bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of the fast plasma sheet flow on the geosynchronous magnetic configuration: Geotail and GOES coordinated study
Authors: Ohtani S. , Singer H. J. , Mukai T.
Published: January 2006

The present study statistically examines how (or if) the geosynchronous (GOES) magnetic field responds to fast earthward flow observed by the Geotail satellite in the plasma sheet. The change of the GOES H (north‐south) component within 15 min of the detection of fast flows, ΔH, is used as a primary measure of the geosynchronous response. It is found that following the detection of fast flows, the geosynchronous magnetic field rarely dipolarizes, but it often becomes more stretched, which is manifested by negative ΔH. This H decrease is not accompanied by any correlated variation of the D (azimuthal) component, suggesting that the associated stretching is not an edge effect of the substorm current wedge formation, but it can be attributed to the intensification of the local tail current. No systematic dependence of ΔH on the satellite separation can be found. On the other hand, the geosynchronous magnetic field tends to dipolarize if it is already stretched significantly, although the associated changes in the H and V (radial) components are not much larger than those in events that are not preconditioned. The flow intensity does not seem to be a controlling factor, either. However, caution needs to be exercised because the present study is not able to address the azimuthal structure of the fast flow. It is concluded that in most events the fast plasma flow does not reach geosynchronous orbit and that the generation of the fast plasma flow in the plasma sheet is not sufficient for causing geosynchronous dipolarization.
Geochemical and mineralogical indicators for aqueous processes in the Columbia Hills of Gusev crater, Mars
Authors: Ming D. W. , Mittlefehldt D. W. , Morris R. V. , Golden D. C. , Gellert R. , Yen A. , Clark B. C. , Squyres S. W. , Farrand W. H. , Ruff S. W. , Arvidson R. E. , Klingelhöfer G. , McSween H. Y. , Rodionov D. S. , Schröder C. , de Souza P. A. , Wang A.
Published: January 2006

Water played a major role in the formation and alteration of rocks and soils in the Columbia Hills. The extent of alteration ranges from moderate to extensive. Five distinct rock compositional classes were identified; the order for degree of alteration is Watchtower ≅ Clovis > Wishstone ≅ Peace > Backstay. The rover's wheels uncovered one unusual soil (Paso Robles) that is the most S‐rich material encountered. Clovis class rocks have compositions similar to Gusev plains soil but with higher Mg, Cl, and Br and lower Ca and Zn; Watchtower and Wishstone classes have high Al, Ti, and P and low Cr and Ni; Peace has high Mg and S and low Al, Na, and K; Backstay basalts have high Na and K compared to plains Adirondack basalts; and Paso Robles soil has high S and P. Some rocks are corundum‐normative, indicating that their primary compositions were changed by loss and/or gain of rock‐forming elements. Clovis materials consist of magnetite, nanophase ferric‐oxides (npOx), hematite, goethite, Ca‐phosphates, Ca‐ and Mg‐sulfates, pyroxene, and secondary aluminosilicates. Wishstone and Watchtower rocks consist of Fe‐oxides/oxyhydroxides, ilmenite, Ca‐phosphate, pyroxene, feldspar, Mg‐sulfates, and secondary aluminosilicates. Peace consists of magnetite, npOx, Mg‐ and Ca‐sulfates, pyroxene, olivine, feldspar, apatite, halides, and secondary aluminosilicates. Paso Robles consists of Fe3+‐, Mg‐, Ca‐, and other sulfates, Ca‐phosphates, hematite, halite, allophane, and amorphous silica. Columbia Hills outcrops and rocks may have formed by the aqueous alteration of basaltic rocks, volcaniclastic materials, and/or impact ejecta by solutions that were rich in acid‐volatile elements.
An improved and simplified method for the large‐scale purification of pediocin PA‐1 produced by Pediococcus acidilactici
Authors: Beaulieu Lucie , Aomari Hafida , Groleau Denis , Subirade Muriel
Published: January 2006

The bacteriocin pediocin PA‐1 produced by Pediococcus acidilactici PAC 1.0 offers significant potential as a food preservative and as an antimicrobial agent in the medical area. However, low production yields and difficulties in obtaining significant amounts of pure pediocin PA‐1 have limited, in part, its biochemical and physical characterization. In the present study, we describe a simple and more efficient purification strategy for pediocin PA‐1. A hydrophobic interaction chromatography step using an octyl‐Sepharose column was introduced for final purification and polishing. The new method is a scalable one, uses only two steps and yields highly purified pediocin PA‐1 with a recovery as high as 73%, which is at least two to three times more than that of the methods reported so far. Highly purified, biologically active pediocin PA‐1 of the correct molecular mass (4624 Da, with two disulphide bridges) was obtained. Fourier‐transform infrared analysis runs at p2H 6 indicated that pediocin PA‐1 was more structured than similar pediocin PA‐1 samples purified using the earlier purification scheme.
Peroxynitrite‐modified DNA: a better antigen for systemic lupus erythematosus anti‐DNA autoantibodies
Authors: Habib Safia , Moinuddin  , Ali Rashid
Published: January 2006

Native human DNA was modified by ONOO− (peroxynitrite), generated by the synergistic action of sodium nitroprusside [Na2Fe(CN)5NO], an NO donor, and pyrogallol, a superoxide donor. The modifications were analysed by UV absorption characteristics, fluorescence emission transitions, nuclease S1 digestibility and melting‐temperature studies. Modified DNA was found to be highly immunogenic, inducing high‐titre immunogen‐specific antibodies in experimental animals. A maximum of 85% inhibition of the antibody binding was observed in competition ELISA with immunogen as inhibitor. SLE (systemic lupus erythematosus) anti‐DNA autoantibodies recognized modified DNA as a better antigen than the native analogue. Modification of native DNA by ONOO−, forming neoepitopes on the molecule, may be one of the factors for the induction of the autoimmune response seen in SLE.
Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells
Authors: Beitz Eric , Liu Kun , Ikeda Masahiro , Guggino William B. , Agre Peter , Yasui Masato
Published: January 2006

Background information. Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid‐secreting type‐A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans‐epithelial water movement.

Results. We show by mutational analyses and immunofluorescence that the N‐terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP—AQP6 or HA—AQP6) to the N‐terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin—Darby canine kidney cells. In contrast, addition of a GFP tag to the C‐terminus (AQP6–GFP) caused the protein to remain intracellular, similar to untagged wild‐type AQP6. Replacement of the N‐terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N‐terminus of AQP6 retained AQP1 in cytosolic sites.

Conclusion. Our results suggest that the N‐terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid‐secreting type‐A intercalated cells.
Forgotten and novel aspects in pancreas development
Authors: Pieler Tomas , Chen Yonglong
Published: January 2006

Diseases related to the pancreas are of highest importance in public health. It is anticipated that a detailed understanding of the molecular events that govern the embryonic development of this organ will have an immediate impact on clinical research relating to this issue. One major aim is the reconstruction of embryonic development in vitro with appropriate precursor cells, a second strategy is aimed at understanding the transdifferentiation of non‐pancreatic into pancreatic tissue, and a third avenue is defined by the stimulation of the intrinsic ability of the pancreas to regenerate. Recent progress in developmental biology with respect to these different topics is reviewed in the present article. In addition, we also address evolutionary aspects of pancreas development, emphasizing the role of the South African clawed frog, Xenopus laevis, as an additional useful model system to study the molecular control of pancreas development.
Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family
Authors: Gäde Gerd , Šimek Petr , Clark Kevin D. , Auerswald Lutz
Published: January 2006

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MSn (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.
Domain II plays a crucial role in the function of ribosome recycling factor
Authors: Guo Peng , Zhang Liqiang , Zhang Hongjie , Feng Yanming , Jing Guozhong
Published: January 2006

RRF (ribosome recycling factor) consists of two domains, and in concert with EF-G (elongation factor-G), triggers dissociation of the post-termination ribosomal complex. However, the function of the individual domains of RRF remains unclear. To clarify this, two RRF chimaeras, EcoDI/TteDII and TteDI/EcoDII, were created by domain swaps between the proteins from Escherichia coli and Thermoanaerobacter tengcongensis. The ribosome recycling activity of the RRF chimaeras was compared with their wild-type RRFs by using in vivo and in vitro activity assays. Like wild-type TteRRF (T. tengcongensis RRF), the EcoDI/TteDII chimaera is non-functional in E. coli, but both wild-type TteRRF, and EcoDI/TteDII can be activated by coexpression of T. tengcongensis EF-G in E. coli. By contrast, like wild-type E. coli RRF (EcoRRF), TteDI/EcoDII is fully functional in E. coli. These findings suggest that domain II of RRF plays a crucial role in the concerted action of RRF and EF-G for the post-termination complex disassembly, and the specific interaction between RRF and EF-G on ribosomes mainly depends on the interaction between domain II of RRF and EF-G. This study provides direct genetic and biochemical evidence for the function of the individual domains of RRF.
Multimers of the fibroblast growth factor (FGF)–FGF receptor–saccharide complex are formed on long oligomers of heparin
Authors: Harmer Nicholas J. , Robinson Christopher J. , Adam Lucy E. , Ilag Leopold L. , Robinson Carol V. , Gallagher John T. , Blundell Tom L.
Published: January 2006

The minimal signalling unit for tyrosine kinase receptors is two protomers dimerized by one or more ligands. However, it is clear that maximal signalling requires the formation of larger complexes of many receptors at discrete foci on the cell surface. The biological interactions that lead to this are likely to be diverse and have system specific components. In the present study, we demonstrate that, in the FGF (fibroblast growth factor)–FGFR (FGF receptor) system, multimers of the minimal complex composed of two FGF1 and two FGFR2 protomers can form on a single chain of the co-receptor heparin. Using size-exclusion chromatography, we show that two complexes can form on heparin chains as small as 16 saccharide units. We also show by MS that discrete complexes containing exactly two copies of the minimal signalling unit are formed. However, the doublet of complexes appears to be less co-operative than the formation of the 2:2:1 FGF1:FGFR2:heparin complex, suggesting that this mechanism is one of a number of weaker interactions that might be involved in the formation of a focal complex on the cell surface.
Structure of the zinc finger domain encompassing residues 13–51 of the nucleocapsid protein from simian immunodeficiency virus
Authors: Morellet Nelly , Meudal Hervé , Bouaziz Serge , Roques Bernard Pierre
Published: January 2006

The NCps (nucleocapsid proteins) of HIV-1 (HIV type 1), HIV-2 and SIV (simian immunodeficiency virus) are small highly basic proteins, characterized by the presence of two CCHC ZF (zinc finger) domains. NCps, closely associated with the dimeric RNA genome in the core of the virus particle, were shown to promote the specific encapsidation of the viral RNA and are implicated in reverse transcription. Solution structure of the HIV-1 NCp7 and complexes of NCp7 with RNA or DNA showed the critical relationships between the structure and its various functions. HIV-1 and HIV-2 have resulted respectively from transmissions of SIV from chimpanzees and sooty mangabeys. It has been shown that the SIVlhoest (SIV from l'Hoest monkeys) also has the potential to infect human populations. Since monkeys are of great interest for clinical studies of antiviral drugs, the structure of (13-51)NCp8 (zinc finger domain of NCp8, encompassing residues 13–51) from SIVlhoest was determined by NMR to appraise the influence of major differences in the sequence, since Glu21, Gly43 and Met46 in NCp7 are replaced by Pro, Glu and Phe respectively in this particular NCp8. The structure of (13-51)NCp8 is very well defined, and surprisingly the structure of each ZF is similar in NCp7 and NCp8. Moreover, contrary to NCp7, the two ZFs are strongly locked to each other in this NCp8. This first reported structure of a simian NCp8 compared with that of NCp7 shows that the main structural differences occur at the flexible linker between the two ZFs but the essential residues responsible for the interaction with oligonucleotides adopt the same orientation in the two proteins.
Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes
Authors: Bruzzone Santina , Moreschi Iliana , Guida Lucrezia , Usai Cesare , Zocchi Elena , De Flora Antonio
Published: January 2006

β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.
Kalata B8, a novel antiviral circular protein, exhibits conformational flexibility in the cystine knot motif
Authors: Daly Norelle L. , Clark Richard J. , Plan Manuel R. , Craik David J.
Published: January 2006

The cyclotides are a family of circular proteins with a range of biological activities and potential pharmaceutical and agricultural applications. The biosynthetic mechanism of cyclization is unknown and the discovery of novel sequences may assist in achieving this goal. In the present study, we have isolated a new cyclotide from Oldenlandia affinis, kalata B8, which appears to be a hybrid of the two major subfamilies (Möbius and bracelet) of currently known cyclotides. We have determined the three-dimensional structure of kalata B8 and observed broadening of resonances directly involved in the cystine knot motif, suggesting flexibility in this region despite it being the core structural element of the cyclotides. The cystine knot motif is widespread throughout Nature and inherently stable, making this apparent flexibility a surprising result. Furthermore, there appears to be isomerization of the peptide backbone at an Asp-Gly sequence in the region involved in the cyclization process. Interestingly, such isomerization has been previously characterized in related cyclic knottins from Momordica cochinchinensis that have no sequence similarity to kalata B8 apart from the six conserved cysteine residues and may result from a common mechanism of cyclization. Kalata B8 also provides insight into the structure–activity relationships of cyclotides as it displays anti-HIV activity but lacks haemolytic activity. The ‘uncoupling’ of these two activities has not previously been observed for the cyclotides and may be related to the unusual hydrophilic nature of the peptide.
TOPs and their regulation
Authors: Hamilton T.L. , Stoneley M. , Spriggs K.A. , Bushell M.
Published: January 2006

Upon cell-cycle arrest or nutrient deprivation, the cellular rate of ribosome production is reduced significantly. In mammalian cells, this effect is achieved in part through a co-ordinated inhibition of RP (ribosomal protein) synthesis. More specifically, translation initiation on RP mRNAs is inhibited. Translational regulation of RP synthesis is dependent on cis-elements within the 5′-UTRs (5′-untranslated regions) of the RP mRNAs. In particular, a highly conserved 5′-TOP (5′-terminal oligopyrimidine tract) appears to play a key role in the regulation of RP mRNA translation. This article explores recent developments in our understanding of the mechanism of TOP mRNA regulation, focusing on upstream signalling pathways and trans-acting factors, and highlighting some interesting observations which have come to light following the recent development of cDNA microarray technology coupled with polysome analysis.
Control of mRNA stability in eukaryotes
Authors: Newbury S.F.
Published: January 2006

mRNA turnover plays a key role in the control of gene expression. Recent work has shown that proteins involved in mRNA turnover are located in multicomponent complexes which are tightly regulated. The control of mRNA stability is also intimately linked with translational processes. This article reviews the pathways and enzymes that control mRNA turnover in eukaryotic cells and discusses their mechanisms of control.
Increased expression of glial fibrillary acidic protein fragments and μ-calpain activation within the hippocampus of prion-infected mice
Authors: Gray B.C. , Skipp P. , O'Connor V.M. , Perry V.H.
Published: January 2006

Prion diseases are characteristically accompanied by marked astrocytic activation, which is initiated relatively early in the disease process. Using the intracerebrally injected ME7 strain of prion agent to model disease, we identified an expected increase in GFAP (glial fibrillary acidic protein) but additionally noted an accumulation of GFAP cleavage fragments in hippocampal homogenates. A time-dependent increase in hippocampal μ-calpain immunoreactivity within astrocytes suggests that its proteolytic activity may account for the cleavage of GFAP that is observed in the ME7 model. It may therefore contribute to the reactive gliosis that is characteristic of prion diseases.
Glycine transporters: essential regulators of synaptic transmission
Authors: Betz H. , Gomeza J. , Armsen W. , Scholze P. , Eulenburg V.
Published: January 2006

Glycine is a major inhibitory neurotransmitter in the mammalian CNS (central nervous system). Glycinergic neurotransmission is terminated by the uptake of glycine into glycinergic nerve terminals and neighbouring glial cells. This uptake process is mediated by specific Na+/Cl−-dependent GlyTs (glycine transporters), GlyT1 and GlyT2. GlyT1, in addition, is thought to regulate the concentration of glycine at excitatory synapses containing NMDARs (N-methyl-D-aspartate receptors), which require glycine as a co-agonist. We have analysed the physiological roles and regulation of GlyT1 and GlyT2 by generating transporter-deficient mice and searching for interacting proteins. Our genetic results indicate that at glycinergic synapses, the glial transporter GlyT1 catalyses the removal of glycine from the synaptic cleft, whereas GlyT2 is required for the re-uptake of glycine into nerve terminals, thereby allowing for neurotransmitter reloading of synaptic vesicles. Both GlyT1 and GlyT2 are essential for CNS function, as revealed by the lethal phenotypes of the respective knockout mice. Mice expressing only a single GlyT1 allele are phenotypically normal but may have enhanced NMDAR function. GlyT2 is highly enriched at glycinergic nerve terminals, and Ca2+-triggered exocytosis and internalization are thought to regulate GlyT2 numbers in the pre-synaptic plasma membrane. We have identified different interacting proteins that may play a role in GlyT2 trafficking and/or pre-synaptic localization.
Building a synapse: genetic analysis of glutamatergic neurotransmission
Authors: Brockie P.J. , Maricq A.V.
Published: January 2006

Ionotropic glutamate receptors (iGluRs) are a critical component of the vertebrate central nervous system and mediate the majority of rapid excitatory neurotransmission. However, iGluRs are not self-regulating molecules and require additional proteins in order to function properly. Understanding the molecular architecture of functional glutamatergic synapses is therefore an important challenge in neurobiology. To address this question, we combine the techniques of genetics, molecular biology and electrophysiology in the nematode Caenorhabditis elegans. To date, genetic analysis has identified a number of genes required to build a glutamatergic synapse, including the CUB-domain transmembrane protein, SOL-1, which is thought to act as an auxiliary subunit that directly modifies iGluR function. Identifying and characterizing new proteins, such as SOL-1, in the relatively simple nervous system of the worm can contribute to our understanding of how more complex vertebrate nervous systems function.
Temperature-sensitive paralytic mutants: insights into the synaptic vesicle cycle
Authors: Vijayakrishnan N. , Broadie K.
Published: January 2006

Forward genetic screens have identified numerous proteins with critical roles in neurotransmission. One particularly fruitful screening target in Drosophila has been TS (temperature-sensitive) paralytic mutants, which have revealed proteins acutely required in neuronal signalling. In the present paper, we review recent insights and current questions from one recently cloned TS paralytic mutant, rbo (rolling blackout). The rbo mutant identifies a putative integral lipase of the pre-synaptic plasma membrane that is required for the SV (synaptic vesicle) cycle. Identification of this mutant adds to a growing body of evidence that lipid-modifying enzymes locally control specialized lipid microenvironments and lipid signalling pathways with key functions regulating neurotransmission strength. The RBO protein is absolutely required for phospholipase C signalling in phototransduction. We posit that RBO might be required to regulate the availability of fusogenic lipids such as phosphatidylinositol 4,5-bisphosphate and diacylglycerol that may directly modify membrane properties and/or activate lipid-binding fusogenic proteins mediating SV exocytosis.
Biochemical and regulatory properties of a respiratory island encoded by a conjugative plasmid in the extreme thermophile Thermus thermophilus
Authors: Cava F. , Berenguer J.
Published: January 2006

In the present paper, we summarize the current knowledge on the first step of the denitrification pathway in the ancestral extreme thermophilic bacterium Thermus thermophilus. In this organism, nitrate respiration is performed by a mobilizable respiratory island that encodes a new type of respiratory NADH dehydrogenase as electron donor, a tetrameric membrane nitrate reductase as final electron acceptor, two nitrate/nitrite transporters and the transcription factors required for their expression in response to nitrate and anoxia.
Microarray analysis of gene regulation by oxygen, nitrate, nitrite, FNR, NarL and NarP during anaerobic growth of Escherichia coli: new insights into microbial physiology
Authors: Overton T.W. , Griffiths L. , Patel M.D. , Hobman J.L. , Penn C.W. , Cole J.A. , Constantinidou C.
Published: January 2006

RNA was isolated from cultures of Escherichia coli strain MG1655 and derivatives defective in fnr, narXL, or narXL with narP, during aerobic growth, or anaerobic growth in the presence or absence of nitrate or nitrite, in non-repressing media in which both strain MG1655 and an fnr deletion mutant grew at similar rates. Glycerol was used as the non-repressing carbon source and both trimethylamine-N-oxide and fumarate were added as terminal electron acceptors. Microarray data supplemented with bioinformatic data revealed that the FNR (fumarate and nitrate reductase regulator) regulon includes at least 104, and possibly as many as 115, operons, 68 of which are activated and 36 are repressed during anaerobic growth. A total of 51 operons were directly or indirectly activated by NarL in response to nitrate; a further 41 operons were repressed. Four subgroups of genes implicated in management of reactive nitrogen compounds, NO and products of NO metabolism, were identified; they included proteins of previously unknown function. Global repression by the nitrate- and nitrite-responsive two-component system, NarQ-NarP, was shown for the first time. In contrast with the frdABCD, aspA and ansB operons that are repressed only by NarL, the dcuB-fumB operon was among 37 operons that are repressed by NarP.
The Bradyrhizobium japonicum napEDABC genes are controlled by the FixLJ-FixK2-NnrR regulatory cascade
Authors: Robles E.F. , Sánchez C. , Bonnard N. , Delgado M.J. , Bedmar E.J.
Published: January 2006

Nitrate respiration by the N2-fixing symbiotic bacteria Bradyrhizobium japonicum USDA110 is mediated by a Nap (periplasmic nitrate reductase) encoded by the napEDABC genes. Expression of a transcriptional fusion of the nap promoter region to the reporter gene lacZ, PnapE-lacZ, was very low in aerobically grown cells of USDA110, but expression was induced approx. 3-fold when the cells were cultured under microaerobic conditions, and 12-fold when nitrate was added to the microaerobic incubation medium. The PnapE-lacZ fusion was not expressed in the fixL 7403, fixJ 7360 and fixK2 9043 mutant strains. Microaerobic induction of the PnapE-lacZ fusion was retained in the nnrR 8678 mutant, but no increase in β-galactosidase activity was observed upon nitrate addition. Western-blot and Methyl Viologen-dependent nitrate reductase activity assays showed that synthesis and activity of the catalytic NapA subunit in USDA110 was similar to that in the napC 0906 and nirK GRK308 mutant strains incubated microaerobically with nitrate. These results suggest that nitrate and nitrite, which are not reduced by the napC 0906 and nirK GRK308 mutant cells respectively, induced the synthesis and activity of NapA; conversely, formation of endogenous NO was not required for induction of Nap expression.
Organization of the respiratory chain of Neisseria meningitidis
Authors: Deeudom M. , Rock J. , Moir J.
Published: January 2006

The ability of Neisseria meningitidis to utilize both oxygen and nitrogen oxides as respiratory substrates allows it to thrive in the diverse environment of the human host. Genome analysis highlighted genes encoding a cbb3 cytochrome oxidase, the aniA nitrite reductase gene and the norB nitric oxide reductase gene. In the present study, we used myxothiazol as an inhibitor of the bc1 complex in intact cells and demonstrated that electron flow to nitrite reductase and the cytochrome oxidase, but not NO reductase, passes via the cytochrome bc1 complex. UV–visible spectrophotometry of intact cells demonstrated that oxygen oxidizes c-type and b-type cytochromes. Oxidation of cytochromes by nitrite was only seen in microaerobically precultured whole cells, and the predominant oxidizable cytochromes were b-type. These are likely to be associated with the oxidation of a b-haem-containing nitric oxide reductase. Nitrite inhibits the oxidation of cytochromes by oxygen in a nitrite reductase-independent manner, indicating that nitrite may inhibit oxidase activity directly, as well as via the intermediate of denitrification, nitric oxide.
Cyanide metabolism of Pseudomonas pseudoalcaligenes CECT5344: role of siderophores
Authors: Huertas M.-J. , Luque-Almagro V.M. , Martínez-Luque M. , Blasco R. , Moreno-Vivián C. , Castillo F. , Roldán M.-D.
Published: January 2006

Cyanide is one of the most potent and toxic chemicals produced by industry. The jewellery industry of Córdoba (Spain) generates a wastewater (residue) that contains free cyanide, as well as large amounts of cyano–metal complexes. Cyanide is highly toxic to living systems because it forms very stable complexes with transition metals that are essential for protein function. In spite of its extreme toxicity, some organisms have acquired mechanisms to avoid cyanide poisoning. The biological assimilation of cyanide needs the concurrence of three separate processes: (i) a cyanide-insensitive respiratory chain, (ii) a system for iron acquisition (siderophores) and (iii) a cyanide assimilation pathway. Siderophores are low-molecular-mass compounds (600–1500 Da) that scavenge iron (Fe3+) ions (usually with extremely high affinity) from the environment under iron-limiting conditions. There are two main classes of siderophores: catechol and hydroxamate types. The catechol-type siderophores chelate ferric ion via a hydroxy group, whereas the hydroxamate-type siderophores bind iron via a carbonyl group with the adjacent nitrogen. In the presence of cyanide, bacterial proliferation requires this specific metal uptake system because siderophores are able to break down cyano–metal complexes. Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide or cyano–metal complexes as nitrogen source. A proteomic approach was used for the isolation and identification, in this strain, of a protein that was induced in the presence of cyanide, namely CN0, that is involved in siderophore biosynthesis in response to cyanide. An overview of bacterial cyanide degradation pathways and the involvement of siderophores in this process are presented.
Role of the central region of NifL in conformational switches that regulate nitrogen fixation
Authors: Little R. , Martinez-Argudo I. , Dixon R.
Published: January 2006

The NifL regulatory protein is an anti-activator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA through the formation of a protein–protein complex. NifL modulates the activity of NifA in response to the redox, carbon and nitrogen status to ensure that nitrogen fixation occurs only under physiological conditions that are appropriate for nitrogenase activity. The domain architecture of NifL is similar to that of some histidine protein kinases, with two N-terminal PAS (PER, ARNT, SIM) domains, one of which contains an FAD cofactor that senses the redox status, and a C-terminal domain containing conserved residues that constitutes the nucleotide-binding domain of the GHKL (gyrase, Hsp90, histidine kinase, MutL) superfamily of ATPases. We have evidence that the central region of NifL, which is located between the PAS domains and the C-terminal GHKL nucleotide-binding domain, plays a crucial role in the propagation of signals perceived in response to the redox and fixed nitrogen status and that this region participates in conformational changes that switch NifL between active and inactive states. We have identified a critical arginine residue in the central region of NifL that participates in the conformational switch that activates NifL. Mutations in the central region of NifL that disable the redox-sensing function of NifL but leave the protein competent to respond to the nitrogen signal conveyed by the signal transduction protein GlnK have also been isolated. Our results suggest that the topological relationship between the central region and the GHKL domain may alter as a consequence of conformational changes induced in response to signal perception.
Role of nitrogen oxides in the metabolism of ammonia-oxidizing bacteria
Authors: Kampschreur M.J. , Tan N.C.G. , Picioreanu C. , Jetten M.S.M. , Schmidt I. , van Loosdrecht M.C.M.
Published: January 2006

Ammonia-oxidizing bacteria (AOB) can use oxygen and nitrite as electron acceptors. Nitrite reduction by Nitrosomonas is observed under three conditions: (i) hydrogen-dependent denitrification, (ii) anoxic ammonia oxidation with nitrogen dioxide (NO2) and (iii) NOx-induced aerobic ammonia oxidation. NOx molecules play an important role in the conversion of ammonia and nitrite by AOB. Absence of nitric oxide (NO), which is generally detectable during ammonia oxidation, severely impairs ammonia oxidation by AOB. The lag phase of recovery of aerobic ammonia oxidation was significantly reduced by NO2 addition. Acetylene inhibition tests showed that NO2-dependent and oxygen-dependent ammonia oxidation can be distinguished. Addition of NOx increased specific activity of ammonia oxidation, growth rate and denitrification capacity. Together, these findings resulted in a hypothetical model on the role of NOx in ammonia oxidation: the NOx cycle.
NO-dependent transcriptional activation of gene expression in Ralstonia eutropha H16
Authors: Cramm R. , Büsch A. , Strube K.
Published: January 2006

The σ54-dependent transcriptional regulator NorR of Ralstonia eutropha H16 activates gene expression in response to nitric oxide (NO). The N-terminal domain of NorR is thought to be involved in signal perception. A C112S exchange within this domain abolished promoter activation by the mutated protein, indicating that Cys112 is essential for the signalling mechanism of NorR. The DNA region recognized by NorR contains three copies of a conserved element termed the NorR-box. Alteration of bases within any of the NorR-boxes resulted in a significant decrease in promoter activation. Therefore all three boxes have to be recognized by NorR to activate its target promoter. NorR controls expression of an operon that encodes a redox-active non-haem-iron protein NorA and an NO reductase NorB. NorA exerts a negative effect on signal-dependent promoter activation by NorR. Optical spectroscopy of purified NorA indicates that the reduced protein can react with NO to form a ferrous nitrosyl adduct. Hence, NO binding by NorA opens up the possibility that NorA and NorR compete for NO in the cytoplasm.
N-oxide sensing and denitrification: the DNR transcription factors
Authors: Rinaldo S. , Giardina G. , Brunori M. , Cutruzzolà F.
Published: January 2006

All denitrifiers can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels by controlling the expression of denitrification gene clusters by redox signalling through transcriptional regulators belonging to the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulator) superfamily.
Proton transfer in bacterial nitric oxide reductase
Authors: Flock U. , Reimann J. , Ädelroth P.
Published: January 2006

The NOR (nitric oxide reductase) from Paracoccus denitrificans catalyses the two-electron reduction of NO to N2O (2NO+2H++2e−→N2O+H2O). The NOR is a divergent member of the superfamily of haem-copper oxidases, oxygen-reducing enzymes which couple the reduction of oxygen with translocation of protons across the membrane. In contrast, reduction of NO catalysed by NOR is non-electrogenic which, since electrons are supplied from the periplasmic side of the membrane, implies that the protons needed for NO reduction are also taken from the periplasm. Thus NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site. The proton pathway has not been identified, and the mechanism and timing of proton transfer during NO reduction is unknown. To address these questions, we have studied the reaction between NOR and the chemically less reactive oxidant O2 [Flock, Watmough and Ädelroth (2005) Biochemistry 44, 10711–10719]. When fully reduced NOR reacts with O2, proton-coupled electron transfer occurs in a reaction that is rate-limited by internal proton transfer from a group with a pKa of 6.6. This group is presumably an amino acid residue close to the active site that acts as a proton donor also during NO reduction. The results are discussed in the framework of a structural model that identifies possible candidates for the proton donor as well as for the proton-transfer pathway.
Effect of combined oxidative and nitrosative stress on Neisseria meningitidis
Authors: Dyet K. , Moir J.
Published: January 2006

Reactive oxygen and nitrogen species are produced by the human immune system in response to infection. Methods to detoxify these reactive species are vital to the survival of human pathogens, such as Neisseria meningitidis, which is the major aetiological agent of bacterial meningitis. Following activation, macrophages produce superoxide (O2−), hydrogen peroxide (H2O2) and nitric oxide (NO). The toxicity of O2−, generated using X/Xo (xanthine/xanthine oxidase), and H2O2 was investigated in the presence and absence of the NO donor DEA-NONOate [2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt]. Most of the toxicity from X/Xo was due to H2O2. In N. meningitidis, NO decreased the toxicity of the H2O2. In contrast, in the enteric bacterium Escherichia coli, NO increased the toxicity of the H2O2.
Neurotrophin signalling in health and disease
Authors: Chao Moses V. , Rajagopal Rithwick , Lee Francis S.
Published: January 2006

Neurotrophins are a unique family of polypeptide growth factors that influence the proliferation, differentiation, survival and death of neuronal and non-neuronal cells. They are essential for the health and well-being of the nervous system. NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3) and NT-4 (neurotrophin-4) also mediate additional higher-order activities, such as learning, memory and behaviour, in addition to their established functions for cell survival. The effects of neurotrophins depend upon their levels of availability, their affinity of binding to transmembrane receptors and the downstream signalling cascades that are stimulated after receptor activation. Alterations in neurotrophin levels have been implicated in neurodegenerative disorders, such as Alzheimer's disease and Huntington's disease, as well as psychiatric disorders, including depression and substance abuse. Difficulties in administering trophic factors have led to the consideration of using small molecules, such as GPCR (G-protein-coupled receptor) ligands, which can participate in transactivation events. In this review, we consider the signalling pathways activated by neurotrophins in both health and disease states.
Effect of chronic N-acetylcysteine treatment on the development of spontaneous hypertension
Authors: Pecháňová Olga , Zicha Josef , Kojšová Stanislava , Dobešová Zdenka , Jendeková Lýdia , Kuneš Jaroslav
Published: January 2006

The imbalance between NO (nitric oxide) and ROS (reactive oxygen species) is an important factor in the development of hypertension. The aim of the present study was to determine the preventive and therapeutic effects of NAC (N-acetylcysteine) in SHRs (spontaneously hypertensive rats). Young and adult SHRs and WKY (Wistar–Kyoto) rats were treated with NAC (20 g/l in the drinking water). After 8 weeks of treatment, BP (blood pressure) and NOS (NO synthase) activity, conjugated dienes and GSH (reduced glutathione) in the kidney and left ventricle were determined. Protein expression of eNOS (endothelial NOS), inducible NOS and NF-κB (nuclear factor κB) were also determined in the left ventricle and kidney. Chronic NAC treatment partially attenuated the rise in BP in young SHRs (179±6 compared with 210±8 mmHg in untreated animals), but it had no significant effect on BP in adult SHRs. The antioxidant action of NAC, measured as a decrease of the concentration of conjugated dienes or inhibition of NF-κB expression, was greater in young than in adult SHRs. Similarly, eNOS protein expression was attenuated more in young than in adult SHRs, although NAC treatment increased NOS activity to a similar extent in both young and adult rats. In conclusion, both decreased ROS production and increased NOS activity appear to participate in the BP changes after NAC treatment in young SHRs. In adult SHRs with established hypertension, however, the secondary alterations (such as pronounced structural remodelling of resistance vessels) might attenuate the therapeutic effect of NAC.
Neuroendocrine and metabolic effects of adipocyte-derived hormones
Authors: Jackson Malaka B. , Ahima Rexford S.
Published: January 2006

Obesity is characterized by an increase in adipose tissue mass. Contrary to the previous view of adipose tissue as simply an inert tissue devoted to energy storage, studies over the past decade have shown that adipose tissue is actively involved in regulating physiological processes and participates in disease. Adipose tissue secretes factors that exert local and systemic effects. Leptin, pro-inflammatory cytokines, resistin and proteins involved in haemodynamic regulation and coagulation are increased in obesity while adiponectin is reduced. The production of active corticosteroids is also increased in obesity. There is now growing evidence that adipocyte secretory factors regulate energy homoeostasis, as well as cardiovascular and immune systems. Some adipocyte hormones, most notably leptin, act in the brain to influence the neuroendocrine axis and energy balance, whereas adiponectin and resistin exert opposing effects on glucose and lipids. Understanding the actions of adipocyte hormones will provide novel insights into the pathophysiology and treatment of obesity.
RhoA/Rho-kinase in erectile tissue: mechanisms of disease and therapeutic insights
Authors: Jin Liming , Burnett Arthur L.
Published: January 2006

Penile erection is a complicated event involving the regulation of corpus cavernosal smooth muscle tone. Recently, the small monomeric G-protein RhoA and its downstream effector Rho-kinase have been proposed to be important players for mediating vasoconstriction in the penis. RhoA/Rho-kinase increases MLC (myosin light chain) phosphorylation through inhibition of MLCP (MLC phosphatase) thereby increasing Ca2+ sensitivity. This review will outline the RhoA/Rho-kinase signalling pathway, including the upstream regulators, guanine nucleotide exchange factors, GDP dissociation inhibitors and GTPase-activating proteins. We also summarize the current knowledge about the physiological roles of RhoA/Rho-kinase in both male and female erectile tissues and its aberrations contributing to erectile dysfunction in several disease states. Understanding the RhoA/Rho-kinase signalling pathway in the regulation of erection is important for the development of therapeutic interventions for erectile dysfunction.
Endothelial dysfunction in growth hormone transgenic mice
Authors: Andersson Irene J. , Johansson Maria E. , Wickman Anna , Bohlooly-Y Mohammad , Klintland Natalia , Caidahl Kenneth , Gustafsson Maria , Borén Jan , Gan Li-ming , Bergström Göran
Published: January 2006

Acromegaly [overproduction of GH (growth hormone)] is associated with cardiovascular disease. Transgenic mice overexpressing bGH (bovine GH) develop hypertension and hypercholesterolaemia and could be a model for cardiovascular disease in acromegaly. The aims of the present study were to investigate the effects of excess GH on vascular function and to test whether oxidative stress affects endothelial function in bGH transgenic mice. We studied the ACh (acetylcholine)-induced relaxation response in aortic and carotid rings of young (9–11 weeks) and aged (22–24 weeks) female bGH transgenic mice and littermate control mice, without and with the addition of a free radical scavenger {MnTBAP [Mn(III)tetrakis(4-benzoic acid)porphyrin chloride]}. We also measured mRNA levels of eNOS (endothelial nitric oxide synthase) and EC-SOD (extracellular superoxide dismutase). Intracellular superoxide anion production in the vascular wall was estimated using a dihydroethidium probe. Carotid arteries from bGH transgenic mice had an impaired ACh-induced relaxation response (young, 46±7% compared with 69±8%; aged, 52±5% compared with 80±3%; P<0.05), whereas endothelial function in aorta was intact in young but impaired in aged bGH transgenic mice. Endothelial dysfunction was corrected by addition of MnTBAP in carotid arteries from young mice and in aortas from aged mice; however, MnTBAP did not correct endothelial dysfunction in carotid arteries from aged bGH transgenic mice. There was no difference in intracellular superoxide anion production between bGH transgenic mice and control mice, whereas mRNA expression of EC-SOD and eNOS was increased in aortas from young bGH transgenic mice compared with control mice (P<0.05). We interpret these data to suggest that bGH overexpression is associated with a time- and vessel-specific deterioration in endothelial function, initially caused by increased oxidative stress and later by other alterations in vascular function.