oxidative stress events in the cytoplasm due to formation of H2O2 may also be important. Since the major pool of low-mass redox-active intracellular iron seems to reside within lysosomes, arising from the continuous intralysosomal autophagocytotic degradation of ferruginous materials, formation of H2O2 inside and outside these organelles may cause lysosomal labilization with release to the cytosol of lytic enzymes and low-mass iron. If of limited magnitude, such release may induce ‘reparative autophagocytosis’, causing additional accumulation of redox-active iron within the lysosomal compartment. We have used radio-resistant histiocytic lymphoma (J774) cells to assess the importance of intralysosomal iron and lysosomal rupture in radiation-induced cellular injury. We found that a 40 Gy radiation dose increased the ‘loose’ iron content of the (still viable) cells approx. 5-fold when assayed 24 h later. Cytochemical staining revealed that most redox-active iron was within the lysosomes. The increase of intralysosomal iron was associated with ‘reparative autophagocytosis’, and sensitized cells to lysosomal rupture and consequent apoptotic/necrotic death following a second, much lower dose of radiation (20 Gy) 24 h after the first one. A high-molecular-mass derivative of desferrioxamine, which specifically localizes intralysosomally following endocytic uptake, added to the culture medium before either the first or the second dose of radiation, stabilized lysosomes and largely prevented cell death. These observations may provide a biological rationale for fractionated radiation.
Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli
Authors: Bertoldi Mariarita , Cellini Barbara , Laurents Douglas V. , Borri Voltattorni Carla
Published: July 2005

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation.
Crystal structure of the proximal BAH domain of the polybromo protein
Authors: Oliver Antony W. , Jones Sarah A. , Roe Stephen Mark , Matthews Steve , Goodwin Graham H. , Pearl Laurence H.
Published: July 2005

The BAH domain (bromo-associated homology domain) was first identified from a repeated motif found in the nuclear protein polybromo – a large (187 kDa) modular protein comprising six bromodomains, two BAH domains and an HMG box. To date, the BAH domain has no ascribed function, although it is found in a wide range of proteins that contain additional domains involved in either transcriptional regulation (e.g. SET, PHD and bromodomain) and/or DNA binding (HMG box and AT hook). The molecular function of polybromo itself also remains unclear, but it has been identified as a key component of an SWI/SNF (switching/sucrose non-fermenting)-related, ATP-dependent chromatin-remodelling complex PBAF (polybromo, BRG1-associated factors; also known as SWI/SNF-B or SWI/SNFβ). We present in this paper the crystal structure of the proximal BAH domain from chicken polybromo (BAH1), at a resolution of 1.6 Å (1 Å=0.1 nm). Structure-based sequence analysis reveals several features that may be involved in mediating protein–protein interactions.
Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells
Authors: Baty James W. , Hampton Mark B. , Winterbourn Christine C.
Published: July 2005

Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 μM H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1β, HSP90β (heat-shock protein 90β) and proteosome components. At 20 μM H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.
Carbohydrate-remodelled acid α-glucosidase with higher affinity for the cation-independent mannose 6-phosphate receptor demonstrates improved delivery to muscles of Pompe mice
Authors: Zhu Yunxiang , Li Xuemei , Mcvie-Wylie Alison , Jiang Canwen , Thurberg Beth L. , Raben Nina , Mattaliano Robert J. , Cheng Seng H.
Published: July 2005

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid α-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.
Resolving the energy paradox of chaperone/usher-mediated fibre assembly
Authors: Zavialov Anton V. , Tischenko Vladimir M. , Fooks Laura J. , Brandsdal Bjørn O. , Åqvist Johan , Zav'yalov Vladimir P. , Macintyre Sheila , Knight Stefan D.
Published: July 2005

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone–subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone–subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone–fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone–subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.
Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells
Authors: Matic Sandra , Geisler Daniela A. , Møller Ian M. , Widell Susanne , Rasmusson Allan G.
Published: July 2005

The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage of NAD(P)+. After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD+). The capacities of different pathways in the respiratory electron-transport chain could thus be determined directly. We conclude that AlaM permeabilization provides a very useful tool for monitoring metabolic pathways or individual enzymes in their native proteinaceous environment with controlled cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed.
NR2A induction and NMDA receptor‐dependent neuronal death by neurotrophin‐4/5 in cortical cell culture
Authors: Choi So‐Young , Hwang Jung Jin , Koh Jae‐Young
Published: July 2005

We have previously shown that prolonged exposure to neurotrophins induces oxidative neuronal death. In the present study, we further examined the cascades involved in neurotrophin‐4/5 (NT‐4/5)‐induced neuronal death. Exposure of mature cortical cultures for 48 h to NT‐4/5 induced neuronal death through TrkB activation. The NT‐4/5‐induced neuronal death was largely attenuated by addition of MK‐801, indicating a critical role for NMDA receptors. Western blots revealed the induction of NR2A by NT‐4/5. In addition, levels of phospho‐NR2A and 2B increased, suggesting the upregulation of the NMDA receptor function. Whereas glutamate levels in the media changed little, levels of d‐serine and l‐glycine, co‐agonists at NMDA receptors, increased significantly following NT‐4/5 treatment. Exposure to NT‐4/5 resulted in the activation of Src and extracellular signal‐regulated kinase‐1/2 (Erk‐1/2). Their inhibitors blocked NR2A induction and phosphorylation as well as neuronal death induced by NT‐4/5. In addition, Egr‐1 was induced in an Src‐ and Erk‐1/2‐dependent manner. Anti‐sense oligodeoxynucleotides to egr‐1 attenuated NR2A induction as well as neuronal death. Although induction of NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributes to NT‐4/5‐induced neuronal death, inhibition of their activity did not reduce NR2A induction. Conversely, blockade of NMDA receptors did not attenuate induction of NADPH oxidase or nNOS. These results indicate that two events are largely independent of each other. Our results demonstrate that the signaling cascade of TrkB leads to increase in NMDA receptor activity. Whereas this cascade may play an important role in the modulation of NMDA receptors in physiologic conditions, in the context of TrkB overactivation, it may contribute to neuronal death.
Characterization of the restricted rotation of the dimethyl groups in chemically N-terminal C13-labeled antifreeze glycoproteins: A temperature-dependent study in water to ice through the supercooled state
Authors: Krishnan V. V. , Lau Edmond Y. , Tsvetkova Nelly M. , Feeney Robert E. , Fink William H. , Yeh Yin
Published: July 2005

Site-specific chemical modification, especially with isotopically enriched groups, allows one to study the structure and dynamics of proteins for which uniform enrichment is difficult. When the N-terminal alanine in antifreeze glycoprotein (AFGP) is replaced with an N,N-dimethyl alanine the methyl groups show signatures of slow rotation about the C–N bond. In order to separate the local dynamics of the N-terminus from the overall protein dynamics, we present a complete characterization of this dynamics. Temperature-dependent nuclear magnetic-resonance experiments from room temperature to subzero temperatures, including the supercooled state and in the presence of ice, are presented. Quantum chemical calculations are also performed on a localized N-terminus of the AFGP. Our results show that in the solution state at room temperature and in the super cooled regime, the dimethyl groups undergo a slow, restricted rotation with an unequal distribution of population between two major conformations. At lower temperatures in the presence of ice, the dynamics become much more complex due to freezing out of several conformational states. Based on these results, we conclude that the segmental dynamics of the N-terminus are local to the first residue and do not affect the overall dynamics of the protein.
Vibrational dynamics of the floppy LiNC∕LiCN molecular system
Authors: Arranz F. J. , Benito R. M. , Borondo F.
Published: July 2005

Modern spectroscopical techniques allow the efficient experimental investigation of highly excited vibrational states in molecular systems. On the theoretical side, powerful computational methods have also been developed for the calculation of the corresponding energy levels and wave functions, and their interpretation. In this paper we use a combination of two such methods, namely, the distribution of zeroes in the Husimi function and energy-level correlation diagrams, to discuss a classification scheme, for the lowest hundred vibrational levels of the LiNC∕LiCN floppy molecular system, based on their dynamical characteristics.
Photoluminescence of oxygen-containing surface defects in germanium oxides: A theoretical study
Authors: Zyubin A. S. , Mebel A. M. , Lin S. H.
Published: July 2005

Photoabsorption and photoluminescence properties of nonbridging oxygen –O–Ge(NBO), –OO–Ge (peroxy radical), OGe, and (O2)Ge defects in germanium oxides have been investigated by high-level ab initio calculations. Geometry optimization for excited electronic states of model clusters simulating these defects was carried out at the complete-active-space self-consistent-field level, and relative energies were calculated by various methods including time-dependent density-functional theory, outer-valence Green’s functions, equation-of-motion coupled cluster theory with single and double excitations, symmetry-adapted cluster configuration interaction, multireference second-order perturbation theory, and multireference configuration interaction. The results demonstrate that the considered excited states of the aforementioned defects normally exhibit large Stokes shifts and that, with few exceptions, UV photoabsorption is accompanied by red or IR photoluminescence.
Photofragment angular momentum distribution beyond the axial recoil approximation: The role of molecular axis rotation
Authors: Kuznetsov Vladislav V. , Vasyutinskii Oleg S.
Published: July 2005

We present the quantum-mechanical expressions for the recoil angle dependence of the photofragment multipole moments which explicitly treat the role of molecular axis rotation on the electronic angular momentum polarization of the fragments. The paper generalizes the result of Siebbeles et al. [J. Chem. Phys. 100, 3610 (1994)] to the case of dissociation of rotating molecules. The electronic wave function of the molecule was used in the adiabatic body-frame representation. The obtained rigorous expressions for the fragment state multipoles have been explicitly derived from the scattering wave-function formalism and then simplified using the quasiclassical approximation in the high-J limit. Possible radial and Coriolis nonadiabatic interactions have been taken into consideration. It is shown that the rotation of the molecular axis is described by a number of rotation factors which depend on the rank of the incident-photon polarization matrix, on the dissociation mechanism, and on the classical angle of rotation of the molecular axis γ.
Acoustic emission and nonergodic states of the electric-field-induced-phase transition of PbMg1∕3Nb2∕3O3
Authors: Dul’kin E. , Roth M. , Dkhil B. , Kiat J. M.
Published: July 2005

Acoustic emission (AE) method was used to investigate the electric-field-induced-phase transition in the well-known relaxor Pb(Mg1∕3Nb2∕3)O3 (PMN). We show that in the field cooling process the induced-phase transition is accompanied by an AE signal, which reflects relaxation of the strong stress, associated with arising of a macroscopic domains structure similar to martensitic phase transition. However, using a procedure in which the field is applied after a zero field cooling, no AE signal is detected even after a very long time higher than expected for the appearance of the induced-phase transition. The comparison with previous x-ray-diffraction results allowed to get in the last case interesting insights on the local and long-range polar order of the induced ferroelectric state because of absence of the relaxation of the strong stress due to percolation mechanism of this phase transition.
Reconstruction of electrostatic force microscopy images
Authors: Strassburg E. , Boag A. , Rosenwaks Y.
Published: July 2005

An efficient algorithm to restore the actual surface potential image from Kelvin probe force microscopy measurements of semiconductors is presented. The three-dimensional potential of the tip-sample system is calculated using an integral equation-based boundary element method combined with modeling the semiconductor by an equivalent dipole-layer and image-charge model. The derived point spread function of the measuring tip is then used to restore the actual surface potential from the measured image, using noise filtration and deconvolution algorithms. The model is then used to restore high-resolution Kelvin probe microscopy images of semiconductor surfaces.
Numerical simulation of dual frequency etching reactors: Influence of the external process parameters on the plasma characteristics
Authors: Georgieva V. , Bogaerts A.
Published: July 2005

A one-dimensional particle-in-cell/Monte Carlo model is used to investigate Ar∕CF4∕N2 discharges sustained in capacitively coupled dual frequency reactors, with special emphasis on the influence of the reactor parameters such as applied voltage amplitudes and frequencies of the two voltage sources. The presented calculation results include plasma density, ion current, average sheath potential and width, electron and ion average energies and energy distributions, and ionization rates. The simulations were carried out for high frequencies (HFs) of 27, 40, 60, and 100MHz and a low frequency (LF) of 1 or 2MHz, varying the LF voltage and keeping the HF voltage constant and vice versa. It is observed that the decoupling of the two sources is possible by increasing the applied HF to very high values (above 60MHz) and it is not defined by the frequency ratio. Both voltage sources have influence on the plasma characteristics at a HF of 27MHz and to some extent at 40MHz. At HFs of 60 and 100MHz, the plasma density and ion flux are determined only by the HF voltage source. The ion energy increases and the ion energy distribution function (IEDF) becomes broader with HF or LF voltage amplitude, when the other voltage is kept constant. The IEDF is broader with the increase of HF or the decrease of LF.
Instability and mixing flux in frontal displacement of viscous fluids from porous media
Authors: Smirnov N. N. , Nikitin V. F. , Maximenko A. , Thiercelin M. , Legros J. C.
Published: July 2005

The goal of the present study is to investigate the instability of viscous fluid displacement by a less viscous one in a two-dimensional channel, and to the determine the characteristics of displacement quality and entrapment zones. Experiments on miscible displacement of fluids in Hele-Shaw cells were conducted under microgravity conditions. Extensive direct numerical simulations allowed to investigate the sensitivity of the displacement process to the variation of values of the main governing parameters. Validation of the code was performed by comparing the results of model problems simulations with experiments and with the existing solutions published in literature. Numerical simulations allowed to explain new experimental results on the pear shape of fingers and periodical separation of their tip elements from the main body of displacing fluid. These separated blobs of less viscous fluid move much faster than the mean flow of the displaced viscous fluid. The results of numerical simulations processed on the basis of dimensions analysis allow to introduce parameters characterizing the quality of displacement and the mixing flux induced by instability. Functional forms describing additional mixing induced by displacement instability were developed. The influence of inhomogeneity of porous matrix on displacement instability is being investigated. The modified Hele-Shaw cell containing regular and randomized obstacles is used to study the effect of inhomogeneity on displacement instability. The result of numerical simulations as well as physical experiment shows that the presence of inhomogeneity of a definite length scale could stabilize unstable displacement and could destabilize a stable one.
Enhancement of 1.54-μm emission from Er-doped sol-gel SiO2 films by Au nanoparticles doping
Authors: Fukushima Masanori , Managaki Nobuto , Fujii Minoru , Yanagi Hisao , Hayashi Shinji
Published: July 2005

A 1.54-μm emission from erbium-doped sol-gel SiO2 was strongly enhanced by doping Au nanoparticles. The enhancement factor depended on Au concentration and was the largest at the concentration of 1.0mol%. The photoluminescence enhancement was accompanied by the lengthening of the lifetime, suggesting that the enhancement is caused by the decrease of hydroxyl groups. In addition, enhancement due to strong fields induced by localized surface plasmon of Au nanoparticles was observed.
Superconducting and transport properties of YBa2Cu3O7∕La0.7Sr0.3MnO3 bilayers
Authors: Lin J. G. , Cheng S. L. , Chang C. R. , Xing D. Y.
Published: July 2005

Temperature-dependent resistivity, thermoelectric power, and magnetoresistance (MR) are investigated in YBa2Cu3O7∕La0.7Sr0.3MnO3 (YBCO/LSMO) bilayers with the thickness of YBCO fixed at 150 nm and that of LSMO varying from t=0 to 50 nm. It is found that the onset temperature Tc of the superconducting transition retains at a constant value of 74 K for t<50nm, but reduces to 20 K at t=50nm. In conjunction with the existence of a critical thickness of 50 nm for delocalizing charge carriers in the LSMO layer, we attribute the Tc suppression to the diffusion of spin-polarized carriers from the metallic LSMO to the YBCO layer. Furthermore, we observe a sign reversal of MR with temperature in the YBCO(150 nm)/LSMO(50 nm) bilayer, indicating a competition between a positive MR of YBCO and a negative MR of LSMO.
Enhancement of the 90° domain-wall mobility in sol-gel (Pb,La)TiO3 thin films prepared by multiple deposition and crystallization
Authors: Poyato R. , Algueró M. , Calzada M. L. , Pardo L.
Published: July 2005

Sol-gel lanthanum-modified lead titanate thin films deposited onto Si-based substrates with two different processing conditions have been studied. The films have been processed with a single crystallization or with a multiple deposition and crystallization process. A preferential ⟨001⟩,⟨100⟩-mixed orientation is present in the two types of films. The dependence of the dielectric permittivity on frequency and field amplitude at subswitching fields, permittivity versus temperature curves, and ferroelectric hysteresis loops have been measured in the films. Both types of films present Rayleigh-type behavior with no differences between them, which suggests a similar density of defects in both films. Higher values of remanent polarization and dielectric permittivity, when approaching the transition temperature, have been achieved for the film prepared with the multiple deposition and crystallization process when compared with the obtained ones in the film prepared with a single crystallization. This is due to a lower clamping effect and an enhancement of the 90° domain-wall mobility in the former, as a consequence of the lower residual stress present in this film, achieved by means of the multiple deposition and crystallization process.
Optimal determination of the equilibrium displacement of a damped harmonic oscillator in the presence of thermal noise
Authors: Moore Michael W. , Steffen Jason H. , Boynton Paul E.
Published: July 2005

From the title, one might assume that issues raised in this article must have been settled long ago. In a sense this is true. This may be one of those cases of failed communication between mathematicians and physicists, experimental physicists in particular. In the Introduction we show that a common approach to this linear, single-parameter estimation problem converges to the minimum variance, unbiased estimate only when analyzing measurements of pendulum motion over intervals much shorter than a single oscillation period (<1∕Q periods) where Q is the oscillator quality factor, or much longer (>Q periods). In the practical regime for long-period torsion oscillators used in gravitation studies (between 1∕Q and Q periods), the estimator variance may exceed the optimal value by a factor of order Q. Moreover, we have found no discussion in the experimental literature of the optimal estimation procedure appropriate to this measurement interval. In this article, using a matched filter technique, we derive the minimum variance, unbiased estimator for the equilibrium displacement of a damped harmonic oscillator in thermal equilibrium when interactions with the thermal bath are the leading source of noise. We compare the variance in this optimal estimator with the variance in other, commonly used estimators in the presence of thermal noise and white noise. We also compare the variance in these estimators for a mixture of thermal and white noise. This result has implications for experimental design, for the collection and analysis of data, and for the extension of this method to the nonlinear, multiparameter case.
Low-energy ion beamline scattering apparatus for surface science investigations
Authors: Gordon M. J. , Giapis K. P.
Published: July 2005

We report on the design, construction, and performance of a high current (monolayers∕s), mass-filtered ion beamline system for surface scattering studies using inert and reactive species at collision energies below 1500 eV. The system combines a high-density inductively coupled plasma ion source, high-voltage floating beam transport line with magnet mass-filter and neutral stripping, decelerator, and broad based detection capabilities (ions and neutrals in both mass and energy) for products leaving the target surface. The entire system was designed from the ground up to be a robust platform to study ion-surface interactions from a more global perspective, i.e., high fluxes (>100μA∕cm2) of a single ion species at low, tunable energy (50–1400±5eV full width half maximum) can be delivered to a grounded target under ultrahigh vacuum conditions. The high current at low energy problem is solved using an accel-decel transport scheme where ions are created at the desired collision energy in the plasma source, extracted and accelerated to high transport energy (20 keV to fight space charge repulsion), and then decelerated back down to their original creation potential right before impacting the grounded target. Scattered species and those originating from the surface are directly analyzed in energy and mass using a triply pumped, hybrid detector composed of an electron impact ionizer, hemispherical electrostatic sector, and rf/dc quadrupole in series. With such a system, the collision kinematics, charge exchange, and chemistry occurring on the target surface can be separated by fully analyzing the scattered product flux. Key design aspects of the plasma source, beamline, and detection system are emphasized here to highlight how to work around physical limitations associated with high beam flux at low energy, pumping requirements, beam focusing, and scattered product analysis. Operational details of the beamline are discussed from the perspective of available beam current, mass resolution, projectile energy spread, and energy tunability. As well, performance of the overall system is demonstrated through three proof-of-concept examples: (1) elastic binary collisions at low energy, (2) core-level charge exchange reactions involving Ne+20 with Mg∕Al∕Si∕P targets, and (3) reactive scattering of CF2+∕CF3+ off Si. These studies clearly demonstrate why low, tunable incident energy, as well as mass and energy filtering of products leaving the target surface is advantageous and often essential for studies of inelastic energy losses, hard-collision charge exchange, and chemical reactions that occur during ion-surface scattering.
Compact and transferable threefold evaporator for molecular beam epitaxy in ultrahigh vacuum
Authors: Kury P. , Hild R. , Thien D. , Günter H.-L. , Meyer zu Heringdorf F.-J. , Hoegen M. Horn-von
Published: July 2005

A very compact molecular beam epitaxy (MBE) source is presented. It contains three Knudsen-type crucibles, two resistively and one electron bombardment heated. An efficient water cooling is implemented to allow MBE deposition under ultrahigh-vacuum conditions. Due to its small size, the evaporator fits inside a DN 36 CF T-piece which makes a separate bakeout possible. Refills of the crucibles as well as changes of the source materials and even repairs of the source can be carried out within a few hours without breaking the vacuum of the main chamber. The design and the usage of the MBE source are described in detail and its functionality is demonstrated exemplarily with data of silver deposition on the silicon (111) surface.
Characteristics of HfO2 thin films grown by plasma atomic layer deposition
Authors: Kim Jinwoo , Kim Seokhoon , Jeon Hyeongtag , Cho M.-H. , Chung K.-B. , Bae Choelhwyi
Published: July 2005

The characteristics of HfO2 films grown on Si substrates using a tetrakis-diethyl-amino-hafnium precursor by the remote plasma atomic layer deposition (RPALD) and direct plasma ALD (DPALD) methods were investigated by physical and electrical measurement techniques. The as-deposited HfO2 layer from RPALD exhibits an amorphous structure, while the HfO2 layer from DPALD exhibits a clearly visible polycrystalline structure. Medium energy ion scattering measurement results indicate that the interfacial layer consists of the interfacial SiO2−x and silicate layers. These results suggested that the stoichiometric change in the depth direction could be related to the energetic reactant in a state of plasma used in the plasma ALD process, resulting in damage to the Si surface and interactions between Hf and SiO2−x. The as-deposited HfO2 films using RPALD have the better interfacial layer characteristics than those using DPALD. A metal-oxide-semiconductor capacitor fabricated using the RPALD method exhibits electrical characteristics such as equivalent oxide thickness (EOT) of 1.8 nm with an effective fixed oxide charge density (Qf,eff) of ∼4.2×1011q∕cm2 and that for DPALD has a EOT (2.0 nm), and Qf,eff(∼−1.2×1013q∕cm2).
Liquid as a required catalyst phase for carbon single-walled nanotube growth
Authors: Harutyunyan A. R. , Tokune T. , Mora E.
Published: July 2005

The evolution of the iron catalyst during carbon single-walled nanotube growth is studied using calorimetry, temperature-programmed oxidation and Raman measurements. Carbon-induced solid-liquid, and solid-liquid-solid phase transitions of the nanocatalyst during the synthesis were observed. We found that liquid phase is favored for the growth of nanotubes, while the solidification of the catalyst nearly terminates the growth. No growth was observed below the eutectic point, when the catalyst is in solid phase. Our results support a growth mechanism of single-walled carbon nanotubes on liquid catalyst particles.