bluuebunny/crossref_metadata_2025 huggingface dataset:
From the trenches
Authors: PEDERSEN PETER NØRGAARD
Published: January 2006

This rubric is intended for occasional contributions from on-the-ground practitioners in Geneva and national capitals. Our hope is that this category will inspire other practitioners to submit notes and articles – typically in the range of 2,000 to 10,000 words – to the World Trade Review. As with all notes and articles submitted to the World Trade Review, manuscripts in this category will be reviewed by independent referees. However, the focus is intended to be practice oriented and at least one of the two referees will be a fellow practitioner.
Anomalous resistivity and the nonlinear evolution of the ion‐acoustic instability
Authors: Petkaki P. , Freeman M. P. , Kirk T. , Watt C. E. J. , Horne R. B.
Published: January 2006

Collisionless magnetic reconnection requires the violation of ideal MHD by various kinetic‐scale effects whose relative importance is uncertain. Recent research has highlighted the potential importance of wave‐particle interactions by showing that Vlasov simulations of unstable ion‐acoustic waves predict an anomalous resistivity that can be at least an order of magnitude higher than a popular analytical quasi‐linear estimate. Here, we investigate the nonlinear evolution of the ion‐acoustic instability and its resulting anomalous resistivity by examining the properties of a statistical ensemble of Vlasov simulations. The simulations differ in their initial electric noise field but are otherwise identical with a Maxwellian electron‐ion plasma of low number density and low electron to ion temperature ratio, appropriate to collisionless space plasmas. By studying the evolution of an ensemble of 104 Vlasov simulations with reduced mass ratio mi/me = 25, we show that (1) the probability distribution of anomalous resistivity values produced during the linear, quasi‐linear, and nonlinear evolution of the ion‐acoustic instability is approximately Gaussian, (2) the ensemble mean of the ion‐acoustic resistivity during the nonlinear regime is higher than estimates at quasi‐linear saturation, and (3) the ensemble standard deviation is comparable to the ensemble mean. We argue that the large variability during the nonlinear phase is due to electron and ion bounce motion which is sensitive to the initial conditions. We demonstrate that the results are essentially similar for a real mass ratio simulation.
Excess pore pressure resulting from methane hydrate dissociation in marine sediments: A theoretical approach
Authors: Xu Wenyue , Germanovich Leonid N.
Published: January 2006

This study quantifies the excess pore pressure resulting from gas hydrate dissociation in marine sediments. The excess pore pressure in confined pore spaces can be up to several tens of megapascals due to the tendency for volume expansion associated with gas hydrate dissociation. On the other hand, the magnitude of excess pore pressure in well‐connected sediment pores is generally smaller, depending primarily on the hydrate dissociation rate and the sediment permeability. Volume expansion due to gas hydrate dissociation in well‐connected pore spaces is related via Darcy's law to an increase in pore pressure and its gradient in sediment, which drives an additional upward fluid flow through the sediment layer overlying the gas hydrate dissociation area. The magnitude of this excess pore pressure is found to be proportional to the rate of gas hydrate dissociation and the depth below seafloor and inversely proportional to sediment permeability and the depth below sea level. The excess pore pressure is the greatest at low initial pressures and decreases rapidly with increasing initial pressure. Excess pore pressure may be the result of gas hydrate dissociation due to continuous sedimentation, tectonic uplift, sea level fall, heating or inhibitor injection. The excess pore pressure is found to be potentially able (1) to facilitate or trigger submarine landslides in shallow water environments, (2) to result in the formation of vertical columns of focused fluid flow and gas migration, and (3) to cause the failure of a sediment layer confined by low‐permeability barriers in relatively deep water environments.
An effective elastic thickness map of Australia from wavelet transforms of gravity and topography using Forsyth's method
Authors: Swain C. J. , Kirby J. F.
Published: January 2006

We have developed a wavelet version of Forsyth's coherence method for estimating the effective elastic thickness (Te) of the lithosphere from gravity and topography. We have previously shown that the ‘fan’ wavelet is particularly well suited to making localised coherence estimates and here we progress the method by showing how the coherence can be modelled by using the wavelet transforms of gravity and topography to infer the initial loads that flex the plate. The effectiveness of the method is demonstrated on synthetic data from a model having a rectangular area of large Te embedded in a uniform low Te surrounding, and a load ratio that increases with wave number. The results for the Australia region show a ‘core’ of high Te under north and central Australia, with much lower values to the east and also, unexpectedly, under the western parts of the Yilgarn and Pilbara cratons.
Amplification of streamflow impacts of El Niño by increased atmospheric greenhouse gases
Authors: Maurer Edwin P. , Gibbard Seran , Duffy Philip B.
Published: January 2006

Some El Niño events produce unusually large precipitation amounts in Northern and Central California. We use a high‐resolution global model of the atmosphere coupled to a physically‐based model of surface hydrology to investigate effects of increased atmospheric CO2 and this type of El Niño, both individually and in combination, on monthly river flows in California. Increased CO2 changes the seasonal timing of river flows and increases their variability. SST anomalies typical of a strong El Niño SST increase monthly‐mean flows. The two perturbations together result in increased mean flows and increased variability, raising the possibility of both increased flood risk and water shortages. The river flow response to this strong El Niño in an increased CO2 climate is significantly different from the sum of the responses to the individual perturbations.
Micropatterned avidin arrays on silicon substrates via photolithography, self‐assembly and bioconjugation
Authors: Vail Timothy L. , Cushing Kevin W. , Ingram Jani C. , Omer Ingrid St.
Published: January 2006

Processes for micropatterning protein arrays on inorganic substrates have gained attention in the development of biosensors and clinical diagnostics. This study demonstrates a chemically selective method based on photolithographic deposition of gold patterns with the subsequent attachment of functionalized alkanethiols via molecular self‐assembly. Selective capping of carboxy groups on alkanethiols by N‐hydroxysuccinimide esters allowed the deposition of methoxypoly(ethylene glycol) silane as a blocking agent on unpatterned regions. Carboxylates were used to couple a form of avidin to create a microarray of protein. This microarray was successfully probed with biotinylated quantum dots. In‐process characterization methods included grazing‐angle Fourier‐transform IR spectroscopy, ellipsometry, contact‐angle goniometry, atomic‐force microscopy and fluorescence microscopy.
Fusion protein between protein ZZ and red fluorescent protein DsRed and its application to immunoassays
Authors: Huang Qi‐Lai , Chen Cheng , Chen Yun‐Zi , Gong Chen‐Guang , Wang Jin , Hua Zi‐Chun
Published: January 2006

In the present study, a red fluorescent protein (DsRed) from the coral Discosoma was fused to the C‐terminus of protein ZZ, a synthetic artificial IgG‐Fc‐fragment‐binding protein derived from the B‐domain of staphylococcal Protein A. The chimaeric protein, tagged with six histidine residues at the N‐terminus, was expressed in Escherichia coli and easily purified by one‐step Ni2+‐chelating affinity chromatography. Its fluorescence and IgG‐binding activities were validated using fluorescence‐spectrum analysis, ELISA and dot‐blot analysis. Furthermore, in subsequent dot‐blotting immunoanalysis of glutathione S‐transferase and tumour necrosis factor‐α, and immunofluorescent microscopy assay of interferon regulatory factor 3, the chimaeric protein enabled effective detection of target molecules. Compared with fluorescence‐conjugated antibodies, ZZ–DsRed is less susceptible to photobleaching and easy to produce. In addition, unlike HRP (horseradish peroxidase)‐conjugated antibodies, using ZZ‐DsRed needs no addition of a chromogenic reagent. Our results indicate that ZZ–DsRed shows a wide and promising application potential in immunological detection as a substitute for fluorescent or HRP‐conjugated anti‐IgGs.
Effect of lobe pumping on human albumin: investigating the underlying mechanisms of aggregate formation1
Authors: Gomme Peter T. , Hunt Ben M. , Tatford Owen C. , Johnston Anna , Bertolini Joseph
Published: January 2006

A common problem in the manufacture of liquid protein therapeutics is the tendency for aggregation and particle formation on extended storage. One aspect of processing that might contribute to particle formation is pumping. In the present study, we demonstrate that lobe pumps can promote aggregation in albumin preparations. This is accentuated where the clearance between the pump housing and lobes is increased. Under these conditions, the pump efficiency decreases, resulting in increased exposure of the protein to the pump environment. Depending on the inherent physicochemical stability of the protein, this can lead to aggregate formation, which can influence the long‐term stability characteristics of the product.
Shiga toxin B‐subunit binds to the chaperone BiP and the nucleolar protein B23
Authors: Falguières Thomas , Johannes Ludger
Published: January 2006

Background information. In many cell lines, such as HeLa cells, STxB (Shiga toxin B‐subunit) is transported from the plasma membrane to the ER (endoplasmic reticulum), via early/recycling endosomes and the Golgi apparatus, bypassing the late endocytic pathway. In human monocyte‐derived macrophages and dendritic cells that are not sensitive to Shiga toxin‐induced protein biosynthesis inhibition, STxB is not detectably targeted to the retrograde route and is degraded in late endosomes/lysosomes.

Results. We have identified B‐subunit interacting proteins in HeLa cells and macrophages. In HeLa cells, the ER‐localized chaperone BiP (binding protein) was co‐immunoprecipitated with the B‐subunit. This interaction was not observed in macrophages, consistent with our previous trafficking results. In both cell types, the B‐subunit also interacted with the nucleolar protein B23. Consistently, the B‐subunit could be detected on nucleoli, suggesting that it could serve to bring the holotoxin to the site of synthesis of its molecular target, rRNA. The nucleolar localization data are critically discussed.

Conclusion. The interaction of STxB with BiP, involved in the retrotranslocation process to the cytosol and nucleolar B23, as described in this study, might be of relevance for explaining the efficiency of even low doses of Shiga toxin to inactivate cellular ribosomes, and for the use of STxB as a vector for targeting antigens to cytosolic proteasomes of the MHC I‐restricted antigen presentation pathway.
Alternative trans‐splicing: a novel mode of pre‐mRNA processing
Authors: Horiuchi Takayuki , Aigaki Toshiro
Published: January 2006

Alternative splicing is an important process contributing to proteome diversity without involving an increase in the number of genes. In some cases, alternative splicing is carried out under ‘trans‐mode’, called alternative trans‐splicing, in which exons located on separate pre‐mRNA molecules are selectively joined to produce mature mRNAs encoding proteins with distinct structures and functions. However, it is not known how widespread or how frequently trans‐splicing occurs in vivo. Recently, trans‐allelic trans‐splicing has been unambiguously demonstrated in Drosophila using a SNP (single nucleotide polymorphism) as a marker. In this review, we provide an overview of alternative trans‐splicing in Drosophila and mammals, and discuss its mechanisms.
PKB/AKT is involved in resumption of meiosis in mouse oocytes
Authors: Kalous Jaroslav , Solc Petr , Baran Vladimir , Kubelka Michal , Schultz Richard M. , Motlik Jan
Published: January 2006

Background information. In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin‐dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes.

Results. We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY‐294002, an inhibitor of phosphoinositide 3‐kinase—PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)‐sensitive phosphatases are involved in PKB‐activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser473 is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr308 is localized on centrosome only.

Conclusions. The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.
Identification of a novel amidase motif in neutral ceramidase
Authors: Galadari Sehamuddin , Wu Bill X. , Mao Cungui , Roddy Patrick , El Bawab Samer , Hannun Yusuf A.
Published: January 2006

Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Δypc1Δydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis–Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.
Transcriptional regulation of lysophosphatidic acid-induced interleukin-8 expression and secretion by p38 MAPK and JNK in human bronchial epithelial cells
Authors: Saatian Bahman , Zhao Yutong , He Donghong , Georas Steve N. , Watkins Tonya , Spannhake Ernst Wm , Natarajan Viswanathan
Published: January 2006

HBEpCs (human bronchial epithelial cells) contribute to airway inflammation by secreting a variety of cytokines and chemokines in response to allergens, pathogens, viruses and environmental toxins and pollutants. The potent neutrophil chemoattractant, IL-8 (interleukin-8), is a major cytokine secreted by HBEpCs. We have recently demonstrated that LPA (lysophosphatidic acid) stimulated IL-8 production in HBEpCs via protein kinase C δ dependent signal transduction. However, mechanisms of IL-8 expression and secretion are complex and involve multiple protein kinases and transcriptional factors. The present study was undertaken to investigate MAPK (mitogen-activated protein kinase) signalling in the transcriptional regulation of IL-8 expression and secretion in HBEpCs. Exposure of HBEpCs to LPA (1 μM) enhanced expression and secretion of IL-8 by 5–8-fold and stimulated threonine/tyrosine phosphorylation of ERK (extracellular-signal-regulated kinase), p38 MAPK and JNK (c-Jun N-terminal kinase). The LPA-induced secretion of IL-8 was blocked by the p38 MAPK inhibitor SB203580, by p38 MAPK siRNA (small interfering RNA), and by the JNK inhibitor JNKi II, but not by the MEK (MAPK/ERK kinase) inhibitor, PD98059. LPA enhanced the transcriptional activity of the IL-8 gene; that effect relied on activation of the transcriptional factors NF-κB (nuclear factor κB) and AP-1 (activator protein-1). Furthermore, SB203580 attenuated LPA-dependent phosphorylation of IκB (inhibitory κB), NF-κB and phospho-p38 translocation to the nucleus, NF-κB transcription and IL-8 promoter-mediated luciferase reporter activity, without affecting the JNK pathway and AP-1 transcription. Similarly, JNKi II only blocked LPA-mediated phosphorylation of JNK and c-Jun, AP-1 transcription and IL-8 promoter-mediated luciferase reporter activity, without blocking p38 MAPK-dependent NF-κB transcription. Additionally, siRNA for LPA1–3 receptors partially blocked LPA-induced IL-8 production and activation of MAPKs. The LPA1 and LPA3 receptors, as compared with LPA2, were most efficient in transducing LPA-mediated IL-8 production. These results show an independent role for p38 MAPK and JNK in LPA-induced IL-8 expression and secretion via NF-κB and AP-1 transcription respectively in HBEpCs.
Computational evidence for protein-mediated fatty acid transport across the sarcolemma
Authors: Musters Mark W. J. M. , Bassingthwaighte James B. , van Riel Natal A. W. , van der Vusse Ger J.
Published: January 2006

Long-chain fatty acids (FAs) are important substrates used by the heart to fulfil its energy requirements. Prior to mitochondrial oxidation, blood-borne FAs must pass through the cell membrane of the cardiac myocyte (sarcolemma). The mechanism underlying the sarcolemmal transport of FAs is incompletely understood. The aim of the present study was to estimate the trans-sarcolemmal FA uptake rate using a comprehensive computer model, in which the most relevant mechanisms proposed for cardiac FA uptake were incorporated. Our in silico findings show that diffusion of FA, present in its unbound form (uFA) in close proximity to the outer leaflet of the sarcolemma and serving as sole FA source, is insufficient to account for the physiological FA uptake rate. The inclusion of a hypothetical membrane-associated FA-TFPC (FA-transport-facilitating protein complex) in the model calculations substantially increased the FA uptake rate across the sarcolemma. The model requires that the biological properties of the FA-TFPC allow for increasing the rate of absorption of FA into the outer leaflet and the ‘flip-flop’ rate of FA from the outer to the inner leaflet of the sarcolemma. Experimental studies have identified various sarcolemma-associated proteins promoting cardiac FA uptake. It remains to be established whether these proteins possess the properties predicted by our model. Our findings also indicate that albumin receptors located on the outer leaflet of the sarcolemma facilitate the transfer of FA across the membrane to a significant extent. The outcomes of the computer simulations were verified with physiologically relevant FA uptake rates as assessed in the intact, beating heart in experimental studies.
Spectroscopic analyses of the binding kinetics of 15d-PGJ2 to the PPARγ ligand-binding domain by multi-wavelength global fitting
Authors: Shiraki Takuma , Kodama Takashi S. , Shiki Sayaka , Nakagawa Tatsuo , Jingami Hisato
Published: January 2006

PPARγ (peroxisome proliferator-activated receptor γ) is a nuclear receptor that is activated by natural lipid metabolites, including 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2). We previously reported that several oxidized lipid metabolites covalently bind to PPARγ through a Michael-addition to activate transcription. To separate the ligand-entering (dock) and covalent-binding (lock) steps in PPARγ activation, we investigated the binding kinetics of 15d-PGJ2 to the PPARγ LBD (ligand-binding domain) by stopped-flow spectroscopy. We analysed the spectral changes of 15d-PGJ2 by multi-wavelength global fitting based on a two-step chemical reaction model, in which an intermediate state represents the 15d-PGJ2–PPARγ complex without covalent binding. The extracted spectrum of the intermediate state in wild-type PPARγ was quite similar to the observed spectrum of 15d-PGJ2 in the C285S mutant, which cannot be activated by 15d-PGJ2, indicating that the complex remains in the inactive, intermediate state in the mutant. Thus ‘lock’ rather than ‘dock’ is one of the critical steps in PPARγ activation by 15d-PGJ2.
Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation
Authors: Wingate Andrew D. , Campbell David G. , Peggie Mark , Arthur J. Simon C.
Published: January 2006

Nur77 is a nuclear orphan receptor that is able to activate transcription independently of exogenous ligand, and has also been shown to promote apoptosis on its localization to mitochondria. Phosphorylation of Nur77 on Ser354 has been suggested to reduce ability of Nur77 to bind DNA; however, the kinase responsible for this phosphorylation in cells has not been clearly established. In the present study, we show that Nur77 is phosphorylated on this site by RSK (ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase), but not by PKB (protein kinase B) or PKA (protein kinase A), in vitro. In cells, phosphorylation of Nur77 in vivo is catalysed by RSK, which is activated downstream of the classical MAPK (mitogen-activated protein kinase) cascade. Phosphorylation of Nur77 by RSK is able to promote the binding of Nur77 to 14-3-3 proteins in vitro, however, no evidence could be seen for this interaction in cells. We have established that two related proteins, Nurr1 and Nor1, are also phosphorylated on the equivalent site by RSK in cells in response to mitogenic stimulation.
Dual promoters control the cell-specific expression of the human cell death-inducing DFF45-like effector B gene
Authors: Da Liang , Li Dong , Yokoyama Kazunari K. , Li Tsaiping , Zhao Mujun
Published: January 2006

CIDE-B [cell death-inducing DFF45 (DNA fragmentation factor 45)-like effector B] is a member of the CIDE family of apoptosis-inducing factors. The highly restricted pattern of expression of CIDE-B in the liver and spleen suggests that a mechanism exists for the tissue- and cell-specific regulation of transcription of this gene. We have analysed the promoters of the human CIDE-B gene, particularly the mechanism of cell-specific transcription. Expression of CIDE-B is driven by two promoters which are responsible for the synthesis of two types of transcript, and Sp1 and Sp3 are key regulators of basal transcription from both the upstream and the internal promoter, as indicated by EMSAs (electrophoretic mobility-shift assays) and site-directed mutagenesis. Bisulphite sequencing analysis demonstrated that the upstream promoter was hypermethylated in cells that did not express the long transcript of CIDE-B, but was hypomethylated in cells that expressed this transcript. Furthermore, methylation of this region in vitro reduced the promoter activity to ∼5% of the control. Thus methylation at CpG sites in the upstream promoter region appeared to be important for cell-specific synthesis of the long transcript. By contrast, HNF4α (hepatocyte nuclear factor-4α) bound to the internal promoter and enhanced its activity. Moreover, the short transcript of CIDE-B gene was expressed in cells which do not normally express this transcript upon introduction of exogenous HNF4α, demonstrating the involvement of HNF4α in the cell-specific synthesis of the short transcript. Thus our analysis revealed a novel mechanism for the cell-specific transcription of the human CIDE-B gene, which involves epigenetic and genetic control at separate respective promoters.
Ligand specificities and structural requirements of two Tachypleus plasma lectins for bacterial trapping
Authors: Kuo Tun-Hsun , Chuang Shiao-Cheng , Chang Sing-Yang , Liang Po-Huang
Published: January 2006

TPL (Tachypleus plasma lectin)-1 was purified by using a Sepharose column and TPL-2 was purified from an LPS–Sepharose (LPS coupled to Sepharose matrix) affinity column, as described previously [Chiou, Chen, Y.-W., Chen, S.-C., Chao and Liu (2000) J. Biol. Chem. 275, 1630–1634] and the corresponding genes were cloned [Chen, Yen, Yeh, Huang and Liu (2001) J. Biol. Chem. 276, 9631–9639]. In the present study, TPL-1 and -2 were produced in yeast, and the recombinant proteins secreted into the media were purified and characterized. The proteins show specific PGN (peptidoglycan)- and LPS-binding activity, suggesting a role in trapping Gram-positive and Gram-negative bacteria respectively in innate immunity. Using BIAcore® assays, the dissociation constant for the TPL-1–PGN complex was measured as 8×10−8 M. Replacement of Asn74, the N-glycosylation site of TPL-1, with Asp abolishes the PGN-binding affinity, whereas the unglycosylated TPL-2 N3D mutant retains LPS-binding activity. DTT (dithiothreitol) treatment to break disulphide linkages abrogates TPL-2 activity but does not interfere with TPL-1 function. Cys4 in TPL-2 may form an intermolecular disulphide bond, which is essential for activity. As a result, the TPL-2 C4S mutant is inactive and is eluted as a monomer on a non-reducing gel. TPL-2 C6S is active and forms a non-covalently linked dimer. A model describing TPL-2 binding with LPS is proposed. These two plasma lectins that have different ligand specificities can be used for the detection and discrimination of bacteria and removal of endotoxins.
Binding of 7-methoxy-4-(aminomethyl)-coumarin to wild-type and W128F mutant cytochrome P450 2D6 studied by time-resolved fluorescence spectroscopy
Authors: Stortelder Aike , Keizers Peter H. J. , Oostenbrink Chris , De Graaf Chris , De Kruijf Petra , Vermeulen Nico P. E. , Gooijer Cees , Commandeur Jan N. M. , Van Der Zwan Gert
Published: January 2006

Enzyme structure and dynamics may play a main role in substrate binding and the subsequent steps in the CYP (cytochrome P450) catalytic cycle. In the present study, changes in the structure of human CYP2D6 upon binding of the substrate are studied using steady-state and time-resolved fluorescence methods, focusing not only on the emission of the tryptophan residues, but also on emission of the substrate. As a substrate, MAMC [7-methoxy-4-(aminomethyl)-coumarin] was selected, a compound exhibiting native fluorescence. As well as the wild-type, the W128F (Trp128→Phe) mutant of CYP2D6 was studied. After binding, a variety of energy transfer possibilities exist, and molecular dynamics simulations were performed to calculate distances and relative orientations of donors and acceptors. Energy transfer from Trp128 to haem appeared to be important; its emission was related to the shortest of the three average tryptophan fluorescence lifetimes observed for CYP2D6. MAMC to haem energy transfer was very efficient as well: when bound in the active site, the emission of MAMC was fully quenched. Steady-state anisotropy revealed that besides the MAMC in the active site, another 2.4% of MAMC was bound outside of the active site to wild-type CYP2D6. The tryptophan residues in CYP2D6 appeared to be less accessible for the external quenchers iodide and acrylamide in presence of MAMC, indicating a tightening of the enzyme structure upon substrate binding. However, the changes in the overall enzyme structure were not very large, since the emission characteristics of the enzyme were not very different in the presence of MAMC.
The life and death of translation elongation factor 2
Authors: Jørgensen R. , Merrill A.R. , Andersen G.R.
Published: January 2006

eEF2 (eukaryotic elongation factor 2) occupies an essential role in protein synthesis where it catalyses the translocation of the two tRNAs and the mRNA after peptidyl transfer on the 80 S ribosome. Recent crystal structures of eEF2 and the cryo-electron microscopy reconstruction of its 80 S complex now provide a substantial structural framework for dissecting the functional properties of this factor. The factor can be modified by either phosphorylation or ADP-ribosylation, which results in cessation of translation. We review the structural and functional properties of eEF2 with particular emphasis on the unique diphthamide residue, which is ADP-ribosylated by diphtheria toxin from Corynebacterium diphtheriae and exotoxin A from Pseudomonas aeruginosa.
Coping with stress: eIF2 kinases and translational control
Authors: Wek R.C. , Jiang H.-Y. , Anthony T.G.
Published: January 2006

In response to environmental stresses, a family of protein kinases phosphorylate eIF2 (eukaryotic initiation factor 2) to alleviate cellular injury or alternatively induce apoptosis. Phosphorylation of eIF2 reduces global translation, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions. Accompanying this general protein synthesis control, eIF2 phosphorylation induces translation of specific mRNAs, such as that encoding the bZIP (basic leucine zipper) transcriptional regulator ATF4 (activating transcription factor 4). ATF4 also enhances the expression of additional transcription factors, ATF3 and CHOP (CCAAT/enhancer-binding protein homologous protein)/GADD153 (growth arrest and DNA-damage-inducible protein), that assist in the regulation of genes involved in metabolism, the redox status of the cells and apoptosis. Reduced translation by eIF2 phosphorylation can also lead to activation of stress-related transcription factors, such as NF-κB (nuclear factor κB), by lowering the steady-state levels of short-lived regulatory proteins such as IκB (inhibitor of NF-κB). While many of the genes induced by eIF2 phosphorylation are shared between different environmental stresses, eIF2 kinases function in conjunction with other stress-response pathways, such as those regulated by mitogen-activated protein kinases, to elicit gene expression programmes that are tailored for the specific stress condition. Loss of eIF2 kinase pathways can have important health consequences. Mice devoid of the eIF2 kinase GCN2 [general control non-derepressible-2 or EIF2AK4 (eIF2α kinase 4)] show sensitivity to nutritional deficiencies and aberrant eating behaviours, and deletion of PEK [pancreatic eIF2α kinase or PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase) or EIF2AK3] leads to neonatal insulin-dependent diabetes, epiphyseal dysplasia and hepatic and renal complications.
Aberrant termination triggers nonsense-mediated mRNA decay
Authors: Amrani N. , Dong S. , He F. , Ganesan R. , Ghosh S. , Kervestin S. , Li C. , Mangus D.A. , Spatrick P. , Jacobson A.
Published: January 2006

NMD (nonsense-mediated mRNA decay) is a cellular quality-control mechanism in which an otherwise stable mRNA is destabilized by the presence of a premature termination codon. We have defined the set of endogenous NMD substrates, demonstrated that they are available for NMD at every round of translation, and showed that premature termination and normal termination are not equivalent biochemical events. Premature termination is aberrant, and its NMD-stimulating defects can be reversed by the presence of tethered poly(A)-binding protein (Pab1p) or tethered eRF3 (eukaryotic release factor 3) (Sup35p). Thus NMD appears to be triggered by a ribosome's failure to terminate adjacent to a properly configured 3′-UTR (untranslated region), an event that may promote binding of the UPF/NMD factors to stimulate mRNA decapping.
Molecules of the mind: integrating synaptic biochemistry to understand brain function
Authors: Holden-Dye L.M. , O'Connor V.M. , Stephenson F.A.
Published: January 2006

The focused meeting entitled ‘Molecular Determinants of Synaptic Function: Molecules and Models’ brought together several molecules and experimental models that are furthering our understanding of the biochemical basis of integrative brain function. Invited speakers and short communications from more junior scientists highlighted how individual molecules or protein networks underlie defined subcellular functions (e.g. transmitter release, receptor activation and transmitter uptake) can be used to unravel integrative function at cellular, systems and behavioural levels.
The synapse proteome and phosphoproteome: a new paradigm for synapse biology
Authors: Grant S.G.N.
Published: January 2006

Synapse proteomics has recently resulted in a quantum leap in knowledge of the protein composition of brain synapses and its phosphorylation. We now have the first draft picture of the synapse, comprising ∼1000 proteins. This is not matched by available methods of functional analysis either in reduced systems or in whole animals. Fewer than 20% of synapse proteome proteins have a known function in the nervous system. A concerted effort is required to establish new technical approaches before we can understand the diversity of functions conferred by the synapse proteome on the synapse, the neuron and the animal. This review will highlight this change in knowledge and discuss current technical and interpretative limitations challenged by synapse proteomics.
WAVE signalling: from biochemistry to biology
Authors: Soderling S.H. , Scott J.D.
Published: January 2006

The small GTPases Rho, Rac and Cdc42 (cell-division cycle 42) function as molecular switches to modulate the actin cytoskeleton. They achieve this by modulating the activity of downstream cellular targets. One group of Rho GTPase effectors, WAVE (Wiskott–Aldrich syndrome protein verprolin homologous)-1, WAVE-2 and WAVE-3, function as scaffolds for actin-based signalling complexes. The present review highlights current knowledge regarding the biochemistry of the WAVE signalling complexes and their biological significance.
Respiratory nitrate and nitrite pathway in the denitrifier haloarchaeon Haloferax mediterranei
Authors: Martínez-Espinosa R.M. , Richardson D.J. , Butt J.N. , Bonete M.J.
Published: January 2006

Haloferax mediterranei cells are able to use high nitrate or nitrite concentrations as electron acceptors under anoxic conditions. The nar operon, which has eight open reading frames, has been sequenced and its regulation has been characterized at the transcriptional level. The narG and narH genes encode the Nar (respiratory nitrate reductase) catalytic subunit (NarG) and the electron transfer Nar subunit (NarH) respectively. Nar has been purified and characterized in vitro. This characterization has included protein-film voltammetry and preliminary EPR studies.
Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus
Authors: Dridge E.J. , Richardson D.J. , Lewis R.J. , Butler C.S.
Published: January 2006

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.
Evolution of the soluble nitrate reductase: defining the monomeric periplasmic nitrate reductase subgroup
Authors: Jepson B.J.N. , Marietou A. , Mohan S. , Cole J.A. , Butler C.S. , Richardson D.J.
Published: January 2006

Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.
The assimilatory nitrate reduction system of the phototrophic bacterium Rhodobacter capsulatus E1F1
Authors: Pino C. , Olmo-Mira F. , Cabello P. , Martínez-Luque M. , Castillo F. , Roldán M.D. , Moreno-Vivián C.
Published: January 2006

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates nitrate under anaerobic phototrophic growth conditions. A 17 kb DNA region encoding the nitrate assimilation (nas) system of this bacterium has been cloned and sequenced. This region includes the genes coding for a putative ABC (ATP-binding cassette)-type nitrate transporter (nasFED) and the structural genes for the enzymes nitrate reductase (nasA), nitrite reductase (nasB) and hydroxylamine reductase (hcp). Three genes code for putative regulatory proteins: a nitrite-sensitive repressor (nsrR), a transcription antiterminator (nasT) and a nitrate sensor (nasS). Other genes probably involved in nitrate assimilation are also present in this region. The sequence analysis of these genes and the biochemical properties of the purified nitrate, nitrite and hydroxylamine reductases are reviewed.
Investigation into the role of the truncated denitrification chain inRhizobium sullaestrain HCNT1
Authors: Casella S. , Shapleigh J.P. , Toffanin A. , Basaglia M.
Published: January 2006

Most denitrifying bacteria reduce nitrate to the inert gases nitrous oxide or nitrogen. A remarkable exception to this is Rhizobium sullae strain HCNT1, which catalyses only a single step in the denitrification pathway, the reduction of nitrite to the reactive molecule nitric oxide. Further study demonstrated that HCNT1 does not encode the genes for NO reductase. Prolonged incubation of HCNT1 under anoxic conditions revealed that the cells had reduced culturability but not viability when nitrite was present. This may indicate an adaptation to anoxic conditions to provide resistance to environmental stresses. A closely related strain of R. sullae, strain CC1335, which is unable to denitrify, was found to lose culturability but not viability irrespective of the presence of nitrite. When the gene for nitrite reductase was mobilized into CC1335, this increased culturability with or without nitrite. These results indicate that the presence of nitrite reductase can influence the long-term survival of R. sullae strains and may provide an explanation as to why HCNT1 possesses this unusual truncation of its denitrification electron transport chain.
Inhibiting Escherichia coli cytochrome c nitrite reductase: voltammetry reveals an enzyme equipped for action despite the chemical challenges it may face in vivo
Authors: Gwyer J.D. , Richardson D.J. , Butt J.N.
Published: January 2006

Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.
Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans
Authors: Clarke T.A. , Hemmings A.M. , Burlat B. , Butt J.N. , Cole J.A. , Richardson D.J.
Published: January 2006

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.
What is the substrate specificity of the System I cytochrome c biogenesis apparatus?
Authors: Allen J.W.A. , Ferguson S.J.
Published: January 2006

c-Type cytochromes are characterized by covalent attachment of haem to protein through thioether bonds between the vinyl groups of the haem and the thiols of a CXXCH motif. Proteins of this type play crucial roles in the biochemistry of the nitrogen cycle. Many Gram-negative bacteria use the Ccm (cytochrome c maturation) proteins for the post-translational haem attachment to their c-type cytochromes; in the present paper, we discuss the substrate specificity of the Ccm apparatus. The main conclusion is that the feature recognized and required in the apocytochrome is simply the two cysteines and the histidine of the haem-binding motif.
A multitude of CRP/FNR-like transcription proteins in Bradyrhizobium japonicum
Authors: Mesa S. , Hennecke H. , Fischer H.-M.
Published: January 2006

In Bradyrhizobium japonicum, the nitrogen-fixing soya bean endosymbiont and facultative denitrifier, three CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein)-type transcription factors [FixK1, FixK2 and NnrR (nitrite and nitric oxide reductase regulator)] have been studied previously in the context of the regulation of nitrogen fixation and denitrification. The gene expression of both fixK1 and nnrR depends on FixK2, which acts as a key distributor of the ‘low-oxygen’ signal perceived by the two-component regulatory system FixLJ. While the targets for FixK1 are not known, NnrR transduces the nitrogen oxide signal to the level of denitrification gene expression. Besides these three regulators, the complete genome sequence of this organism has revealed the existence of 13 additional CRP/FNR-type proteins whose functions have not yet been studied. Based on sequence similarity and phylogenetic analysis, we discuss in this paper the peculiarities of these additional factors.
Metabolic regulation of nitrogen fixation in Rhodospirillum rubrum
Authors: Wang H. , Norén A.
Published: January 2006

Nitrogenase activity in Rhodospirillum rubrum is post-translationally regulated by DRAG (dinitrogenase reductase glycohydrolase) and DRAT (dinitrogenase reductase ADP-ribosylation transferase). When a sudden increase in fixed nitrogen concentration or energy depletion is sensed by the cells, DRAG is inactivated and DRAT activated. We propose that the regulation of DRAG is dependent on its location in the cell and the presence of an ammonium-sensing protein.
Analysis of community composition of biofilms in a submerged filter system for the removal of ammonia and phenol from industrial wastewater
Authors: Cortés-Lorenzo C. , Molina-Muñoz M.L. , Gómez-Villalba B. , Vilchez R. , Ramos A. , Rodelas B. , Hontoria E. , González-López J.
Published: January 2006

The bacterial diversity of a submerged filter, used for the removal of ammonia and phenol from an industrial wastewater with high salinity, was studied by a cultivation-independent approach based on PCR/TGGE (temperature-gradient gel electrophoresis). The wastewater treatment plant (laboratory scale) combined the nitrification and denitrification processes and consisted of two separated columns (one anoxic and one aerated) connected through a valve. The spatial diversity of bacterial communities in the plant biofilms was analysed by taking samples at four different heights in the system. TGGE profiles of PCR-amplified sequences of the 16 S rRNA gene (V3-hypervariable region) showed significant variations of the bacterial diversity, mainly depending on the concentration of O2 along the system. Several bands separated by TGGE were reamplified and sequenced, in order to explore the composition of the microbial communities in the biofilms. Most of the sequenced bands (10 out of 13) were closely related to the 16 S rRNA gene of marine α-proteobacteria, mainly grouping in the periphery of the genus Roseobacter. Other sequences were related to those of γ-proteobacteria, the nitrite oxidizer Nitrospira marina and anaerobic phenol-degrading bacteria of the Desulfobacteraceae.
Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria
Authors: Op den Camp H.J.M. , Kartal B. , Guven D. , van Niftrik L.A.M.P. , Haaijer S.C.M. , van der Star W.R.L. , van de Pas-Schoonen K.T. , Cabezas A. , Ying Z. , Schmid M.C. , Kuypers M.M.M. , van de Vossenberg J. , Harhangi H.R. , Picioreanu C. , van Loosdrecht M.C.M. , Kuenen J.G. , Strous M. , Jetten M.S.M.
Published: January 2006

In the anaerobic ammonium oxidation (anammox) process, ammonia is oxidized with nitrite as primary electron acceptor under strictly anoxic conditions. The reaction is catalysed by a specialized group of planctomycete-like bacteria. These anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. The reactions are assumed to be carried out in a unique prokaryotic organelle, the anammoxosome. This organelle is surrounded by ladderane lipids, which make the organelle nearly impermeable to hydrazine and protons. The localization of the major anammox protein, hydrazine oxidoreductase, was determined via immunogold labelling to be inside the anammoxosome. The anammox bacteria have been detected in many marine and freshwater ecosystems and were estimated to contribute up to 50% of oceanic nitrogen loss. Furthermore, the anammox process is currently implemented in water treatment for the low-cost removal of ammonia from high-strength waste streams. Recent findings suggested that the anammox bacteria may also use organic acids to convert nitrate and nitrite into dinitrogen gas when ammonia is in short supply.