bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1 6)-galactanase gene
Authors: KOTAKE Toshihisa , KANEKO Satoshi , KUBOMOTO Aya , HAQUE Md. Ashraful , KOBAYASHI Hideyuki , TSUMURAYA Yoichi
Published: November 2007

A gene encoding endo-β-(1→6)-galactanase from Trichoderma viride was cloned by reverse transcriptase–PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His6 tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed β-(1→6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse α-l-arabinofuranosidase-treated arabinogalactan protein from radish. It produced β-(1→6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and β-(1→6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-β-(1→6)-galactanase. As far as we know, this is the first time an endo-β-(1→6)-galactanase has been cloned.
A common mechanism for the regulation of vesicular SNAREs on phospholipid membranes
Authors: HU Kuang , RICKMAN Colin , CARROLL Joe , DAVLETOV Bazbek
Published: November 2007

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) family of proteins is essential for membrane fusion in intracellular traffic in eukaryotic organisms. v-SNAREs (vesicular SNAREs) must engage target SNAREs in the opposing membrane to form the fusogenic SNARE complex. Temporal and spatial control of membrane fusion is important for many aspects of cell physiology and may involve the regulation of the SNAREs resident on intracellular membranes. Here we show that the v-SNARE synaptobrevin 2, also known as VAMP (vesicle-associated membrane protein) 2, is restricted from forming the SNARE complex in chromaffin granules from adrenal medullae to the same degree as in brain-purified synaptic vesicles. Our analysis indicates that the previously reported synaptophysin–synaptobrevin interaction is not likely to be involved in regulation of the v-SNARE. Indeed, the restriction can be reproduced for two distinct v-SNARE homologues, synaptobrevin 2 and cellubrevin/VAMP3, by reconstituting them in pure liposomal membranes. Overall, our data uncover a common mechanism for the control of SNARE engagement where intact phospholipid membranes rather than proteins down-regulate vesicular SNAREs in different cellular organelles.
Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate
Authors: ITO Mikiko , IIDAWA Sachi , IZUKA Michiyo , HAITO Sakiko , SEGAWA Hiroko , KUWAHATA Masashi , OHKIDO Ichiro , OHNO Hiroshi , MIYAMOTO Ken-ichi
Published: November 2007

Treatment with PTH (parathyroid hormone) or a high-Pi diet causes internalization of the type IIa sodium-dependent phosphate (Na/Pi IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/Pi IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/Pi IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/Pi IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/Pi IIa co-transporter.
MSKs are required for the transcription of the nuclear orphan receptors Nur77, Nurr1 and Nor1 downstream of MAPK signalling
Authors: Darragh Joanne , Soloaga Ana , Beardmore Victoria A. , Wingate Andrew D. , Wiggin Giselle R. , Peggie Mark , Arthur J. Simon C.
Published: November 2007

MSK (mitogen- and stress-activated protein kinase) 1 and MSK2 are kinases activated downstream of either the ERK (extracellular-signal-regulated kinase) 1/2 or p38 MAPK (mitogen-activated protein kinase) pathways in vivo and are required for the phosphorylation of CREB (cAMP response element-binding protein) and histone H3. Here we show that the MSKs are involved in regulating the transcription of the immediate early gene Nur77. Stimulation of mouse embryonic fibroblasts with PMA, EGF (epidermal growth factor), TNF (tumour necrosis factor) or anisomycin resulted in induction of the Nur77 mRNA. The induction of Nur77 by TNF and anisomycin was abolished in MSK1/2 double-knockout cells, whereas induction was significantly reduced in response to PMA or EGF. The MSK responsive elements were mapped to two AP (activator protein)-1-like elements in the Nur77 promoter. The induction of Nur77 was also blocked by A-CREB, suggesting that MSKs control Nur77 transcription by phosphorylating CREB bound to the two AP-1-like elements. Consistent with the decrease in Nur77 mRNA levels in the MSK1/2-knockout cells, it was also found that MSKs were required for the induction of Nur77 protein by PMA and TNF. MSKs were also found to be required for the transcription of two genes related to Nur77, Nurr1 and Nor1, which were also transcribed in a CREB- or ATF1 (activating transcription factor-1)-dependent manner. Downstream of anisomycin signalling, a second ERK-dependent pathway, independent of MSK and CREB, was also required for the transcription of Nurr1 and Nor1.
Conjugation of fatty acids with different lengths modulates the antibacterial and antifungal activity of a cationic biologically inactive peptide
Authors: Malina Amir , Shai Yechiel
Published: November 2007

Many studies have shown that an amphipathic structure and a threshold of hydrophobicity of the peptidic chain are crucial for the biological function of AMPs (antimicrobial peptides). However, the factors that dictate their cell selectivity are not yet clear. In the present study, we show that the attachment of aliphatic acids with different lengths (10, 12, 14 or 16 carbon atoms) to the N-terminus of a biologically inactive cationic peptide is sufficient to endow the resulting lipopeptides with lytic activity against different cells. Mode-of-action studies were performed with model phospholipid membranes mimicking those of bacterial, mammalian and fungal cells. These include determination of the structure in solution and membranes by using CD and ATR-FTIR (attenuated total reflectance Fourier-transform infrared) spectroscopy, membrane leakage experiments and by visualizing bacterial and fungal damage via transmission electron microscopy. The results obtained reveal that: (i) the short lipopeptides (10 and 12 carbons atoms) are non-haemolytic, active towards both bacteria and fungi and monomeric in solution. (ii) The long lipopeptides (14 and 16 carbons atoms) are highly antifungal, haemolytic only at concentrations above their MIC (minimal inhibitory concentration) values and aggregate in solution. (iii) All the lipopeptides adopt a partial α-helical structure in 1% lysophosphatidylcholine and bacterial and mammalian model membranes. However, the two short lipopeptides contain a significant fraction of random coil in fungal membranes, in agreement with their reduced antifungal activity. (iv) All the lipopeptides have a membranolytic effect on all types of cells assayed. Overall, the results reveal that the length of the aliphatic chain is sufficient to control the pathogen specificity of the lipopeptides, most probably by controlling both the overall hydrophobicity and the oligomeric state of the lipopeptides in solution. Besides providing us with basic important information, these new lipopeptides are potential candidates that can target bacteria and/or fungi, especially in cases where the bacterial flora should not be harmed.
Binding and repression of translation of the cognate mRNA by Trichinella spiralis thymidylate synthase differ from the corresponding interactions of the human enzyme
Authors: Cieśla Joanna , Jagielska Elżbieta , Skopiński Tomasz , Dąbrowska Magdalena , Maley Frank , Rode Wojciech
Published: November 2007

Thymidylate synthase (TS) of Trichinella spiralis, a parasitic nematode causing trichinellosis, was found to bind its own mRNA and repress translation of the latter, similar to its human counter-part [Chu, Koeller, Casey, Drake, Chabner, Elwood, Zinn and Allegra (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8977–8981]. However, in striking contrast with human TS, the parasite enzyme's interaction with mRNA was not affected by any of the substrate (deoxyuridylate or N5,10-methylenetetrahydrofolate) nor by the inhibitor (fluorodeoxyuridylate; used alone or in the presence of N5,10-methylenetetrahydrofolate) similar to that shown for the bifunctional enzyme from Plasmodium falciparum [Zhang and Rathod (2002) Science 296, 545–547]. Moreover, repression of the translation of the parasite enzyme was enhanced by the same ligands that were shown by others (Chu et al., 1991) to prevent human TS from impairing its translation. On comparing the capacity of TS to bind to its cognate mRNA, relative to its ability to inhibit its translation, the same enzyme preparation was active as translational repressor at a considerably lower protein/mRNA ratio, suggesting the two phenomena to be disconnected. Of interest is the fact that the presence of the enzyme protein N-terminal methionine proved to be critical for binding, but not for repression of its translation, indicating that mRNA binding requires a methionine or an adduct (i.e. methionine–histidine) at the N-terminus of TS, but that the translational repression effect does not. Notably, chicken liver dihydrofolate reductase, which is incapable of binding to T. spiralis TS mRNA, repressed the translation of TS.
Functional expression of a low-affinity zinc uptake transporter (FrZIP2) from pufferfish (Takifugu rubripes) in MDCK cells
Authors: Qiu Andong , Hogstrand Christer
Published: November 2007

Zinc is a vital micronutrient to all organisms and it is therefore very important to determine the mechanisms that regulate cellular zinc uptake. Previously, we reported on zinc uptake transporters from zebrafish (Danio rerio; DrZIP1) and Fugu pufferfish (Takifugu rubripes; FrZIP1) that facilitated cellular zinc uptake of high affinity (Km<0.5 μM) in both CHSE214 [chinook salmon (Oncorhynchus tshawytscha) embryonic 214] cells and Xenopus laevis oocytes. To investigate additional biochemical pathways of zinc uptake in fish, we molecularly cloned the second fish member (FrZIP2) of the SLC39 subfamily II from Fugu pufferfish gill. Functional characterization suggests that FrZIP2 stimulated zinc uptake in a temperature-, time-, concentration- and pH-dependent manner when overexpressed in MDCK cells (Madin–Darby canine kidney cells). In comparison with FrZIP1 and DrZIP1 (<0.5 μM), FrZIP2 appears to represent a low-affinity zinc uptake transporter (Km=13.6 μM) in pufferfish. FrZIP2 protein was selective for zinc, but it might also transport Cu2+, since 20 times excess of Cu2+ completely abolished its zinc uptake activity. The zinc uptake by FrZIP2 was stimulated in a slightly acidic medium (pH 5.5–6.5) and was completely blocked at pH 7.5 and above, suggesting that an inward H+ gradient might provide a driving force for zinc transport by FrZIP2. Furthermore, FrZIP2-mediated zinc uptake activity was slightly inhibited by 0.5 mM HCO3−, indicating that FrZIP2 may employ a different mechanism of zinc translocation from the assumed HCO3−-coupled zinc transport used by human SLC39A2. The FrZIP2 gene was expressed in all the tissues studied herein, with especially high levels in the ovary and intestines. Thus FrZIP2 may be a prominent zinc uptake transporter of low affinity in many cell types of Fugu pufferfish.
Analysis of COX2 mutants reveals cytochrome oxidase subassemblies in yeast
Authors: Horan Susannah , Bourges Ingrid , Taanman Jan-Willem , Meunier Brigitte
Published: November 2007

Cytochrome oxidase catalyses the reduction of oxygen to water. The mitochondrial enzyme contains up to 13 subunits, 11 in yeast, of which three, Cox1p, Cox2p and Cox3p, are mitochondrially encoded. The assembly pathway of this complex is still poorly understood. Its study in yeast has been so far impeded by the rapid turnover of unassembled subunits of the enzyme. In the present study, immunoblot analysis of blue native gels of yeast wild-type and Cox2p mutants revealed five cytochrome oxidase complexes or subcomplexes: a, b, c, d and f; a is likely to be the fully assembled enzyme; b lacks Cox6ap; d contains Cox7p and/or Cox7ap; f represents unassembled Cox1p; and c, observed only in the Cox2p mutants, contains Cox1p, Cox3p, Cox5p and Cox6p and lacks the other subunits. The identification of these novel cytochrome oxidase subcomplexes should encourage the reexamination of other yeast mutants.
High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse
Authors: Hong Jie , Qian Zhikang , Shen Shuiyuan , Min Taishan , Tan Chang , Xu JianFeng , Zhao Yingchun , Huang Weida
Published: November 2007

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.
PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner
Authors: Landsverk Helga B. , Kirkhus Marie , Bollen Mathieu , Küntziger Thomas , Collas Philippe
Published: November 2007

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309–691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309–691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401→Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.
Cytochrome P450/NADPH-dependent biosynthesis of 5,6-trans-epoxyeicosatrienoic acid from 5,6-trans-arachidonic acid
Authors: Roy Uzzal , Joshua Robert , Stark Russell L. , Balazy Michael
Published: November 2007

5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis–trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (ω−1)/(ω−2) hydroxylations. We found that the C5–C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5–C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.
Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus
Authors: Sheldrick E. Linda R. , Derecka Kamila , Marshall Elaine , Chin Evonne C. , Hodges Louise , Wathes D. Claire , Abayasekara D. Robert E. , Flint Anthony P. F.
Published: November 2007

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARα and PPARδ (also known as PPARβ) (but not PPARγ). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARα (but not PPARδ or PPARγ) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4β-PMA and PGF2α, and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4β-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4β-PMA in the absence of a PPAR ligand was decreased by the NF-κB (nuclear factor κB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-κB in addition to PPAR phosphorylation. Use of NF-κB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARα to increase PTGS2 levels in bovine endometrial stromal cells.
Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm
Authors: Ono Nobuhito , Van der Heijden Ingrid , Scheffer George L. , Van de Wetering Koen , Van Deemter Elizabeth , De Haas Marcel , Boerke Arjan , Gadella Bart M. , De Rooij Dirk G. , Neefjes Jacques J. , Groothuis Tom A. M. , Oomen Lauran , Brocks Lenny , Ishikawa Toshihisa , Borst Piet
Published: November 2007

The human and murine genes for MRP9 (multidrug resistance-associated protein 9; ABCC12) yield many alternatively spliced RNAs. Using a panel of monoclonal antibodies, we detected full-length Mrp9 only in testicular germ cells and mouse sperm; we obtained no evidence for the existence of the truncated 100 kDa MRP9 protein reported previously. In contrast with other MRPs, neither murine Mrp9 nor the human MRP9 produced in MRP9-transfected HEK-293 cells (human embryonic kidney cells) appears to contain N-linked carbohydrates. In mouse and boar sperm, Mrp9 localizes to the midpiece, a structure containing all sperm mitochondria. However, immunolocalization microscopy and cell fractionation studies with transfected HEK-293 cells and mouse testis show that MRP9/Mrp9 does not localize to mitochondria. In HEK-293 cells, it is predominantly localized in the endoplasmic reticulum. We have been unable to demonstrate transport by MRP9 of substrates transported by other MRPs, such as drug conjugates and other organic anions.
The crystal structure of the pentahaem c-type cytochrome NrfB and characterization of its solution-state interaction with the pentahaem nitrite reductase NrfA
Authors: Clarke Thomas A. , Cole Jeffrey A. , Richardson David J. , Hemmings Andrew M.
Published: November 2007

NrfB is a small pentahaem electron-transfer protein widely involved in the respiratory reduction of nitrite or nitric oxide to ammonia, processes that provide energy for anaerobic metabolism in many enteric bacteria and also serve to detoxify these reactive nitrogen species. The X-ray crystal structure of Escherichia coli NrfB is presented at 1.74 Å (1 Å=0.1 nm) resolution. The architecture of the protein is that of a 40 Å ‘nanowire’ in which the five haems are positioned within 6 Å of each other along a polypeptide scaffold. During nitrite reduction, the physiological role of NrfB is to mediate electron transfer to another pentahaem protein, NrfA, the enzyme that catalyses periplasmic nitrite or nitric oxide reduction. Protein–protein interaction studies suggest NrfA and NrfB can form a 20-haem NrfA2–NrfB2 heterotetrameric complex.
Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates
Authors: Wiktelius Eric , Stenberg Gun
Published: November 2007

In the present paper, we report a novel class of GSTs (glutathione transferases), called the Chi class, originating from cyanobacteria and with properties not observed previously in prokaryotic enzymes. GSTs constitute a widespread multifunctional group of proteins, of which mammalian enzymes are the best characterized. Although GSTs have their origin in prokaryotes, few bacterial representatives have been characterized in detail, and the catalytic activities and substrate specificities observed have generally been very modest. The few well-studied bacterial GSTs have largely unknown physiological functions. Genome databases reveal that cyanobacteria have an extensive arsenal of glutathione-associated proteins. We have studied two cyanobacterial GSTs which are the first examples of bacterial enzymes that are as catalytically efficient as the best mammalian enzymes. GSTs from the thermophile Thermosynechococcus elongatus BP-1 and from Synechococcus elongatus PCC 6301 were found to catalyse the conjugation of naturally occurring plant-derived isothiocyanates to glutathione at high rates. The cyanobacterial GSTs studied are smaller than previously described members of this enzyme family, but display many of the typical structural features that are characteristics of GSTs. They are also active towards several classical substrates, but at the same moderate rates that have been observed for other GSTs derived from prokaryotes. The cloning, expression and characterization of two cyanobacterial GSTs are described. The possible significance of the observed catalytic properties is discussed in the context of physiological relevance and GST evolution.
Inhibition of MMP-2 gelatinolysis by targeting exodomain–substrate interactions
Authors: Xu Xiaoping , Chen Zhihua , Wang Yao , Bonewald Lynda , Steffensen Bjorn
Published: November 2007

MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715–721 in human α1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2E404A, a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2E404A. In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2ΔCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited ∼90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2ΔCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.
MptpB, a virulence factor from Mycobacterium tuberculosis, exhibits triple-specificity phosphatase activity
Authors: Beresford Nicola , Patel Sumayya , Armstrong Jane , Szöor Balázs , Fordham-Skelton Anthony P. , Tabernero Lydia
Published: November 2007

Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity.
Blood haemoglobin is an independent predictor of B-type natriuretic peptide (BNP)
Authors: WOLD KNUDSEN Cathrine , VIK-MO Harald , OMLAND Torbjørn
Published: November 2007

BNP (B-type natriuretic peptide) and anaemia are both associated with adverse outcome in patients with chronic heart failure. Whether low haemoglobin levels are independently predictive of elevated BNP levels in subjects without heart failure is unknown. In the present study, we examined the relationship between haemoglobin and BNP levels in 234 patients with suspected coronary heart disease without a history of chronic heart failure, adjusting for known predictors of BNP levels. By univariate analysis, haemoglobin levels were inversely related to logarithmically transformed BNP values (r=−0.30, P<0.0001). After adjustment for patient age, gender, body mass index, history of myocardial infarction, use of diuretics, angiotensin-converting enzyme inhibitors and β-blockers, estimated creatinine clearance rate, extent of coronary disease, left ventricular ejection fraction and left ventricular end-diastolic pressure, blood haemoglobin remained an independent predictor of plasma BNP (standardized β-coefficient=−0.253, P<0.0001). A similar relationship was observed between haematocrit and BNP (standardized β-coefficient −0.215, P<0.0001). We conclude that haemoglobin levels are independently predictive of plasma BNP levels in patients with suspected coronary heart disease without heart failure. Anaemia may contribute to elevated BNP levels in the absence of heart failure, and may represent an important confounder of the relationship between BNP, cardiac function and prognosis.
Methylglyoxal administration induces diabetes-like microvascular changes and perturbs the healing process of cutaneous wounds
Authors: BERLANGA Jorge , CIBRIAN Danay , GUILLÉN Isabel , FREYRE Freya , ALBA José S. , LOPEZ-SAURA Pedro , MERINO Nelson , ALDAMA Alfredo , QUINTELA Ana M. , TRIANA Maria E. , MONTEQUIN Jose F. , AJAMIEH Hussam , URQUIZA Dioslaida , AHMED Naila , THORNALLEY Paul J.
Published: November 2007

Increased formation of MG (methylglyoxal) and related protein glycation in diabetes has been linked to the development of diabetic vascular complications. Diabetes is also associated with impaired wound healing. In the present study, we investigated if prolonged exposure of rats to MG (50–75 mg/kg of body weight) induced impairment of wound healing and diabetes-like vascular damage. MG treatment arrested growth, increased serum creatinine, induced hypercholesterolaemia (all P<0.05) and impaired vasodilation (P<0.01) compared with saline controls. Degenerative changes in cutaneous microvessels with loss of endothelial cells, basement membrane thickening and luminal occlusion were also detected. Acute granulation appeared immature (P<0.01) and was associated with an impaired infiltration of regenerative cells with reduced proliferative rates (P<0.01). Immunohistochemical staining indicated the presence of AGEs (advanced glycation end-products) in vascular structures, cutaneous tissue and peripheral nerve fibres. Expression of RAGE (receptor for AGEs) appeared to be increased in the cutaneous vasculature. There were also pro-inflammatory and profibrotic responses, including increased IL-1β (interleukin-1β) expression in intact epidermis, TNF-α (tumour necrosis factor-α) in regions of angiogenesis, CTGF (connective tissue growth factor) in medial layers of arteries, and TGF-β (transforming growth factor-β) in glomerular tufts, tubular epithelial cells and interstitial endothelial cells. We conclude that exposure to increased MG in vivo is associated with the onset of microvascular damage and other diabetes-like complications within a normoglycaemic context.
Size of myocardial infarction induced by ischaemia/reperfusion is unaltered in rats with metabolic syndrome
Authors: Thim Troels , Bentzon Jacob F. , Kristiansen Steen B. , Simonsen Ulf , Andersen Heidi L. , Wassermann Karsten , Falk Erling
Published: November 2007

Obesity is associated with metabolic syndrome and increased incidence of and mortality from myocardial infarction. The aim of the present study was to develop an animal model with metabolic syndrome and examine how that influences size of myocardial infarcts induced by occlusion and reperfusion of the left anterior descending coronary artery. Sprague–Dawley rats (n=105) were fed either LF (low-fat) or MHF (moderately high-fat) diets for 13 weeks before coronary occlusion for 45 min, followed by reperfusion for 60 min. Compared with LF-fed and lean MHF-fed rats, obese MHF-fed rats developed metabolic disturbances similar to those seen in the metabolic syndrome, including being overweight by 24% (compared with lean MHF-fed rats), having 74% more visceral fat (compared with LF-fed rats), 15% higher blood pressure (compared with LF-fed rats), 116% higher plasma insulin (compared with lean MHF-fed rats), 10% higher fasting plasma glucose (compared with LF-fed rats), 35% higher non-fasting plasma glucose (compared with lean MHF-fed rats), 36% higher plasma leptin (compared with lean MHF-fed rats) and a tendency to lower plasma adiponectin and higher plasma non-esterified fatty acids. Infarct size was similar in the three groups of rats (36±14, 42±18 and 41±14% in obese MHF-fed, lean MHF-fed and LF-fed rats respectively). In conclusion, rats fed a MHF diet developed metabolic syndrome, but this did not influence myocardial infarct size.
Alveolar recruitment during prone position: time matters
Authors: Reutershan Jörg , Schmitt Andre , Dietz Klaus , Unertl Klaus , Fretschner Reinhold
Published: November 2007

Alveolar recruitment is one of the beneficial effects of prone positioning in patients with ARDS (acute respiratory distress syndrome). However, responses vary among patients and, therefore, we hypothesized that alveolar recruitment is an individual time-dependent process and its measurement might be helpful to ‘dose’ prone positioning individually. In 13 patients diagnosed with ARDS, EELV (end-expiratory lung volume) was measured in the supine position, immediately after turning to the prone position, at 1, 2, 4 and 8 h in the prone position and after returning to the supine position. Responders were defined based on a 30% increase in oxygenation. EELV increased in responders, whereas it remained constant in non-responders. The time course was different in individual patients. In some responders, a plateau was reached as early as 2–4 h, whereas, in others, 8 h of prone positioning was not sufficient to allow complete recruitment. The increase in lung volume was associated with both an increase in arterial oxygenation and a decrease in venous admixture. Furthermore, responders had significantly lower baseline EELVs than non-responders. In conclusion, alveolar recruitment during prone positioning has been characterized as an individual time-dependent process. Its measurement might be useful to apply prone positioning more individually and might also help to identify responders.
Stringent test of the statistical quasiclassical trajectory model for the H3+ exchange reaction: A comparison with rigorous statistical quantum mechanical results
Authors: Aoiz F. J. , González-Lezana T. , Sáez Rábanos V.
Published: November 2007

A complete formulation of a statistical quasiclassical trajectory (SQCT) model is presented in this work along with a detailed comparison with results obtained with the statistical quantum mechanical (SQM) model for the H++D2 and H++H2 reactions. The basic difference between the SQCT and the SQM models lies in the fact that trajectories instead of wave functions are propagated in the entrance and exit channels. Other than this the two formulations are entirely similar and both comply with the principle of detailed balance and conservation of parity. Reaction probabilities, and integral and differential cross sections (DCS’s) for these reactions at different levels of product’s state resolution and from various initial states are shown and discussed. The agreement is in most cases excellent and indicates that the effect of tunneling through the centrifugal barrier is negligible. Some differences are found, however, between state resolved observables calculated by the SQCT and the SQM methods which makes use of the centrifugal sudden (coupled states) approximation (SQM-CS). When this approximation is removed and the full close coupling treatment is used in the SQM model (SQM-CC), an almost perfect agreement is achieved. This shows that the SQCT is sensitive enough to show the relatively small inaccuracies resulting from the decoupling inherent to the CS approximation. In addition, the effect of ignoring the parity conservation is thoroughly examined. This effect is in general minor except in particular cases such as the DCS from initial rotational state j=0. It is shown, however, that in order to reproduce the sharp forward and backward peaks the conservation of parity has to be taken into account.
Correlated double-proton transfer. I. Theory
Authors: Smedarchina Zorka , Siebrand Willem , Fernández-Ramos Antonio
Published: November 2007

The dynamics of double-proton transfer reactions is studied on a model of transfer along two identical hydrogen bonds represented by quartic double-minimum potentials. Correlation between the proton motions is introduced by a coupling term that is bilinear in the two proton coordinates; it is shown that this form properly accounts for the polarity and symmetry of the interaction and correctly reproduces the observed transfer behavior in the strong- and weak-coupling limits. The model allows a universal description of double-proton transfer mechanisms in symmetric systems in terms of the variation of a single parameter, the (dimensionless) coupling between the two hydrogen bonds. The corresponding two-dimensional (2D) transfer potential has up to nine stationary points, depending on the coupling strength. The resulting dynamics and its dependence on temperature and isotopic substitution are studied analytically by instanton techniques for the full range of the correlation parameter whereby the potential has multiple saddle points. For any coupling, the dynamics at high temperatures is dominated by classical transitions over the saddle point of lowest barrier. Strong coupling leads exclusively to synchronous transfer along a single collective coordinate, weak coupling to competition between this synchronous transfer, and stepwise transfer along local coordinates, the relative contributions of these mechanisms being governed by the temperature. Below a certain crossover temperature, transfer dynamics is dominated by the instanton, i.e., the trajectory with maximum tunneling probability. Two types of instanton are found on the 2D potential. The well-known one-dimensional instanton, corresponding to synchronous motion, exists for any coupling. It dominates at low temperatures and is responsible for any observed tunneling splittings, independent of the number of saddle points of the symmetric potential. An alternative 2D instanton, corresponding to asynchronous motion, exists for weak coupling. It is shown that under conditions where 2D tunneling dominates, it is much slower than stepwise transfer. Therefore 2D tunneling trajectories do not contribute significantly to the rate of transfer and can be ignored. The favorable quantitative aspects of the model are illustrated by an application to double-proton rate constants in porphine, which have been measured in a wide range of temperatures.
Estimating intermittency exponent in neutrally stratified atmospheric surface layer flows: A robust framework based on magnitude cumulant and surrogate analyses
Authors: Basu Sukanta , Foufoula-Georgiou Efi , Lashermes Bruno , Arnéodo Alain
Published: November 2007

This study proposes a novel framework based on magnitude cumulant and surrogate analyses to reliably detect the presence of intermittency and estimate the intermittency coefficient from short-length coarse-resolution turbulent time series. Intermittency coefficients estimated from a large number of neutrally stratified atmospheric surface layer turbulent series from various field campaigns are shown to remarkably concur with well-known laboratory experimental results. In addition, surrogate-based hypothesis testing significantly reduces the likelihood of detecting a spurious nonzero intermittency coefficient from nonintermittent series. The discriminatory power of the proposed framework is promising for addressing the unresolved question of how atmospheric stability affects the intermittency properties of boundary layer turbulence.
Cationic and anionic vacancies on the NiO(100) surface: DFT+U and hybrid functional density functional theory calculations
Authors: Ferrari Anna Maria , Pisani Cesare , Cinquini Fabrizio , Giordano Livia , Pacchioni Gianfranco
Published: November 2007

The electronic structure of oxygen and nickel vacancies at the surface of NiO(100) has been investigated theoretically by means of density functional theory (DFT) comparing plane wave density functional theory with the Hubbard correction DFT+U with atomic-orbital-hybrid-functional (20% exact-exchange) calculations. The two approaches provide a similar description of the nature of the oxygen vacancy. At variance with the same defect center in MgO, where two electrons are trapped in the vacancy, on NiO the charge is more delocalized, partly over the Ni ions around the vacancy, the rest either trapped in the vacancy or delocalized over other Ni ions. Concerning the nickel vacancy, both methods clearly show that the removal of a neutral Ni atom does not result in the oxidation of other nickel ions from Ni2+ to Ni3+ but rather in the formation of two holes in the O 2p valence band. However, the description is slightly different with the approaches, the hybrid functional indicates that the holes are essentially localized on the oxygen ions nearest to the vacancy, while they result much more delocalized from the DFT+U calculation. Comparison with the corresponding results, obtained with the two methods for the case of the Mg vacancy in MgO, suggests that the DFT+U approach does not adequately correct for the self-interaction of the unpaired electrons in this case. However, the overall picture that emerges clearly from the present calculations is that both defects affect the electronic structure in a much wider region in NiO than in MgO.
Impinging axisymmetric turbulent fountains
Authors: Cooper Paul , Hunt Gary R.
Published: November 2007

The behavior of a turbulent fountain that impinges perpendicularly on a horizontal plate a distance H from the fountain origin in a quiescent environment of uniform density is investigated. Images from light induced fluorescence visualization of the flow provide a clear picture of the internal structure of the vertical fountain, impinging radial outflow and the subsequent plumelike flow. For source-plate separations H∕Lm0⩽1.5, where Lm0 is the source jet length, and source Froude number Fr0>10, the impingement of the fountain results in a buoyant radial wall jet that spreads horizontally before separating from the plate at a radius Rsp. The re-entrainment into the fountain and wall jet of buoyant fluid released on separation leads to the formation of a toroidal vortex of outer radius ∼Rsp. The correlation Rsp∕H=0.03Rirad−0.75, deduced from theoretical scalings and the results of our experiments, successfully relates the maximum nondimensional radial spread to the Richardson number Rirad that characterizes the source of the buoyant radial wall jet. The transient development of the flow and influence of the buoyant plume above the impingement zone are discussed.
Rotating magnetic field current drive of high-temperature field reversed configurations with high ζ scaling
Authors: Guo H. Y. , Hoffman A. L. , Milroy R. D.
Published: November 2007

Greatly reduced recycling and impurity ingestion in the Translation, Confinement, and Sustainment—Upgrade (TCSU) device has allowed much higher plasma temperatures to be achieved in the field reversed configurations (FRC) under rotating magnetic field (RMF) formation and sustainment. The hotter plasmas have higher magnetic fields and much higher diamagnetic electron rotation rates so that the important ratio of average electron rotation frequency to RMF frequency, called ζ, approaches unity, for the first time, in TCSU. A large fraction of the RMF power is absorbed by an as yet unexplained (anomalous) mechanism directly proportional to the square of the RMF magnitude. It becomes of relatively lesser significance as the FRC current increases, and simple resistive heating begins to dominate, but the anomalous absorption is useful for initial plasma heating. Measurements of total absorbed power, and comparisons of applied RMF torque to torque on the electrons due to electron-ion friction under high-ζ operation, over a range of temperatures and fields, have allowed the separation of the classical Ohmic and anomalous heating to be inferred, and cross-field plasma resistivities to be calculated.
Power dependence of the photocurrent lineshape in a semiconductor quantum dot
Authors: Russell A. , Fal’ko Vladimir I.
Published: November 2007

We propose a kinetic theory to describe the power dependence of the photocurrent lineshape in optically pumped quantum dots at low temperatures in both zero and finite magnetic fields. We show that there is a crossover power Pc, determined by the electron and hole tunneling rates, where the photocurrent no longer reflects the exciton lifetime. For P>Pc, we show that the photocurrent saturates due to the slow hole escape rate, whereas the linewidth increases with power. We analyze the spin-doublet lineshape in high magnetic fields and determine to what measure it reflects the degree of circular polarization of incident light.
The development of Thomson scattering system on HL-2A tokamak
Authors: Huang Y. , Zhang P. , Feng Z. , Liu C. H. , Shi P. L. , Ding X. T. , Liu Yong
Published: November 2007

A new Thomson scattering diagnostic system is successfully developed to measure core plasma electron temperature (Te) and density (ne) of HL-2A tokamak (major radius R=165cm, minor radius a=40cm). In this system, a standard lamp-monochromator combination is utilized for the calibration of spectral responses. By sweeping in the range of 750–1200nm with a step of 2nm, the work can be done automatically for one-point calibration and then for other. Electronic gain calibration and gain monitoring are done by pulsed light emitting diode light. By utilizing an intense Nd:YAG laser of pulse energy up to 4J and employing good quality interference filters in the five-channel filter polychromator to surpress greatly the stray light, the TS system can be routinely used to make measurements with good quality data. After each HL-2A plasma discharge, the measured Te and ne data are transferred to HL-2A database for lookup and analyses.