bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification, cloning and characterization of a plasma membrane zinc efflux transporter, TrZnT-1, from fugu pufferfish (Takifugu rubripes)
Authors: Balesaria Sara , Hogstrand Christer
Published: March 2006

An orthologue of the mammalian ZnT-1 (zinc transporter-1) gene was cloned from the intestine of the torafugu pufferfish (Takifugu rubripes), demonstrating that this gene predates the evolution of land-living vertebrates. TrZnT-1 (T. rubripes ZnT-1) shares overall topology with other members of the ZnT-1 family of zinc transporters, with six TMs (transmembrane domains) including a large histidine-rich intracellular loop between TM IV and V and intracellular C- and N-termini. Expression of TrZnT-1 in a metallothionein acquiescent cell line suggested that this protein reduces intracellular Zn2+ levels. Manipulation of the transporting media showed that several externally applied hydrominerals had no effect on TrZnT-1 activity. However, addition of N-ethylmaleimide increased TrZnT-1-mediated transport, possibly by increasing intracellular free Zn2+ levels by Zn2+ release from carrier proteins. Generation of a specific antibody and subsequent immunocytochemistry on fixed cells overexpressing TrZnT-1 indicated that the protein is localized to the plasma membrane in these cells. The genomic organization of TrZnT-1 is the same as that in mammals with two exons. The upstream regulatory region of the TrZnT-1 gene contains several putative cis-acting elements, including metal-response elements and an Sp1 site. Analysis of the DNA contigs surrounding the TrZnT-1 gene reveal limited synteny between corresponding regions in the rat, mouse and human; however, this was very low, with only two syntenic genes, ZnT-1 and NEK2 (never in mitosis gene A-related kinase).
The intersubunit lock-and-key motif in human glutathione transferase A1-1: role of the key residues Met51 and Phe52 in function and dimer stability
Authors: Alves Carla S. , Kuhnert Diane C. , Sayed Yasien , Dirr Heini W.
Published: March 2006

The dimeric structure of certain cytosolic GSTs (glutathione S-transferases) is stabilized by a hydrophobic lock-and-key motif at their subunit interface. In hGSTA1-1 (human class Alpha GST with two type-1 subunits), the key consists of two residues, Met51 and Phe52, that fit into a hydrophobic cavity (lock) in the adjacent subunit. SEC (size-exclusion chromatography)–HPLC, far-UV CD and tryptophan fluorescence of the M51A and M51A/F52S mutants indicated the non-disruptive nature of these mutations on the global structure. While the M51A mutant retained 80% of wild-type activity, the activity of the M51A/F52S was markedly diminished, indicating the importance of Phe52 in maintaining the correct conformation at the active site. The M51A and M51A/F52S mutations altered the binding of ANS (8-anilinonaphthalene-l-sulphonic acid) at the H-site by destabilizing helix 9 in the C-terminal region. Data from urea unfolding studies show that the dimer is destabilized by both mutations and that the dimer dissociates to aggregation-prone monomers at low urea concentrations before global unfolding. Although not essential for the assembly of the dimeric structure of hGSTA1-1, both Met51 and Phe52 in the intersubunit lock-and-key motif play important structural roles in maintaining the catalytic and ligandin functions and stability of the GST dimer.
Regulation of the sphingosine-recycling pathway for ceramide generation by oxidative stress, and its role in controlling c-Myc/Max function
Authors: Sultan Iyad , Senkal Can E. , Ponnusamy Suriyan , Bielawski Jacek , Szulc Zdzislaw , Bielawska Alicja , Hannun Yusuf A. , Ogretmen Besim
Published: March 2006

In the present study, the regulation of the sphingosine-recycling pathway in A549 human lung adenocarcinoma cells by oxidative stress was investigated. The generation of endogenous long-chain ceramide in response to exogenous C6-cer (C6-ceramide), which is FB1 (fumonisin B1)-sensitive, was employed to probe the sphingosine-recycling pathway. The data showed that ceramide formation via this pathway was significantly blocked by GSH and NAC (N-acetylcysteine) whereas it was enhanced by H2O2, as detected by both palmitate labelling and HPLC/MS. Similar data were also obtained using a novel approach that measures the incorporation of 17Sph (sphingosine containing 17 carbons) of 17C6-cer (C6-cer containing a 17Sph backbone) into long-chain 17C16-cer in cells by HPLC/MS, which was significantly decreased and increased in response to GSH and H2O2 respectively. TNF (tumour necrosis factor)-α, which decreases the levels of endogenous GSH, increased the generation of C16-cer in response to C6-cer, and this was blocked by exogenous GSH or NAC, or by the overexpression of TPx I (thioredoxin peroxidase I), an enzyme that reduces the generation of intracellular ROS (reactive oxygen species). Additional data showed that ROS regulated both the deacylation and reacylation steps of C6-cer. At a functional level, C6-cer inhibited the DNA-binding function of the c-Myc/Max oncogene. Inhibition of the generation of longchain ceramide in response to C6-cer by FB1 or NAC significantly blocked the modulation of the c-Myc/Max function. These data demonstrate that the sphingosine-recycling pathway for the generation of endogenous long-chain ceramide in response to exogenous C6-cer is regulated by ROS, and plays an important biological role in controlling c-Myc function.
Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity
Authors: Wu Jun , Nilsson Åke , Jönsson Bo A. G. , Stenstad Hanna , Agace William , Cheng Yajun , Duan Rui-Dong
Published: March 2006

Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [3H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1–0.25 mM Zn2+. The activity was abolished by site mutation of the predicted metal-binding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The Vmax for PAF hydrolysis was 374 μmol·h−1·(mg of protein)−1. The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer.
Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete
Authors: Montanini Barbara , Viscomi Arturo R. , Bolchi Angelo , Martin Yusé , Siverio José M. , Balestrini Raffaella , Bonfante Paola , Ottonello Simone
Published: March 2006

Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (Km=4.7 μM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate- (or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.
Multiple functions of l0036 in the regulation of the pathogenicity island of enterohaemorrhagic Escherichia coli O157:H7
Authors: Tsai Nien-Pei , Wu Yi-Chih , Chen Jenn-Wei , Wu Chih-Feng , Tzeng Chi-Meng , Syu Wan-Jr
Published: March 2006

Diarrhoeagenic enterohaemorrhagic Escherichia coli and enteropathogenic E. coli attach to human intestinal epithelium and efface brush-border microvilli, forming an A/E (attaching and effacing) lesion. These human pathogens are phenotypically similar to the mouse pathogen Citrobacter rodentium. Genetically, they all have a homologous set of virulent genes involved in the A/E lesion, and these genes are organized on a LEE (locus of enterocyte effacement), a pathogenicity island. This island comprises 41 specific open reading frames, of which most are organized at five operons, LEE1, LEE2, LEE3, LEE4 and tir (LEE5). The expression of the LEE genes is regulated in a complicated manner, and current knowledge is that there are at least two positive regulators, Ler (LEE-encoded regulator) and GrlA (global regulator of LEE activator), and one negative regulator, called GrlR (global regulator of LEE repressor). In enterohaemorrhagic E. coli, GrlA is encoded by l0043, whereas GrlR is encoded by l0044. Here we report a fourth regulatory gene located in LEE3, namely l0036. Its expression is tightly controlled. When overexpressed, this factor, named Mpc (multiple point controller), interacts with Ler and suppresses the expression of the LEE proteins. When the translation is not initiated or terminated before maturation, the type III secretion of effectors is completely abolished. Therefore, together with the fact that several cis elements reside in the region that l0036 spans, l0036 appeared to have multiple functions in the regulation of LEE expression.
In yeast, loss of Hog1 leads to osmosensitivity of autophagy
Authors: Prick Tanja , Thumm Michael , Köhrer Karl , Häussinger Dieter , Vom Dahl Stephan
Published: March 2006

In mammalian liver, proteolysis is regulated by the cellular hydration state in a microtubule- and p38MAPK (p38 mitogen-activated protein kinase)-dependent fashion. Osmosensing in liver cells towards proteolysis is achieved by activation of integrin receptors. The yeast orthologue of p38MAPK is Hog1 (high-osmolarity glycerol 1), which is involved in the hyperosmotic-response pathway. Since it is not known whether starvation-induced autophagy in yeast is osmosensitive and whether Hog1 is involved in this process, we performed fluorescence microscopy experiments. The hog1Δ cells exhibited a visible decrease of autophagy in hypo-osmotic and hyperosmotic nitrogen-starvation medium as compared with normo-osmolarity, as determined by GFP (green fluorescent protein)–Atg8 (autophagy-related 8) fluorescence. Western blot analysis of GFP–Atg8 degradation showed that WT (wild-type) cells maintained a stable autophagic activity over a broad osmolarity range, whereas hog1Δ cells showed an impaired autophagic actitivity during hypo- and hyper-osmotic stress. In [3H]leucine-pre-labelled yeast cells, the proteolysis rate was osmodependent only in hog1Δ cells. Neither maturation of pro-aminopeptidase I nor vitality was affected by osmotic stress in either yeast strain. In contrast, rapamycin-dependent autophagy, as measured by degradation of GFP–Atg8, did not significantly respond to hypo-osmotic or hyperosmotic stress in hog1Δ or WT cells. We conclude that Hog1 plays a role in the stabilization machinery of nitrogen-deprivation-induced autophagy in yeast cells during ambient osmolarity changes. This could be an analogy to the p38MAPK pathway in mammalian liver, where osmosensing towards p38MAPK is required for autophagy regulation by hypo-osmotic or amino-acid-induced cell swelling. A phenotypic difference is observed in rapamycin-induced autophagy, which does not seem to respond to extracellular osmolarity changes in hog1Δ cells.
Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis
Authors: Cao Heping , Deterding Leesa J. , Venable John D. , Kennington Elizabeth A. , Yates John R. , Tomer Kenneth B. , Blackshear Perry J.
Published: March 2006

Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption–ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)–tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.
Effects of peptide C corresponding to the Glu724–Pro760 region of the II–III loop of the DHP (dihydropyridine) receptor α1 subunit on the domain- switch-mediated activation of RyR1 (ryanodine receptor 1) Ca2+ channels
Authors: Bannister Mark L. , Ikemoto Noriaki
Published: March 2006

The Leu720–Leu764 region of the II–III loop of the dihydropyridine receptor is believed to be important for both orthograde and retrograde communications with the RyR (ryanodine receptor), but its actual role has not yet been resolved. Our recent studies suggest that voltage-dependent activation of the RyR channel is mediated by a pair of interacting N-terminal and central domains, designated as the ‘domain switch’. To investigate the effect of peptide C (a peptide corresponding to residues Glu724–Pro760) on domain- switch-mediated activation of the RyR, we measured Ca2+ release induced by DP (domain peptide) 1 or DP4 (which activates the RyR by mediation of the domain switch) and followed the Ca2+ release time course using a luminal Ca2+ probe (chlortetracycline) under Ca2+-clamped conditions. Peptide C produced a significant potentiation of the domain-switch-mediated Ca2+ release, provided that the Ca2+ concentration was sufficiently low (e.g. 0.1 μM) and the Ca2+ channel was only partially activated by the domain peptide. However, at micromolar Ca2+ concentrations, peptide C inhibits activation. Covalent cross-linking of fluorescently labelled peptide C to the RyR and screening of the fluorescently labelled tryptic fragments permitted us to localize the peptide-C-binding site to residues 450–1400, which may represent the primary region involved in physical coupling. Based on the above findings, we propose that the physiological role of residues Glu724–Pro760 is to facilitate depolarization-induced and domain-switch-mediated RyR activation at sub- or near-threshold concentrations of cytoplasmic Ca2+ and to suppress activation upon an increase of cytoplasmic Ca2+.
Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells
Authors: Behnam Joseph T. , Williams Emma L. , Brink Susanne , Rumsby Gill , Danpure Christopher J.
Published: March 2006

Failure to detoxify the intermediary metabolite glyoxylate in human hepatocytes underlies the metabolic pathology of two potentially lethal hereditary calcium oxalate kidney stone diseases, PH (primary hyperoxaluria) types 1 and 2. In order to define more clearly the roles of enzymes involved in the metabolism of glyoxylate, we have established singly, doubly and triply transformed CHO (Chinese-hamster ovary) cell lines, expressing all combinations of normal human AGT (alanine:glyoxylate aminotransferase; the enzyme deficient in PH1), GR/HPR (glyoxylate/hydroxypyruvate reductase; the enzyme deficient in PH2), and GO (glycolate oxidase). We have embarked on the preliminary metabolic analysis of these transformants by studying the indirect toxicity of glycolate as a simple measure of the net intracellular production of glyoxylate. Our results show that glycolate is toxic only to those cells expressing GO and that this toxicity is diminished when AGT and/or GR/HPR are expressed in addition to GO. This finding indicates that we have been able to reconstruct the glycolate→glyoxylate, glyoxylate→glycine, and glyoxylate→glycolate metabolic pathways, catalysed by GO, AGT, and GR/HPR respectively, in cells that do not normally express them. These results are compatible with the findings in PH1 and PH2, in which AGT and GR/HPR deficiencies lead to increased oxalate synthesis, due to the failure to detoxify its immediate precursor glyoxylate. These CHO cell transformants have a potential use as a cell-based bioassay for screening small molecules that stabilize AGT or GR/HPR and might have use in the treatment of PH1 or PH2.
Intracellular composition of fatty acid affects the processing and function of tyrosinase through the ubiquitin–proteasome pathway
Authors: Ando Hideya , Wen Zhi-Ming , Kim Hee-Yong , Valencia Julio C. , Costin Gertrude-E. , Watabe Hidenori , Yasumoto Ken-ichi , Niki Yoko , Kondoh Hirofumi , Ichihashi Masamitsu , Hearing Vincent J.
Published: March 2006

Proteasomes are multicatalytic proteinase complexes within cells that selectively degrade ubiquitinated proteins. We have recently demonstrated that fatty acids, major components of cell membranes, are able to regulate the proteasomal degradation of tyrosinase, a critical enzyme required for melanin biosynthesis, in contrasting manners by relative increases or decreases in the ubiquitinated tyrosinase. In the present study, we show that altering the intracellular composition of fatty acids affects the post-Golgi degradation of tyrosinase. Incubation with linoleic acid (C18:2) dramatically changed the fatty acid composition of cultured B16 melanoma cells, i.e. the remarkable increase in polyunsaturated fatty acids such as linoleic acid and arachidonic acid (C20:4) was compensated by the decrease in monounsaturated fatty acids such as oleic acid (C18:1) and palmitoleic acid (C16:1), with little effect on the proportion of saturated to unsaturated fatty acid. When the composition of intracellular fatty acids was altered, tyrosinase was rapidly processed to the Golgi apparatus from the ER (endoplasmic reticulum) and the degradation of tyrosinase was increased after its maturation in the Golgi. Retention of tyrosinase in the ER was observed when cells were treated with linoleic acid in the presence of proteasome inhibitors, explaining why melanin synthesis was decreased in cells treated with linoleic acid and a proteasome inhibitor despite the abrogation of tyrosinase degradation. These results suggest that the intracellular composition of fatty acid affects the processing and function of tyrosinase in connection with the ubiquitin–proteasome pathway and suggest that this might be a common physiological approach to regulate protein degradation.
Coupling of protein motions and hydrogen transfer during catalysis byEscherichia colidihydrofolate reductase
Authors: Swanwick Richard S. , Maglia Giovanni , Tey Lai-hock , Allemann Rudolf K.
Published: March 2006

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0±0.2 at 5 °C to 2.2±0.2 at 40 °C and an inverse ratio of the pre-exponential factors of 0.108±0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue, Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 °C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.
Mechanism of nitrite oxidation by eosinophil peroxidase: implications for oxidant production and nitration by eosinophils
Authors: van Dalen Christine J. , Winterbourn Christine C. , Kettle Anthony J.
Published: March 2006

Eosinophil peroxidase is a haem enzyme of eosinophils that is implicated in oxidative tissue injury in asthma. It uses hydrogen peroxide to oxidize thiocyanate and bromide to their respective hypohalous acids. Nitrite is also a substrate for eosinophil peroxidase. We have investigated the mechanisms by which the enzyme oxidizes nitrite. Nitrite was very effective at inhibiting hypothiocyanous acid (‘cyanosulphenic acid’) and hypobromous acid production. Spectral studies showed that nitrite reduced the enzyme to its compound II form, which is a redox intermediate containing FeIV in the haem active site. Compound II does not oxidize thiocyanate or bromide. These results demonstrate that nitrite is readily oxidized by compound I, which contains FeV at the active site. However, it reacts more slowly with compound II. The observed rate constant for reduction of compound II by nitrite was determined to be 5.6×103 M−1·s−1. Eosinophils were at least 4-fold more effective at promoting nitration of a heptapeptide than neutrophils. This result is explained by our finding that nitrite reacts 10-fold faster with compound II of eosinophil peroxidase than with the analogous redox intermediate of myeloperoxidase. Nitration by eosinophils was increased 3-fold by superoxide dismutase, which indicates that superoxide interferes with nitration. We propose that at sites of eosinophilic inflammation, low concentrations of nitrite will retard oxidant production by eosinophil peroxidase, whereas at higher concentrations nitrogen dioxide will be a major oxidant formed by these cells. The efficiency of protein nitration will be decreased by the diffusion-controlled reaction of superoxide with nitrogen dioxide.
Gαq binds to p110α/p85α phosphoinositide 3-kinase and displaces Ras
Authors: Ballou Lisa M. , Chattopadhyay Mohar , Li Yan , Scarlata Suzanne , Lin Richard Z.
Published: March 2006

Several studies have reported that activation of Gq-coupled receptors inhibits PI3K (phosphoinositide 3-kinase) signalling. In the present study, we used purified proteins to demonstrate that Gαq directly inhibits p110α/p85α PI3K in a GTP-dependent manner. Activated Gαq binds to the p110α/p85α PI3K with an apparent affinity that is seven times stronger than that for Gαq·GDP as measured by fluorescence spectroscopy. In contrast, Gαq did not bind to the p110γ PI3K. Fluorescence spectroscopy experiments also showed that Gαq competes with Ras, a PI3K activator, for binding to p110α/p85α. Interestingly, co-precipitation studies using deletion mutants showed that Gαq binds to the p85-binding domain of p110α and not to the Ras-binding domain. Expression of constitutively active GαqQ209L in cells inhibited Ras activation of the PI3K/Akt pathway but had no effect on Ras/Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signalling. These results suggest that activation of Gq-coupled receptors leads to increased binding of Gαq·GTP to some isoforms of PI3K, which might explain why these receptors inhibit this signalling pathway in certain cell types.
Identification and characterization of Fep15, a new selenocysteine-containing member of the Sep15 protein family
Authors: Novoselov Sergey V. , Hua Deame , Lobanov Alexey V. , Gladyshev Vadim N.
Published: March 2006

Sec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec. When transiently expressed in mammalian cells, Fep15 incorporated Sec in an SECIS- and SBP2 (SECIS-binding protein 2)-dependent manner and was targeted to the endoplasmic reticulum by its N-terminal signal peptide. Phylogenetic analyses of Sep15 family members suggest that Fep15 evolved by gene duplication.
Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design
Authors: Clark Richard J. , Daly Norelle L. , Craik David J.
Published: March 2006

The cyclotide family of plant proteins is of interest because of their unique topology, which combines a head-to-tail cyclic backbone with an embedded cystine knot, and because their remarkable chemical and biological properties make them ideal candidates as grafting templates for biologically active peptide epitopes. The present study describes the first steps towards exploiting the cyclotide framework by synthesizing and structurally characterizing two grafted analogues of the cyclotide kalata B1. The modified peptides have polar or charged residues substituted for residues that form part of a surface-exposed hydrophobic patch that plays a significant role in the folding and biological activity of kalata B1. Both analogues retain the native cyclotide fold, but lack the undesired haemolytic activity of their parent molecule, kalata B1. This finding confirms the tolerance of the cyclotide framework to residue substitutions and opens up possibilities for the substitution of biologically active peptide epitopes into the framework.
The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation
Authors: Jaleel Mahaboobi , Villa Fabrizio , Deak Maria , Toth Rachel , Prescott Alan R. , van Aalten Daan M. F. , Alessi Dario R.
Published: March 2006

Recent work indicates that the LKB1 tumour suppressor protein kinase, which is mutated in Peutz–Jeghers cancer syndrome, phosphorylates and activates a group of protein kinases that are related to AMPK (AMP-activated protein kinase). Ten of the 14 AMPK-related protein kinases activated by LKB1, including SIK (salt-induced kinase), MARK (microtubule-affinity-regulating kinase) and BRSK (brain-specific kinase) isoforms, possess a ubiquitin-associated (UBA) domain immediately C-terminal to the kinase catalytic domain. These are the only protein kinases in the human genome known to possess a UBA domain, but their roles in regulating AMPK-related kinases are unknown. We have investigated the roles that the UBA domain may play in regulating these enzymes. Limited proteolysis of MARK2 revealed that the kinase and UBA domains were contained within a fragment that was resistant to trypsin proteolysis. SAXS (small-angle X-ray scattering) analysis of inactive and active LKB1-phosphorylated MARK2 revealed that activation of MARK2 is accompanied by a significant conformational change that alters the orientation of the UBA domain with respect to the catalytic domain. Our results indicate that none of the UBA domains found in AMPK-related kinases interact with polyubiquitin or other ubiquitin-like molecules. Instead, the UBA domains appear to play an essential conformational role and are required for the LKB1-mediated phosphorylation and activation of AMPK-related kinases. This is based on the findings that mutation or removal of the UBA domains of several AMPK-related kinases, including isoforms of MARK, SIK and BRSK, markedly impaired the catalytic activity and LKB1-mediated phosphorylation of these enzymes. We also provide evidence that the UBA domains do not function as LKB1–STRAD (STE20-related adaptor)–MO25 (mouse protein 25) docking/interacting sites and that mutations in the UBA domain of SIK suppressed the ability of SIK to localize within punctate regions of the nucleus. Taken together, these findings suggest that the UBA domains of AMPK-related kinases play an important role in regulating the conformation, activation and localization of these enzymes.
Structural properties of liquid N-methylacetamide via ab initio, path integral, and classical molecular dynamics
Authors: Whitfield T. W. , Crain J. , Martyna G. J.
Published: February 2006

In order to better understand the physical interactions that stabilize protein secondary structure, the neat liquid state of a peptidic fragment, N-methylacetamide (NMA), was studied using computer simulation. Three different descriptions of the molecular liquid were examined: an empirical force field treatment with classical nuclei, an empirical force field treatment with quantum mechanical nuclei, and an ab initio density functional theory (DFT) treatment. The DFT electronic structure was evaluated using the BLYP approximate functional and a plane wave basis set. The different physical effects probed by the three models, such as quantum dispersion, many-body polarization, and nontrivial charge distributions on the liquid properties, were compared. Much of the structural ordering in the liquid is characterized by hydrogen bonded chains of NMA molecules. Modest structural differences are present among the three models of liquid NMA. The average molecular dipole in the liquid under the ab initio treatment, however, is enhanced by 60% over the gas phase value.
Quantum phase transitions and the H–T phase diagram of a multisublattice Van Vleck antiferromagnet
Authors: Kalita V. M. , Loktev V. M.
Published: February 2006

The magnetic ordering of an easy-plane Van Vleck magnet with a large anisotropy of the easy-plane type and an antiferromagnetic exchange interaction between spins is considered in the case when the ground state of the integer-spin ions is a singlet and the antiferromagnetic phase is realized only in an external magnetic field. It is found that the phase transformation of the singlet (paramagnetic) phase to the antiferromagnetic phase is a quantum phase transition belonging to the magnetic phase transitions of the displacive type. It is shown that the Landau theory of phase transitions is applicable to such a transformation, with the order parameter being the polarization of the singlet state. Nonstandard manifestations of the paraprocess converting the second-order magnetic phase transition of the displacive type to a first-order transition are noted. The phase diagram is constructed; it differs from those of classical antiferromagnets in that the antiferromagnetic phase on it is surrounded only by the paramagnetic phase.
Improved performance of electrophosphorescent devices based on Re(CO)3Cl-dipyrido[3,2-a:2′,3′-c]phenazine
Authors: Fu Chunyan , Li Mingtao , Su Zhongmin , Hong Ziruo , Li Wenlian , Li Bin
Published: February 2006

Highly efficient orange-yellow organic electroluminescent (EL) devices based on Re(CO)3Cl-dipyrido[3,2-a:2′,3′-c]phenazine (Re-DPPz) have been fabricated. By incorporating Re-DPPz into EL devices with balanced charge injection and efficient host-to-guest energy transfer, the EL performances were significantly improved comparing with previously reports based on the same complex. For EL device with 7wt% Re-DPPz doped emissive layer, turn-on voltage of 6V, maximum efficiency of 6.3cd∕A, and orange-yellow emission with brightness approaching 1000 cd∕m2 were achieved. The mechanism responsible for the significant improvement was discussed.
Fast electrochromic effect obtained from solid-state inorganic thin-film configuration with a carrier accumulation structure
Authors: Yoshimura Hideo , Koshida Nobuyoshi
Published: February 2006

To improve the dynamic response of a solid-state electrochromic (EC) device, a new concept has been introduced into the EC operation. The device is composed of an indium tin oxide-coated glass substrate, a thin amorphous WO3 film, a thin SiO2 film, a thin amorphous Ta2O5 film, and a semitransparent top contact. The EC coloration and bleaching are measured by transient evolution in the optical transmittance under a positive or negative bias voltage Vb. The experimental results show that the response time at which the transmittance becomes 70% of the initial value reaches 100ms at Vb=3V. From voltammometric analyses, it is suggested that the inserted thin SiO2 film acts as a barrier for holes and electrons against diffusion or leakage, and that both the production rate of protons at the SiO2∕Ta2O5 interface and their subsequent intercalation into the WO3 film are significantly accelerated. This approach is promising for the development of practical solid-state inorganic thin-film EC devices, such as a smart window and paperlike passive display.
Velocity map imaging study of BrCl photodissociation at 467 nm: Determination of all odd-rank (K=1 and 3) anisotropy parameters for the Cl(P3∕22) photofragments
Authors: Smolin Andrey G. , Vasyutinskii Oleg S. , Vieuxmaire Olivier P. J. , Ashfold Michael N. R. , Balint-Kurti Gabriel G. , Orr-Ewing Andrew J.
Published: February 2006

Resonance-enhanced multiphoton ionization and velocity map imaging of the Cl(P3∕202) fragments of BrCl photolysis at 467.16 nm have been used to obtain a complete set of orientation parameters (with ranks K=1 and 3) describing the polarization of the electronic angular momentum. The experiments employ two geometries distinguished only by the circular or linear polarization of the photolysis laser beam. Normalized difference images constructed from the data accumulated using a right or left circularly polarized probe-laser beam, counterpropagating with the photolysis laser, were fitted to basis images corresponding to contributions from various odd-rank anisotropy parameters. Expressions are given for the difference images in terms of the K=1 and 3 anisotropy parameters, which describe coherent and incoherent parallel and perpendicular excitation and dissociation mechanisms. The nonzero values of the anisotropy parameters are indicative of nonadiabatic dissociation dynamics, with likely contributions from flux on the AΠ3(1),BΠ3(0+),CΠ1(1), and XΣ+1(0+) states as well as one further Ω=1 state, all of which correlate adiabatically to Cl(P3∕202)+Br(P3∕202) photofragments. The magnitudes of the parameters depend both on the amplitudes of dissociative flux in these states, and also on the phases accumulated by the nuclear wave functions for different dissociation pathways.
Tunneling chemical reactions D+H2→DH+H and D+DH→D2+H in solid D2–H2 and HD–H2 mixtures: An electron-spin-resonance study
Authors: Kumada Takayuki
Published: February 2006

Tunneling chemical reactions D+H2→DH+H and D+DH→D2+H in solid HD–H2 and D2–H2 mixtures were studied in the temperature range between 4 and 8K. These reactions were initiated by UV photolysis of DI molecules doped in these solids for 30s and followed by measuring the time course of electron-spin-resonance (ESR) intensities of D and H atoms. ESR intensity of D atoms produced by the photolysis decreases but that of H atoms increases with time. Time course of the D and H intensities has the fast and slow processes. The fast process, which finishes within ∼300s after the photolysis, is assigned to the reaction of D atom with one of its nearest-neighboring H2 molecules, D(H2)n(HD)12−n→H(H2)n−1(HD)13−n or D(H2)n(D2)12−n→H(HD)(H2)n−1(D2)12−n for 12⩾n⩾1. Rate constant for the D+H2 reaction between neighboring D atom–H2 molecule pair is determined to be (7.5±0.7)×10−3s−1 in solid HD–H2 and (1.3±0.3)×10−2s−1 in D2–H2 at 4.1K, which is very close to that calculated based on the theory of chemical reaction in gas phase by Hancock et al. [J. Chem. Phys. 91, 3492 (1989)] and Takayanagi and Sato [J. Chem. Phys. 92, 2862 (1990)]. This rate constant was found to be independent of temperature up to 7K within experimental error of ±30%. The slow process is assigned to the reaction of D atom produced in a cage fully surrounded by HD or D2 molecules, D(HD)12 or D(D2)12. This D atom undergoes the D+DH reaction with one of its nearest-neighboring HD molecules in solid HD–H2 or diffuses to the neighbor of H2 molecules to allow the D+H2 reaction in solid HD–H2 and D2–H2. The former is the main channel in solid HD–H2 below 6K where D atoms diffuse very slowly, whereas the latter dominates over the former above 6K. Rate for the reactions in the slow process is independent of temperature below 6K but increases with the increase in temperature above 6K. We found that the increase is due to the increase in hopping rate of D atoms to the neighbor of H2 molecules. Rate constant for the D+DH reaction was found to be independent of temperature up to 7K as well.
Crystallographic, electrical, and magnetic properties of the system La0.7Sr0.3Mn1−xFexO3
Authors: Yanchevskii O. Z. , V’yunov O. I. , Belous A. G. , Tovstolytkin A. I.
Published: February 2006

On the basis of x-ray structural data and studies of the electrical, magnetic, and magnetoresistive properties and the ferromagnet resonance spectra of ceramic samples of La0.7Sr0.3Mn1−xFexO3 (0⩽x⩽0.10) it is established that a competition between structural changes influencing the ferromagnetism leads to a monotonic weakening of the ferromagnetism (a lowering of TC by around 5K∕%Fe). The increase of the magnetoresistance in the low-temperature region and the large values of the low-field contribution to the magnetoresistance are indicative of an inhomogeneous character of the electrical conductivity and magnetization of the samples at T<TC, which may be due to the coexistence of the ferro- and paramagnetic phases over a wide temperature region.
Comparison of pressure, magnetic-field, and excess manganese effects on transport properties of film and bulk ceramic La–Ca manganites
Authors: Mikhaylov V. I. , Zubov E. E. , Pashchenko A. V. , Varyukhin V. N. , Shtaba V. A. , Dyakonov V. P. , Szewczyk A. , Abal’oshev A. , Piotrowski K. , Lewandowski S. J. , Szymczak H. , Dyakonov K.
Published: February 2006

The pressure, magnetic-field, and excess manganese effects on transport and magnetoresistance effect (MRE) are studied in both epitaxial films and bulk ceramics of the manganites (La0.7Ca0.3)1−xMn1+xO3−y (x=0–0.2). A comparison of the electrical behavior in both kinds of samples of similar composition at hydrostatic pressures of up to 1.8GPa and in magnetic fields of up to 8kOe is performed. The pressure and magnetic-field effects are shown to increase with increasing manganese content. Experimental data show that the effects of pressure and magnetic field on the temperatures of both the metal-insulator transition (TMD) and the MRE peak (TMR) are considerably stronger in the films than in the ceramics. The hydrostatic pressure increases TMD and TMR. It was shown that for both types of samples the magnetoresistance effect is affected in opposite ways by pressure and magnetic field. A direct correlation is established between TMD and conductivity bandwidth as well as between MRE and concentration of charge carriers at applied pressure. The differences in the values of pressure effect on resistance, MRE and TMD temperature in the films and ceramics are connected with both granular structure of ceramics and the oxygen nonstoichiometry in ceramic and film samples of the same content as well as with the film strain induced by lattice mismatch between the film and the substrate. The origin of pressure-magnetic-field effects is analyzed in the framework of the double exchange interaction and the small polaron hopping and variable-range hopping models.
Effect of doping on the magnetic ordering of quasi-one-dimensional antiferromagnets
Authors: Zvyagin A. A.
Published: February 2006

We have studied theoretically how nonmagnetic dopants, which change the local coupling of spins to the host, affect the low-temperature thermodynamic characteristics of quasi-one-dimensional (Q1D) quantum spin antiferromagnets. Our theory qualitatively and, in some cases, quantitatively describes the behavior of the magnetic susceptibility and specific heat of the Q1D system BaCu2(Si1−xGex)2O7. We have shown that in some cases the strong disorder in the distribution of characteristics of magnetic impurities in quantum antiferromagnetic spin chains can be the cause of magnetic ordering, if such chains are weakly coupled to each other, while for homogeneous chains and chains with a weak disorder a small enough coupling between chains will not produce magnetic ordering. For other values of the parameters, magnetic impurities can decrease the Néel temperature compared to that of the homogeneous Q1D spin system.
Molecular simulation of liquid water confined inside graphite channels: Thermodynamics and structural properties
Authors: Marti J. , Nagy G. , Gordillo M. C. , Guàrdia E.
Published: February 2006

We carried out molecular dynamics simulations to describe the properties of water inside a narrow graphite channel. Two stable phases were found: a low-density one made of water clusters adsorbed on the graphite sheets and a liquid one that fills the entire channel, forming several layers around a bulklike region. We analyzed the interfacial structure, orientational order, water residence times in several regions, and hydrogen bonding of this last water phase, calculating also a quantity of electrochemical interest, the probability of electron tunneling through interfacial water. The results are in good qualitative agreement with the available experimental data.
Growth mechanisms of thin-film columnar structures in zinc oxide on p-type silicon substrates
Authors: Shin J. W. , Lee J. Y. , Kim T. W. , No Y. S. , Cho W. J. , Choi W. K.
Published: February 2006

X-ray diffraction analysis reveals that the crystallinity of (0001)-oriented columnar grains in ZnO thin films grown on p-Si (100) substrates is enhanced with increasing growth temperature, and transmission electron microscopy confirms that the columnar structures become more stable at higher growth temperature. The morphological evolution of the columnar structure in ZnO thin films is described on the basis of experimental measurements.
Nanoscaled electrical homogeneity of indium zinc oxide films
Authors: Kumar Bhupendra , Gong Hao , Gosvami Nitya Nand , Akkipeddi Ramam , O’Shea Sean J.
Published: February 2006

We are able to fabricate both polycrystalline and amorphous indium zinc oxide thin films. All the thin films exhibited an n-type semiconductor behavior with room-temperature conductivities in the range of 2.5×103–1.58×103 (Ωcm)−1. A nanoscaled conductivity inhomogeneity was observed in polycrystalline films by means of conducting atomic force microscopy, with morphology effect excluded by simultaneous topographic mapping. This effect has been explained in the presence of highly conducting In2O3 and Zn2In2O5 nano crystalline phases imbedded in amorphous matrix. On the other hand, excellent electrical homogeneity throughout the amorphous film was observed, suggesting its promising potential in microelectronic device applications.
Indentation size effect in barium titanate with spherical tipped nanoindenters
Authors: Scholz T. , Muñoz-Saldaña J. , Swain M. V. , Schneider G. A.
Published: February 2006

Nanoindentation tests in an 90°-ac-domain area of an {001} orientated barium titanate single crystal were performed using four different indenters (two with cube corner and two with spherical shape) with tip radii from 61nm to 1.9μm. Extensive calibrations of the tips on fused quartz and sapphire defined the penetration depth range for approximately spherical contact prior to indentation of barium titanate (BaTiO3). The measured elastic modulus is independent of the different indenters. The measurements showed plastic deformation after “pop-in”. The calculated mean pressure remained constant for each indenter, but clearly depends upon the indenter radius. The indenter radius dependence of the hardness support the concept of “geometrically necessary dislocations”, proposed by W. D. Nix and H. Gao [J. Mech. Phys. Sol., 46, 411 (1998)] and its extension to spherical tipped indenters [J. G. Swadener, E. P. George, G. M. Pharr, J. Mech. Phys. Solids, 50, 681 (2002)]. The results show this concept fits the data generated with indenter radii which are at least an order of magnitude lower than investigated by Swadener. Furthermore, the results agree with estimates of the statistically stored dislocation density determined for BaTiO3.