bluuebunny/crossref_metadata_2025 huggingface dataset:
Unique structure of Ascaris suum b5-type cytochrome: an additional α-helix and positively charged residues on the surface domain interact with redox partners
Authors: Yokota Takehiro , Nakajima Yoshitaka , Yamakura Fumiyuki , Sugio Shigetoshi , Hashimoto Muneaki , Takamiya Shinzaburo
Published: March 2006

Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 Å (1 Å=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six α-helices and five β-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional α-helix, α4A, between helices α4 and α5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80° relative to the axis formed by haem-Fe and Nϵ atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode metmyoglobin can be hypothesized.
L-Cysteate sulpho-lyase, a widespread pyridoxal 5′-phosphate-coupled desulphonative enzyme purified from Silicibacter pomeroyi DSS-3T
Authors: Denger Karin , Smits Theo H. M. , Cook Alasdair M.
Published: March 2006

Quantitative utilization of L-cysteate (2-amino-3-sulphopropionate) as the sole source of carbon and energy for growth of the aerobic, marine bacterium Silicibacter pomeroyi DSS-3T was observed. The sulphonate moiety was recovered in the medium largely as sulphite, and the appropriate amount of the ammonium ion was also observed. Genes [suyAB (3-sulpholactate sulpho-lyase)] encoding the known desulphonation reaction in cysteate degradation were absent from the genome, but a homologue of a putative sulphate exporter gene (suyZ) was found, and its neighbour, annotated as a D-cysteine desulphhydrase, was postulated to encode pyridoxal 5′-phosphate-coupled L-cysteate sulpho-lyase (CuyA), a novel enzyme. Inducible CuyA was detected in cysteate-grown cells. The enzyme released equimolar pyruvate, sulphite and the ammonium ion from L-cysteate and was purified to homogeneity by anion-exchange, hydrophobic-interaction and gel-filtration chromatography. The N-terminal amino acid sequence of this 39-kDa subunit confirmed the identification of the cuyA gene. The native enzyme was soluble and homomultimeric. The Km-value for L-cysteate was high (11.7 mM) and the enzyme also catalysed the D-cysteine desulphhydrase reaction. The gene cuyZ, encoding the putative sulphite exporter, was co-transcribed with cuyA. Sulphite was exported despite the presence of a ferricyanide-coupled sulphite dehydrogenase. CuyA was found in many bacteria that utilize cysteate.
Evolutionarily conserved structural motifs in bacterial GST (glutathione S-transferase) are involved in protein folding and stability
Authors: Allocati Nerino , Masulli Michele , Pietracupa Marilena , Federici Luca , Di Ilio Carmine
Published: March 2006

The bacterium Proteus mirabilis expresses a cytosolic class beta glutathione S-transferase (PmGST B1-1) that is part of a family of multifunctional detoxication enzymes. Like other cytosolic GSTs, PmGST B1-1 possesses two local structural motifs, an N-capping box and a hydrophobic staple motif, both of which are located between amino acids 151 and 156. The N-capping box consists of a reciprocal hydrogen bonding interaction of Thr152 with Asp155, whereas the hydrophobic staple motif consists of a hydrophobic interaction between Phe151 and Ala156. By contrast with other GSTs, PmGST B1-1 displays distinct hydrogen bond interactions in the N-capping box. In mammalian GSTs these structural elements are critical for protein folding and stability. To investigate the role played by these two motifs in a distantly related organism on the evolutionary scale, site-directed mutagenesis was used to generate several mutants of both motifs in PmGST B1-1. All mutants were efficiently overexpressed and purified, but they were quite unstable, although at different levels, indicating that protein folding was significantly destabilized. The analysis of the T152A and D155G variants indicated that the N-capping box motif plays an important role in the stability and correct folding of the enzyme. The analysis of F151A and A156G mutants revealed that the hydrophobic staple motif influences the structural maintenance of the protein and is implicated in the folding process of PmGST B1-1. Finally, the replacement of Thr152 and Asp155, as well as Phe151 and Ala156 residues influences the catalytic efficiency of the enzyme.
Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase
Authors: Shiryaev Sergey A. , Ratnikov Boris I. , Chekanov Alexei V. , Sikora Sergey , Rozanov Dmitri V. , Godzik Adam , Wang Jun , Smith Jeffrey W. , Huang Ziwei , Lindberg Iris , Samuel Melanie A. , Diamond Michael S. , Strongin Alex Y.
Published: March 2006

Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9–12-mer peptides are potent inhibitors of WNV NS3 with Ki values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.
Class II phosphoinositide 3-kinase α-isoform regulates Rho, myosin phosphatase and contraction in vascular smooth muscle
Authors: Wang Yu , Yoshioka Kazuaki , Azam Mohammed Ali , Takuwa Noriko , Sakurada Sotaro , Kayaba Yuji , Sugimoto Naotoshi , Inoki Isao , Kimura Takaharu , Kuwaki Tomoyuki , Takuwa Yoh
Published: March 2006

We demonstrated previously that membrane depolarization and excitatory receptor agonists such as noradrenaline induce Ca2+-dependent Rho activation in VSM (vascular smooth muscle), resulting in MP (myosin phosphatase) inhibition through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1. In the present study, we show in de-endothelialized VSM strips that the PI3K (phosphoinositide 3-kinase) inhibitors LY294002 and wortmannin inhibited KCl membrane depolarization- and noradrenaline-induced Rho activation and MYPT1 phosphorylation, with concomitant inhibition of MLC (20-kDa myosin light chain) phosphorylation and contraction. LY294002 also augmented de-phosphorylation of MLC and resultantly relaxation in KCl-contracted VSM, whereas LY294002 was much less effective or ineffective under the conditions in which MP was inhibited by either a phosphatase inhibitor or a phorbol ester in Rho-independent manners. VSM express at least four PI3K isoforms, including the class I enzymes p110α and p110β and the class II enzymes PI3K-C2α and -C2β. The dose–response relationships of PI3K-inhibitor-induced inhibition of Rho, MLC phosphorylation and contraction were similar to that of PI3K-C2α inhibition, but not to that of the class I PI3K inhibition. Moreover, KCl and noradrenaline induced stimulation of PI3K-C2α in a Ca2+-dependent manner, but not of p110α or p110β. Down-regulation of PI3K-C2α expression by siRNA (small interfering RNA) inhibited contraction and phosphorylation of MYPT1 and MLC in VSM cells. Finally, intravenous wortmannin infusion induced sustained hypotension in rats, with inhibition of PI3K-C2α activity, GTP-loading of Rho and MYPT1 phosphorylation in the artery. These results indicate the novel role of PI3K-C2α in Ca2+-dependent Rho-mediated negative control of MP and thus VSM contraction.
Cysteine-accessibility analysis of transmembrane domains 11–13 of human concentrative nucleoside transporter 3
Authors: Zhang Jing , Tackaberry Tracey , Ritzel Mabel W. L. , Raborn Taylor , Barron Gerry , Baldwin Stephen A. , Young James D. , Cass Carol E.
Published: March 2006

hCNT3 (human concentrative nucleoside transporter 3) is a nucleoside–sodium symporter that transports a broad range of naturally occurring purine and pyrimidine nucleosides as well as anticancer nucleoside drugs. To understand its uridine binding and translocation mechanisms, a cysteine-less version of hCNT3 was constructed and used for cysteine-accessibility and permeant-protection assays. Cysteine-less hCNT3, with 14 endogenous cysteine residues changed to serine, displayed wild-type properties in a yeast expression system, indicating that endogenous cysteine residues are not essential for hCNT3-mediated nucleoside transport. A series of cysteine-substitution mutants spanning predicted TMs (transmembrane domains) 11–13 was constructed and tested for accessibility to thiol-specific reagents. Mutants M496C, G498C, F563C, A594C, G598C and A606C had no detectable transport activity, indicating that a cysteine substitution at each of these positions was not tolerated. Two functional mutants in putative TM 11 (L480C and S487C) and four in putative TM 12 (N565C, T557C, G567C and I571C) were partially inhibited by MTS (methanethiosulphonate) reagent and high concentrations of uridine protected against inhibition, indicating that TMs 11 and 12 may form part of the nucleoside translocation pathway. The lack of accessibility of MTS reagents to TM 13 mutants suggests that TM 13 is not exposed to the nucleoside translocation pathway. Furthermore, G567C, N565C and I571C mutants were only sensitive to MTSEA (MTS-ethylammonium), a membranepermeant thiol reagent, indicating that these residues may be accessible from the cytoplasmic side of the membrane, providing evidence in support of the predicted orientation of TM 12 in the current putative topology model of hCNT3.
Role of N-terminal residues in the ubiquitin-independent degradation of human thymidylate synthase
Authors: Peña Maria Marjorette O. , Xing Yang Yang , Koli Sangita , Berger Franklin G.
Published: March 2006

Thymidylate synthase (TS) catalyses the reductive methylation of dUMP to form dTMP, a reaction that is essential for maintenance of nucleotide pools during cell growth. Because the enzyme is indispensable for DNA replication in actively dividing cells, it is an important target for cytotoxic drugs used in cancer chemotherapy, including fluoropyrimidines (e.g. 5-fluorouracil and 5-fluoro-2′-deoxyuridine) and anti-folates (e.g. raltitrexed, LY231514, ZD9331 and BW1843U89). These drugs generate metabolites that bind to the enzyme's active site and inhibit catalytic activity, leading to thymidylate deprivation and cellular apoptosis. Ligand binding to TS results in stabilization of the enzyme and an increase in its intracellular concentration. Previously, we showed that degradation of the TS polypeptide is carried out by the 26 S proteasome in a ubiquitin-independent manner. Such degradation is directed by the disordered N-terminal region of the TS polypeptide, and is abrogated by ligand binding. In the present study, we have verified the ubiquitin-independent nature of TS proteolysis by showing that a ‘lysine-less’ polypeptide, in which all lysine residues were replaced by arginine, is still subject to proteasome-mediated degradation. In addition, we have mapped the structural determinants of intracellular TS degradation in more detail and show that residues at the N-terminal end of the molecule, particularly the penultimate amino acid Pro2, play an important role in governing the half-life of the enzyme. This region is capable on its own of destabilizing an evolutionarily distinct TS molecule that normally lacks this domain, indicating that it functions as a degradation signal. Interestingly, degradation of an intrinsically unstable mutant form of TS, containing a Pro→Leu substitution at residue 303, is directed by C-terminal, rather than N-terminal, sequences. The implications of these findings for the control of TS expression, and for the regulation of protein degradation in general, are discussed.
Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin–26 S proteasome system in the living rat
Authors: Dominy John E. , Hirschberger Lawrence L. , Coloso Relicardo M. , Stipanuk Martha H.
Published: March 2006

Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin–26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.
Homology-model-guided site-specific mutagenesis reveals the mechanisms of substrate binding and product-regulation of adenosine kinase from Leishmania donovani
Authors: Datta Rupak , Das Ishita , Sen Banibrata , Chakraborty Anutosh , Adak Subrata , Mandal Chhabinath , Datta Alok K.
Published: March 2006

Despite designating catalytic roles of Asp299 and Arg131 during the transfer of γ-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591–600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study, employing homology-model-guided site-specific protein mutagenesis, we show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in Km (Ado) with a 1000-fold decrease in kcat/Km, implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance of steric complementarity of the binding pocket. Use of 2′or 3′-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme–Ado complex, the binding energy (ΔΔGB) contribution of the former was greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding studies revealed that, unlike the R131A mutant, which showed abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered Km (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.
A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae
Authors: Fernandez Luisa E. , Aimanova Karlygash G. , Gill Sarjeet S. , Bravo Alejandra , Soberón Mario
Published: March 2006

A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI–ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI–ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI–ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI–ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.
A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding
Authors: Altincicek Boran , Shibamiya Aya , Trusheim Heidi , Tzima Eleni , Niepmann Michael , Linder Dietmar , Preissner Klaus T. , Kanse Sandip M.
Published: March 2006

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.
Interactions between the plasma membrane and the antimicrobial peptide HP (2-20) and its analogues derived from Helicobacter pylori
Authors: Lee Kwang H. , Lee Dong G. , Park Yoonkyung , Kang Dong-Il , Shin Song Y. , Hahm Kyung-Soo , Kim Yangmee
Published: March 2006

HP (2-20), a 19-residue peptide derived from the N-terminus of Helicobacter pylori ribosomal protein L1, has antimicrobial activity but is not cytotoxic to human erythrocytes. We synthesized several peptide analogues to investigate the effects of substitutions on structure and antimicrobial activity. Replacement of Gln16 and Asp18 with tryptophan [anal-3 (analogue-3)] caused a dramatic increase in lytic activities against bacteria and fungi. By contrast, a decrease in amphiphilicity caused by replacement of Phe5 or Leu11 with serine was accompanied by a reduction in antimicrobial activity. Analysis of the tertiary structures of the peptides in SDS micelles by NMR spectroscopy revealed that they have a well-defined α-helical structure. Among the analogues, anal-3 has the longest α-helix, from Val4 to Trp18. The enhanced hydrophobicity and increased α-helicity results in enhanced antimicrobial activity in anal-3 without an increase in haemolytic activity. Fluorescence experiments proved that the bacterial-cell selectivity of the anal-3 peptide is due to its high binding affinity for negatively charged phospholipids in bacterial cells. Results showing the effect of spin-labels on the NMR spectra indicated that the side chains in the hydrophobic phase of the amphiphilic α-helix are buried on the surface of the micelle and the tryptophan indole ring is anchored in the membrane surface. Because anal-3 shows high selectivity towards bacterial and fungal cells, it may provide an avenue for the development of new antibiotics.
Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts
Authors: Morty Rory E. , Bulau Patrick , Pellé Roger , Wilk Sherwin , Abe Koji
Published: March 2006

Peptidases of parasitic protozoans are emerging as novel virulence factors and therapeutic targets in parasitic infections. A trypanosome-derived aminopeptidase that exclusively hydrolysed substrates with Glp (pyroglutamic acid) in P1 was purified 9248-fold from the plasma of rats infected with Trypanosoma brucei brucei. The enzyme responsible was cloned from a T. brucei brucei genomic DNA library and identified as type I PGP (pyroglutamyl peptidase), belonging to the C15 family of cysteine peptidases. We showed that PGP is expressed in all life cycle stages of T. brucei brucei and is expressed in four other blood-stream-form African trypanosomes. Trypanosome PGP was optimally active and stable at bloodstream pH, and was insensitive to host plasma cysteine peptidase inhibitors. Native purified and recombinant hyper-expressed trypanosome PGP removed the N-terminal Glp blocking groups from TRH (thyrotrophin-releasing hormone) and GnRH (gonadotropin-releasing hormone) with a kcat/Km value of 0.5 and 0.1 s−1·μM−1 respectively. The half-life of TRH and GnRH was dramatically reduced in the plasma of trypanosome-infected rats, both in vitro and in vivo. Employing an activity-neutralizing anti-trypanosome PGP antibody, and pyroglutamyl diazomethyl ketone, a specific inhibitor of type I PGP, we demonstrated that trypanosome PGP is entirely responsible for the reduced plasma half-life of TRH, and partially responsible for the reduced plasma half-life of GnRH in a rodent model of African trypanosomiasis. The abnormal degradation of TRH and GnRH, and perhaps other neuropeptides N-terminally blocked with a pyroglutamyl moiety, by trypanosome PGP, may contribute to some of the endocrine lesions observed in African trypanosomiasis.
Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme
Authors: Müller Sandra , Disse Jennifer , Schöttler Manuela , Schön Sylvia , Prante Christian , Brinkmann Thomas , Kuhn Joachim , Kleesiek Knut , Götting Christian
Published: March 2006

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.
TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKγ and NF-κB activation
Authors: Bonif Marianne , Meuwis Marie-Alice , Close Pierre , Benoit Valérie , Heyninck Karen , Chapelle Jean-Paul , Bours Vincent , Merville Marie-Paule , Piette Jacques , Beyaert Rudi , Chariot Alain
Published: March 2006

Pro-inflammatory cytokines trigger signalling cascades leading to NF-κB (nuclear factor-κB)-dependent gene expression through IKK [IκB (inhibitory κB) kinase]-dependent phosphorylation and subsequent degradation of the IκB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-κB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKβ upon TNFα stimulation and that this modification negatively regulates TANK binding to NEMO (NF-κB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFα-mediated induction of a subset of NF-κB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFα by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKβ-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-κB activation.
Oxidative folding of hirudin in human serum
Authors: Chang Jui-Yoa , Lu Bao-Yun , Lai Por-Hsiung
Published: March 2006

Human serum contains factors that promote oxidative folding of disulphide proteins. We demonstrate this here using hirudin as a model. Hirudin is a leech-derived thrombin-specific inhibitor containing 65 amino acids and three disulphide bonds. Oxidative folding of hirudin in human serum is shown to involve an initial phase of rapid disulphide formation (oxidation) to form the scrambled isomers as intermediates. This is followed by the stage of slow disulphide shuffling of scrambled isomers to attain the native hirudin. The kinetics of regenerating the native hirudin depend on the concentrations of both hirudin and human serum. Quantitative regeneration of native hirudin in undiluted human serum can be completed within 48 h, without any redox supplement. These results cannot be adequately explained by the existing oxidized thiol agents in human serum or the macromolecular crowding effect, and therefore indicate that human serum may contain yet to be identified potent oxidase(s) for assisting protein folding.
Lipid phosphate phosphatase-1 regulates lysophosphatidic acid- and platelet-derived-growth-factor-induced cell migration
Authors: Long Jaclyn S. , Yokoyama Kazuaki , Tigyi Gabor , Pyne Nigel J. , Pyne Susan
Published: March 2006

LPPs (lipid phosphate phosphatases) are members of a family of enzymes that catalyse the dephosphorylation of lipid phosphates. The only known form of regulation of this family of enzymes is via de novo expression of LPP isoforms in response to growth factors. In this respect, we evaluated the effect of moderate increases in the expression of recombinant LPP1 on signal transduction by both G-protein-coupled receptors and receptor tyrosine kinases. We present evidence for a novel role of LPP1 in reducing PDGF (platelet-derived growth factor)- and lysophosphatidic acid-induced migration of embryonic fibroblasts. We demonstrate that the overexpression of LPP1 inhibits cell migration by reducing the PDGF-induced activation of p42/p44 MAPK (mitogen-activated protein kinase). This appears to occur via a mechanism that involves the LPP1-induced down-regulation of typical PKC (protein kinase C) isoform(s), which are normally required for PDGF-induced activation of p42/p44 MAPK and migration. In this regard, DAG (diacylglycerol) levels are high and sustained in cells overexpressing LPP1, suggesting a dynamic interconversion of phosphatidic acid into DAG by LPP1. This may account for the effects of LPP1 on cell migration, as sustained DAG is known to down-regulate PKC isoforms in cells. Therefore the physiological changes in the expression levels of LPP1 might represent a heterologous desensitization mechanism for attenuating PKC-mediated signalling and regulation of cell migration.
Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4α transcriptional activity
Authors: Guo Hongtao , Gao Chengjiang , Mi Zhiyong , Wai Philip Y. , Kuo Paul C.
Published: March 2006

In IL-1β (interleukin 1β)-stimulated rat hepatocytes exposed to superoxide, we have previously identified an IRX (inflammatory redox)-sensitive DR1 [direct repeat of RG(G/T)TCA with one base spacing] cis-acting activator element (nt –1327 to –1315) in the iNOS (inducible nitric oxide synthase) promoter: AGGTCAGGGGACA. The corresponding transcription factor was identified to be HNF4α (hepatocyte nuclear factor-4α). HNF4α DNA binding activity and transactivation potential are tightly regulated by its state of phosphorylation. However, the functional consequences of IRX-mediated post-translational phosphorylation of HNF4α have not been well characterized. In the setting of IL-1β+H2O2, HNF4α functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4α to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4α, serial mutations were performed to render the target residues phosphorylation-resistant. Electrophoretic mobility-shift assays and transient transfection studies utilizing the iNOS promoter showed that the S158A mutation ablates IRX-mediated HNF4α DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4α vector supports a critical role for Ser158 phosphorylation. In vitro phosphorylation and kinase inhibitor studies implicate p38 kinase activity. Our results indicate that p38 kinase-mediated Ser158 phosphorylation is essential for augmentation of the DNA binding and transactivation potential of HNF4α in the presence of IL-1β+H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.
Unexpected similarity between the cytosolic West Nile virus NS3 and the secretory furin-like serine proteinases
Authors: Seidah Nabil G.
Published: March 2006

Many viral proteins undergo proteolytic processing events that are required for virus infection and virion assembly. In this issue of Biochemical Journal, Strongin and co-workers report that the NS3 protease from West Nile virus unexpectedly cleaves certain substrates at pairs of basic residues, a specificity that resembles that of the furin-like PCs (proprotein convertases). This led to the demonstration that furin/PC inhibitors containing poly(D-arginine) are also potent inhibitors of NS3, and that anthrax toxin protective antigen and myelin basic protein are potential NS3 substrates. Structural modelling based on Dengue virus NS3 provided a possible rationale for the observed cleavage specificity of West Nile virus NS3.
Molecular dynamics study of charged dendrimers in salt-free solution: Effect of counterions
Authors: Gurtovenko Andrey A. , Lyulin Sergey V. , Karttunen Mikko , Vattulainen Ilpo
Published: March 2006

Polyamidoamine dendrimers, being protonated under physiological conditions, represent a promising class of nonviral, nanosized vectors for drug and gene delivery. We performed extensive molecular dynamics simulations of a generic model dendrimer in a salt-free solution with dendrimer’s terminal beads positively charged. Solvent molecules as well as counterions were explicitly included as interacting beads. We find that the size of the charged dendrimer depends nonmonotonically on the strength of electrostatic interactions demonstrating a maximum when the Bjerrum length equals the diameter of a bead. Many other structural and dynamic characteristics of charged dendrimers are also found to follow this pattern. We address such a behavior to the interplay between repulsive interactions of the charged terminal beads and their attractive interactions with oppositely charged counterions. The former favors swelling at small Bjerrum lengths and the latter promotes counterion condensation. Thus, counterions can have a dramatic effect on the structure and dynamics of charged dendrimers and, under certain conditions, cannot be treated implicitly.
Kinetic theory study of steady condensation of a polyatomic gas
Authors: Frezzotti Aldo , Ytrehus Tor
Published: March 2006

The steady one-dimensional flow of a monatomic vapor condensing onto a planar surface kept at constant and uniform temperature has been the subject of a number of investigations based on the kinetic theory of gases. It has been shown that, depending on the upstream value of the Mach number normal to the surface, a steady solution exists only when the problem parameters lie on a surface in the parameters spaces (upstream subsonic flow), or when the problem parameters lie in a proper subregion of the whole parameters space (upstream supersonic flow). Similar detailed studies do not exist for a polyatomic vapor, in spite of their potential relevance for many applications. The present paper aims at describing the effects of internal degrees of freedom on the relationships which determine the existence of steady one-dimensional condensation flows. The study is based on the numerical solution of the Boltzmann equation for a gas with rotational degrees of freedom. Inelastic collision are described by the Borgnakke-Larsen model. A few cases also have been computed by a finite difference discretization of Holway’s model kinetic equation. The role of boundary conditions is also briefly discussed.
An alternative method for determination of the lock-in angle in twinned superconductors
Authors: Keshavarzi Shokat , Horvat Joseph , Pan Alexey V. , Qin Meng Jun , Dou Shi Xue , Yao Xin , Munroe Paul
Published: March 2006

An alternative method for determining the lock-in angle φL for pinning of the vortices on extended defects has been developed. This method does not require any preassumed criterion for defining φL. Highly twinned Sm1+xBa2−xCu3O6+y single crystal was used for demonstrating the method. Appropriate scaling of the hysteresis loops measured for different angles between the field and twin planes in highly twinned SmBaCuO single crystal led to a clear discrimination between two vortex dynamics regimes. From this scaling, the lock-in angle was determined to be 6°±0.1° for the single crystal investigated. This method significantly reduces the uncertainty in determining the lock-in angle when compared to all the other currently employed methods.
Magnetic and transport properties of Sb2−xFexTe3 (0<x<0.02) single crystals
Authors: Zhou Zhenhua , Žabèík Marek , Lošták Petr , Uher Ctirad
Published: March 2006

Magnetization, electrical resistivity, and Hall coefficient of single crystals Sb2−xFexTe3 (0<x<0.02) have been measured from 2 to 300 K. Doping diamagnetic Sb2Te3 with Fe results in a paramagnetic behavior and the exchange interaction between Fe ions is determined to be antiferromagnetic based on low-temperature magnetization investigations. We detect no presence of a ferromagnetic ordering in Fe-doped Sb2Te3 down to 2 K. Fe doping initially increases the concentration of holes in relation to the case of pure Sb2Te3 but it rapidly saturates. The electrical resistivity increases throughout the temperature range studied on account of enhanced carrier scattering.
Nanoscale electron field emissions from the bare, hydrogenated, and graphitelike-layer-covered tetrahedral amorphous carbon films
Authors: Liu Dongping , Benstetter Günther , Frammelsberger Werner
Published: March 2006

We have compared nanoscale electron field emissions from the bare, hydrogenated, and graphitelike-layer-covered tetrahedral amorphous carbon (ta-C) films. The electron field emission is investigated using a combination of atomic force microscopy (AFM)-based nanowear tests and conducting AFM, by simultaneously measuring the topography and the conductivity of the samples. The analysis of Fowler-Nordheim tunneling currents indicates the formation of filamentlike emission channels within ta-C films. The low-field emission from carbon films is primarily due to a field enhancement arising from conducting nanostructures inside the films. The implications of surface structures for electron field emission are discussed. Electrons are easily delocalized within sp2-bonded rings/chains at a film surface, which leads to an increase in the nanotip emission area. At identical emission currents of 60–80 pA, hydrogenated films are much more easily destroyed due to a relatively insulating surface structure. The results suggest that a very high emission site density, required for field-emission display applications, may be produced by locally modifying a film surface layer.
Ablation of thin metal films by short-pulsed lasers coupled through near-field scanning optical microscopy probes
Authors: Hwang David Jen , Chimmalgi Anant , Grigoropoulos Costas P.
Published: March 2006

Short-pulsed lasers have been proven to be useful tools for precise modification of electronic materials. In conventional lens focusing schemes, the minimum feature size is determined by the diffraction limit. Finer resolution is accomplished by combining pulsed laser radiation with near-field scanning optical microscopy (NSOM) probes. In this study, short laser pulses are coupled to a fiber-based NSOM in order to ablate thin metal films. A detailed parametric study on the effects of probe aperture size, laser pulse energy, temporal width, and environment gas is performed. The significance of lateral thermal diffusion is highlighted and the dependence of the ablation process on the imparted near-field distribution is revealed.
Trap generation in cycled hot-electron-injection-programed/hot-hole-erased silicon-oxide-nitride-oxide-silicon memories
Authors: Daniel Ramiz , Ruzin Arie , Roizin Yakov
Published: March 2006

Generation of surface states and bulk traps in the bottom oxide of the silicon-oxide-nitride-oxide-silicon memory cells with thick bottom oxide programed by hot electron injection and erased by hot hole injection is investigated by using charge-pumping technique. It is shown that the degradation of the bottom oxide under the hot hole stress is the responsible mechanism for the shift of the programed state threshold voltage in retention bake. A physical model of the memory cell degradation is proposed that supposes trap generation by breaking the Si–H bonds in the vicinity of Si-bottom oxide interface and explains the observed trap generation kinetics.
Will crystallographic faces of a crystal keep their order in strength and friction coefficient when the contact force is reduced to nano/micro-Newton level?
Authors: Li D. Y. , Chen Q. , Wang X. Y.
Published: March 2006

Properties of a crystal are generally anisotropic, which makes different crystallographic planes behave differently. By choosing a suitable crystallographic plane or textured polycrystalline surface, one may obtain optimum mechanical and tribological properties, e.g., the maximum strength and desired friction coefficient. Up to date, we have had sufficient knowledge about the relationship between mechanical properties and the crystallographic orientation for different crystal systems. However, when the contact force is decreased to nano/micro-Newton level, will the crystallographic faces of a crystal keep their order in strength and other properties? This article reports our recent studies on this issue using copper as a sample material and demonstrates that there are transitions in hardness and friction coefficient between different crystallographic planes of Cu. It has been demonstrated that the closely packed plane (111) is harder and has a smaller friction coefficient than the (001) plane; however, the situation is reversed when the load is reduced to nano/micro-Newton level that only results in distortion of a few atomic layers. Such changes are of particular importance to the application of crystalline materials in nanomachines or nanodevices.
Exchange bias-like phenomenon in SrRuO3
Authors: Pi Li , Zhang Shixiong , Tan Shun , Zhang Yuheng
Published: March 2006

Exchange bias-like phenomenon is observed in the SrRuO3 polycrystalline. After the sample is cooled down to 4K in a magnetic field, the magnetic hysteresis loop shifts in the opposite direction to the applied biasing field. The shift decreases as the sweeping range of the measuring field increases. This phenomenon may arise from the spin glass property of SrRuO3. Since SrRuO3 is widely used in spintronics devices, the observation of exchange bias-like shift in SrRuO3 itself suggests that the application of SrRuO3 should be more carefully considered.
Effect of nitrogen ion bombardment on defect formation and luminescence efficiency of GaNP epilayers grown by molecular-beam epitaxy
Authors: Dagnelund D. , Buyanova I. A. , Mchedlidze T. , Chen W. M. , Utsumi A. , Furukawa Y. , Wakahara A , Yonezu H.
Published: March 2006

Radiative efficiency of GaNP epilayers grown on GaP substrates by solid-source molecular beam epitaxy is significantly improved by reduced nitrogen ion bombardment during the growth. Based on the results of temperature-dependent photoluminescence (PL) and optically detected magnetic resonance studies (ODMR), the observed improvements are attributed to reduced formation of defects, such as a Ga interstitial related defect and an unidentified defect revealed by ODMR. We demonstrate that these defects act as competing recombination centers, which promote thermal quenching of the PL intensity and result in a substantial (34×) decrease in room-temperature PL intensity.
Highly efficient white-light-emitting diodes fabricated with short-wavelength yellow oxynitride phosphors
Authors: Xie Rong-Jun , Hirosaki Naoto , Mitomo Mamoru , Takahashi Kosei , Sakuma Ken
Published: March 2006

We have already reported orangish yellow Ca–α-SiAlON:Eu2+ phosphors, and applied them to fabricate warm white light-emitting diodes (LEDs). In this letter, we report on greenish yellow Li–α-SiAlON:Eu2+ phosphors, and use them to create daylight when coupled to an InGaN blue LED chip (460nm). The newly discovered Li–α-SiAlON:Eu2+ phosphors emit at shorter wavelengths of 573–577nm under the 460nm excitation, and exhibit a smaller Stokes shift than Ca–α-SiAlON:Eu2+ does. By using this short-wavelength yellow oxynitride phosphor, bright daylight emissions from white LEDs can be generated. Thus, highly efficient white LEDs with tunable white light can be fabricated with α-SiAlON:Eu2+ phosphors, enabling them for a wider range of applications.
Raman study of lattice dynamic behaviors in phosphorus-doped ZnO films
Authors: Ye J. D. , Gu S. L. , Zhu S. M. , Liu S. M. , Zheng Y. D. , Zhang R. , Shi Y. , Chen Q. , Yu H. Q. , Ye Y. D.
Published: March 2006

Phosphorus-induced lattice dynamic behaviors in ZnO:P epilayers grown by the metalorganic chemical vapor deposition technique have been studied using the Raman scattering method. Additional modes around 504, 520, 655, and 866cm−1 are attributed to the disorder-activated modes due to the breakdown of translational symmetry by P doping, well supported by the reported ab initio calculations of lattice dynamics in w-ZnO. Two modes around 364 and 478cm−1 are assigned to the local vibrational modes of Zn–P and P–O pairs, respectively. The correlation of transport and vibrational properties demonstrates the complex doping mechanism and the amphoteric nature of P dopant in ZnO. In addition, the redshift of 2 longitudinal optical multiphonon around 1154cm−1 is possibly originated from the variation of short-range forces in ZnO uniaxial lattice caused by P incorporation.
Dynamics of the energy flow through photosystem II under changing light conditions: a model approach
Authors: Porcar-Castell Albert , Bäck Jaana , Juurola Eija , Hari Pertti
Published: March 2006

Several biochemical models of photosynthesis exist that consider the effects of the dynamic adjustment of enzymatic and stomatal processes on carbon assimilation under fluctuating light. However, the rate of electron transport through the light reactions is commonly modelled by means of an empirical equation, parameterised with data obtained at the steady state. A steady-state approach cannot capture the dynamic nature of the adjustment of the light reactions under fluctuating light. Here we present a dynamic model approach for photosystem II that considers the adjustments in the regulative non-photochemical processes. The model is initially derived to account for changes occurring at the seconds-to-minutes time-scale under field conditions, and is parameterised and tested with chlorophyll fluorescence data. Results derived from this model show good agreement with experimentally obtained photochemical and non-photochemical quantum yields, providing evidence for the effect that the dark reactions exert in the adjustment of the energy flows at the light reactions. Finally, we compare the traditional steady-state approach with our dynamic approach and find that the steady-state approach produces an underestimation of the modelled electron transport rate (ETR) under rapidly fluctuating light (1 s or less), whereas it produces overestimations under slower fluctuations of light (5 s or more).