bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of signal transduction mediated by oxidized lipids: the role of the electrophile-responsive proteome
Authors: Ceaser E.K. , Moellering D.R. , Shiva S. , Ramachandran A. , Landar A. , Venkartraman A. , Crawford J. , Patel R. , Dickinson D.A. , Ulasova E. , Ji S. , Darley-Usmar V.M.
Published: June 2004

Cellular redox signalling is mediated by the post-translational modification of proteins by reactive oxygen/nitrogen species or the products derived from their reactions. In the case of oxidized lipids, several receptor-dependent and -independent mechanisms are now emerging. At low concentrations, adaptation to oxidative stress in the vasculature appears to be mediated by induction of antioxidant defences, including the synthesis of the intracellular antioxidant glutathione. At high concentrations apoptosis occurs through mechanisms that have yet to be defined in detail. Recent studies have revealed a mechanism through which electrophilic lipids, formed as the reaction products of oxidation, orchestrate these adaptive responses in the vasculature. Using a proteomics approach, we have identified a subset of proteins in cells that we term the electrophile-responsive proteome. Electrophilic modification of thiol groups in these proteins can initiate cell signalling events through the transcriptional activation of genes regulated by consensus sequences for the antioxidant response element found in their promoter regions. The insights gained from our understanding of the biology of these mechanisms will be discussed in the context of cardiovascular disease.
Exceptionally diverse morphotypes and genomes of crenarchaeal hyperthermophilic viruses
Authors: Prangishvili D. , Garrett R.A.
Published: June 2004

The remarkable diversity of the morphologies of viruses found in terrestrial hydrothermal environments with temperatures >80°C is unprecedented for aquatic ecosystems. The best-studied viruses from these habitats have been assigned to novel viral families: Fuselloviridae, Lipothrixviridae and Rudiviridae. They all have double-stranded DNA genomes and infect hyperthermophilic crenarchaea of the orders Sulfolobales and Thermoproteales. Representatives of the different viral families share a few homologous ORFs (open reading frames). However, about 90% of all ORFs in the seven sequenced genomes show no significant matches to sequences in public databases. This suggests that these hyperthermophilic viruses have exceptional biochemical solutions for biological functions. Specific features of genome organization, as well as strategies for DNA replication, suggest that phylogenetic relationships exist between crenarchaeal rudiviruses and the large eukaryal DNA viruses: poxviruses, the African swine fever virus and Chlorella viruses. Sequence patterns at the ends of the linear genome of the lipothrixvirus AFV1 are reminiscent of the telomeric ends of linear eukaryal chromosomes and suggest that a primitive telomeric mechanism operates in this virus.
Distribution and molecular investigation of highly thermophilic bacteria associated with cool soil environments
Authors: Rahman T.J. , Marchant R. , Banat I.M.
Published: June 2004

In this study, both molecular and culture-based methods were used to characterize thermophilic bacteria associated with the subsurface soil environment in Northern Ireland. A total of 53 thermophilic, aerobic, sporulating and non-sporulating bacteria were isolated from subsurface soil samples obtained from two sites. They were screened by amplified ribosomal DNA restriction analysis prior to 16 S rRNA gene sequencing. The majority of the sequences were associated with Geobacillus thermoleovorans (50%) and Geobacillus caldoxylosilyticus (34.6%). Isolates F10, F20 and Tf exhibited only 93% similarity with Geobacillus toebii strain F70. Hence they may represent a new species of the genus Geobacillus.
Habitat, applications and genomics of the aerobic, thermophilic genus Geobacillus
Authors: McMullan G. , Christie J.M. , Rahman T.J. , Banat I.M. , Ternan N.G. , Marchant R.
Published: June 2004

Thermophilic bacteria belonging to Bacillus genetic group 5 have been reclassified as being members of Geobacillus gen. nov., with G. stearothermophilus as the type strain. Geobacillus species, literally meaning earth or soil Bacillus, are widely distributed and readily isolated from natural and man-made thermophilic biotopes. Work within our group has however shown that an abundance of genetically distinct Geobacillus isolates can be obtained from temperate Irish soils. As with many thermophiles there is considerable interest in potential industrial application of these bacteria and their gene products. This review describes two novel applications for Geobacillus isolates, firstly in the metabolism of the herbicide glyphosate and secondly in the metabolism of quorum-sensing signal molecules from Gram-negative bacteria. Finally the current state of the art is described for Bacillus genomics, with details given of three independent genome-sequencing projects of Geobacillus isolates.
16 S rDNA primers and the unbiased assessment of thermophile diversity
Authors: Baker G.C. , Cowan D.A.
Published: June 2004

Our understanding of thermophile diversity is based predominantly on PCR studies of community DNA. ‘Universal’ and domain-specific rRNA gene PCR primers have historically been used for the assessment of microbial diversity without adequate regard to the degree of specificity of primer pairs to different prokaryotic groups. In a reassessment of the published primers commonly used for ‘universal’ and archaeal 16 S rDNA sequence amplification we note that substantial variations in specificity exist. An unconsidered choice of primers may therefore lead to significant bias in determination of microbial community composition. In particular, Archaea-specific primer sequences typically lack specificity for the Korarchaeota and Nanoarchaea and are often biased towards certain clades. New primer pairs specifically designed for ‘universal’ archaeal 16 S rDNA sequence amplification, with homology to all four archaeal groups, have been designed. Here we present the application of these new primers for preparation of 16 S libraries from thermophile communities.
Structure, function and evolution of the Archaeal class I fructose-1,6-bisphosphate aldolase
Authors: Lorentzen E. , Siebers B. , Hensel R. , Pohl E.
Published: June 2004

FBPA (fructose-1,6-bisphosphate aldolase) catalyses the reversible aldol condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate to form fructose 1,6-bisphosphate. Two classes of FBPA, which rely on different reaction mechanisms, have so far been discovered, class I mainly found in Eucarya and class II mainly in Bacteria. Only recently were genes encoding proteins with FBPA activity identified in Archaea. Archaeal FBPAs do not share any significant overall sequence identity with members of the traditional classes of FBPAs, raising the interesting question of whether they have evolved independently by convergent evolution or diverged from a common ancestor. Biochemical characterization of FBPAs of the two hyperthermophilic Archaea Thermoproteus tenax and Pyrococcus furiosus showed that the enzymes use a Schiff-base mechanism and thus belong to the class I aldolases. The crystal structure of the archaeal FBPA from T. tenax revealed that the protein fold, as for the classical FBPA I and II, is that of a parallel (βα)8 barrel. A substrate-bound crystal structure allowed detailed active-site comparisons which showed the conservation of six important catalytic and substrate-binding residues between the archaeal and the classical FBPA I. This observation provides further evidence that the two sequence families of proteins share a common evolutionary origin. Furthermore, structure and sequence analysis indicate that the class I FBPA shares a common evolutionary origin with several other enzyme superfamilies of the (βα)8 barrel fold.
From hyperthermophiles to psychrophiles: the structural basis of temperature stability of the amino acid dehydrogenases
Authors: Baker P.J.
Published: June 2004

The determination and comparison of the structures of a number of glutamate dehydrogenases that span the spectrum of thermal stability from mesophiles to hyperthermophiles have revealed that the formation of extended ion-pair networks is a major stabilizing feature in the adaptation of the organism to life at 100°C. Analysis of the structures of other hyperthermophilic proteins has shown that ion-pair networks also play roles in their stabilization, including the phosphoglucose isomerase from Pyrococcus furiosus and the maltosyltransferase from Thermotoga maritima.
Exploring and exploiting starch-modifying amylomaltases from thermophiles
Authors: Kaper T. , van der Maarel M.J.E.C. , Euverink G.J.W. , Dijkhuizen L.
Published: June 2004

Starch is a staple food present in water-insoluble granules in many economically important crops. It is composed of two glucose polymers: the linear α-1,4-linked amylose and amylopectin with a backbone of α-1,4-glycosidic bonds and α-1,6-linked side chains. To dissolve starch completely in water it needs to be heated; when it cools down too much the starch solution forms a thermo-irreversible gel. Amylomaltases (EC 2.4.1.25) are enzymes that transfer a segment of an α-1,4-d-glucan to a new 4-position in an acceptor, which may be glucose or another α-1,4-d-glucan. Acting upon starch, amylomaltases can produce cycloamylose or a thermoreversible starch gel, both of which are of commercial interest.
Potential for using thermophilic anaerobic bacteria for bioethanol production from hemicellulose
Authors: Sommer P. , Georgieva T. , Ahring B.K.
Published: June 2004

A limited number of bacteria, yeast and fungi can convert hemicellulose or its monomers (xylose, arabinose, mannose and galactose) into ethanol with a satisfactory yield and productivity. In the present study we tested a number of thermophilic enrichment cultures, and new isolates of thermophilic anaerobic bacterial strains growing optimally at 70–80°C for their ethanol production from d-xylose. The new isolates came from different natural and man-made systems such as hot springs, paper pulp mills and brewery waste water. The test was composed of three different steps; (i) test for conversion of d-xylose into ethanol; (ii) test for viability and ethanol production in pretreated wheat straw hemicellulose hydrolysate; (iii) test for tolerance against high d-xylose concentrations. A total of 86 enrichment cultures and 58 pure cultures were tested and five candidates were selected which successfully fulfilled the criteria defined for the screening test.
Application of thermophilic enzymes in commercial biotransformation processes
Authors: Taylor I.N. , Brown R.C. , Bycroft M. , King G. , Littlechild J.A. , Lloyd M.C. , Praquin C. , Toogood H.S. , Taylor S.J.C.
Published: June 2004

Biocatalysis is a useful tool in the provision of chiral technology and extremophilic enzymes are just one component in that toolbox. Their role is not always attributable to their extremophilic properties; as with any biocatalyst certain other criteria should be satisfied. Those requirements for a useful biocatalyst will be discussed including issues of selectivity, volume efficiency, security of supply, technology integration, intellectual property and regulatory compliance. Here we discuss the discovery and commercialization of an l-aminoacylase from Thermococcus litoralis, the product of a LINK project between Chirotech Technology and the University of Exeter. The enzyme was cloned into Escherichia coli to aid production via established mesophilic fermentation protocols. A simple downstream process was then developed to assist in the production of the enzyme as a genetically modified-organism-free reagent. The fermentation and downstream processes are operated at the 500 litre scale. Characterization of the enzyme demonstrated a substrate preference for N-benzoyl groups over N-acetyl groups. The operational parameters have been defined in part by substrate-concentration tolerances and also thermostabilty. Several examples of commercial biotransformations will be discussed including a process that is successful by virtue of the enzyme's thermotolerance.
Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts
Authors: Bergquist P.L. , Te'o V.S.J. , Gibbs M.D. , Curach N.C. , Nevalainen K.M.H.
Published: June 2004

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.
Triosephosphate isomerase of the hyperthermophile Thermoproteus tenax: thermostability is not everything
Authors: Walden H. , Taylor G. , Lilie H. , Knura T. , Hensel R.
Published: June 2004

The triosephosphate isomerase of the hyperthermophilic crenarchaeum Thermoproteus tenax (TtxTIM) represents a homomeric tetramer. Unlike the triosephosphate isomerases of other hyperthermophiles, however, the association of the TtxTIM tetramers is looser, allowing a reversible dissociation into inactive dimers. The dimer/tetramer equilibrium of TtxTIM is shifted to the tetrameric state through a specific interaction with glycerol-1-phosphate dehydrogenase of T. tenax, suggesting that higher oligomerization of the TtxTIM serves functional rather than stabilizing purposes.
Phosphoinositide 3-kinase: the protein kinase that time forgot
Authors: Foukas L.C. , Shepherd P.R.
Published: June 2004

Class I phosphoinositide 3-kinases were originally characterized as lipid kinases, although more than 10 years ago they were also found to phosphorylate protein serine residues. However, while there is a vast amount of data on the function of this lipid kinase activity, relatively little is known about the function of the protein kinase activity. We discuss the evidence that suggests that the protein kinase activity of phosphoinositide 3-kinases mediates important signalling functions in cells.
Phosphoinositide 3-kinase in T cell activation and survival
Authors: Okkenhaug K. , Bilancio A. , Emery J.L. , Vanhaesebroeck B.
Published: June 2004

PI3Ks (phosphoinositide 3-kinases) regulate diverse signalling pathways involved in growth, proliferation, survival, differentiation and metabolism. In T cells, PI3Ks can be activated by a number of different receptors, including the TcR (T cell receptor), co-stimulatory receptors, cytokine receptors and chemokine receptors. However, the specific roles of PI3Ks downstream of these receptors vary. An inactivating mutation in the leucocyte-specific PI3K isoform p110 δ results in impaired TcR-dependent proliferation under circumstances where CD28 co-stimulation is blocked or not required. Recruitment and activation of PI3K by CD28 promotes survival by inducing increased expression of Bcl-XL. However, CD28 engages additional signals that regulate proliferation and interleukin-2 production independently of PI3K. Thus a model emerges whereby PI3K is involved in both TcR and CD28 signalling, but each receptor may only exploit a subset of the signalling pathways potentially controlled by PI3K activation.
Visualizing signalling by phosphoinositide 3-kinase pathway lipids
Authors: Prestwich G.D.
Published: June 2004

Cells signal through lipids produced by phospholipid and phosphoinositide metabolism that involves three enzymic processes: (i) ester and phosphodiester hydrolysis by phospholipases; (ii) monophosphate hydrolysis by phosphatases; and (iii) phosphorylation of hydroxy groups by kinases. Unregulated enzyme activity correlates with specific pathologies, which are specific targets for therapeutic intervention. Three categories of reagents developed at the University of Utah and at Echelon Biosciences permit monitoring of in vitro enzyme activity and spatiotemporal changes in intracellular lipid concentrations, and identification of lipid–protein interactions.
Acute regulation of the tumour suppressor phosphatase, PTEN, by anionic lipids and reactive oxygen species
Authors: Downes C.P. , Walker S. , McConnachie G. , Lindsay Y. , Batty I.H. , Leslie N.R.
Published: June 2004

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a member of the protein tyrosine phosphatase family that is structurally adapted to facilitate the metabolism of 3-phosphoinositide lipid second messengers, especially PtdIns(3,4,5)P3. Cellular PTEN activity is restrained by the retention of C-terminally phosphorylated enzyme in the cytosol. Dephosphorylation by as yet undefined phosphatases initiates an electrostatic switch which targets PTEN specifically to the plasma membrane, where it binds through multiple positively charged residues in both the C2 and N-terminal domains and is susceptible to feedback regulation through proteolytic degradation. PTEN also forms signalling complexes with PDZ domain-containing adaptors, such as the MAGUK (membrane-associated guanylate kinase) proteins, interactions which appear to be necessary for metabolism of localized pools of PtdIns(3,4,5)P3 involved in regulating actin cytoskeleton dynamics. TPIP [TPTE (transmembrane phosphatase with tensin homology) and PTEN homologous inositol lipid phosphatase] is a novel gene product which exists in multiply spliced forms. TPIPα has PtdIns(3,4,5)P3 3-phosphatase activity and is localized to the endoplasmic reticulum, via two transmembrane spanning regions, where it may metabolize PtdIns(3,4,5)P3 that appears to be unaffected by expressed PTEN. PTEN can be acutely regulated by oxidative stress and by endogenously produced reactive oxygen species. This mechanism provides a novel means to stimulate phosphoinositide 3-kinase-dependent signalling pathways, which may be important in circumstances where PtdIns(3,4,5)P3 and oxidants are produced concurrently.
Physiological functions of protein kinase B/Akt
Authors: Yang Z.-Z. , Tschopp O. , Baudry A. , Dümmler B. , Hynx D. , Hemmings B.A.
Published: June 2004

The genetic manipulation of mice has become an essential and elegant method for studying the function of proteins in physiology, and for testing the veracity of information obtained from cell culture experiments. During the past few years, a variety of transgenic and knockout mouse models of PKB (protein kinase B)/Akt have been generated and investigated. In this paper, we focus on the phenotypes of these PKB/Akt overexpression and mutant mice that may help to elucidate the functions exerted by PKB/Akt in mammals.
Functional analysis of the tumour suppressor gene PTEN in murine B cells and keratinocytes
Authors: Suzuki A. , Sasaki T. , Mak T.W. , Nakano T.
Published: June 2004

To investigate the roles of the PTEN (phosphatase and tensin homologue deleted from chromosome 10)/PI3K (phosphoinositide 3-kinase) signalling pathway in vivo, we have generated a series of mutant mice with null or tissue-specific gene-targeted deletions of Pten. Here we present our investigations of Pten function in B cells and keratinocytes in mice. Mice with a B cell-specific mutation of Pten showed increased serum autoantibodies and elevated numbers of B1a cells. Among conventional B (B2) cells in mutant spleens, numbers of marginal zone B cells were significantly increased, while those of follicular B cells were reciprocally decreased. Immunoglobulin class switch recombination was defective and associated with impaired induction of activation-induced cytidine deaminase. Mice with a keratinocyte-specific mutation of Pten exhibited epidermal hyperplasia, hyperkeratosis and accelerated skin morphogenesis. Within 3 weeks of birth, 90% of these animals died of malnutrition, possibly caused by hyperkeratosis of the oesophageal epithelia. Surviving mutant mice developed spontaneous skin tumours within 8.5 months of birth, and chemical treatment accelerated the onset of tumours. Our data show that PTEN is an important regulator in B cells and keratinocytes.
PI 3-kinase inhibition: a therapeutic target for respiratory disease
Authors: Finan P.M. , Thomas M.J.
Published: June 2004

Asthma and COPD (chronic obstructive pulmonary disease) are a growing major health burden, which, despite improvements in disease management, still require new effective treatments. As our understanding of the cellular and molecular processes which govern respiratory diseases improves, the range of potential therapeutic targets increase. PI 3-kinases (phosphoinositide 3-kinases) are a family of closely related enzymes, which play pivotal roles in a diverse array of cellular mechanisms. In the present paper, we review the evidence for PI 3-kinase involvement in various cellular processes underlying asthma and COPD generated through inhibitor studies and gene-targeting approaches, and discuss the prospects for PI 3-kinase inhibition as a future therapeutic strategy for the treatment of respiratory disease.
Phosphoinositide 3-kinases and the regulation of platelet function
Authors: Jackson S.P. , Yap C.L. , Anderson K.E.
Published: June 2004

A clear understanding of the role of PI (phosphoinositide) 3-kinases in supporting the haemostatic function of platelets has been slow to evolve. In fact, insight into the roles of individual PI 3-kinase isoforms in platelet function remains rudimentary. However, based on in vitro studies using wortmannin and LY294002, there is evidence for an important role for PI 3-kinases in regulating a broad range of functional platelet responses, including primary platelet adhesion, cytoskeletal remodelling and platelet aggregation. One of the critical platelet responses involves affinity regulation of the major platelet integrin αIIbβ3, the primary receptor mediating platelet aggregation and thrombus growth. The input signals regulating integrin αIIbβ3 can be divided into three main groups: (1) Gq-coupled receptors linked to the activation of PLCβ (phospholipase Cβ); (2) Gi-coupled receptors linked to the regulation of adenylate cyclase and Rap1b; and (3) adhesion receptor signalling involving Src kinase-dependent activation of PLCγ isoforms. PI 3-kinases have not been demonstrated to play a critical role in Gq-dependent platelet activation; however, one or more PI 3-kinase isoforms appears to be important for Gi-dependent activation of Rap1b and adhesion receptor activation of PLCγ isoforms. Thus distinct co-operative PI 3-kinase signalling mechanisms appear to play an important role in regulating the adhesive function of integrin αIIbβ3.
Vascular zip codes in angiogenesis and metastasis
Authors: Ruoslahti E.
Published: June 2004

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular differences in the blood vessels of individual normal tissues. Pathological lesions also put their signature on the vasculature; in tumours, both blood and lymphatic vessels differ from normal vessels. The changes that characterize tumour blood vessels include selective expression of certain integrins. Peptides isolated by in vivo phage display for homing to tumours have been shown to be useful in directing therapeutic agents to experimental tumours. The targeting can enhance the efficacy of the therapy while reducing side effects. Phage screening has also revealed lung-specific vascular markers that promote tumour metastasis to the lungs by mediating specific adherence of tumour cells to the lung vasculature. These phage-screening studies have revealed a previously unsuspected degree of vascular specialization and provide potentially useful guidance devices for targeted therapies.
Imaging protein–protein interactions in cell motility using fluorescence resonance energy transfer (FRET)
Authors: Parsons M. , Vojnovic B. , Ameer-Beg S.
Published: June 2004

Protein–protein interactions and signal transduction pathways have traditionally been analysed using biochemical techniques or standard microscopy. Although invaluable in the delineation of protein hierarchy, these methods do not provide information on the true spatial and temporal nature of complex formation within the intact cell. Recent advances in microscopy have allowed the development of new methods to analyse protein–protein interactions at very high resolution in both fixed and live cells. The present paper provides a brief overview of using fluorescence resonance energy transfer to analyse directly molecular interactions and conformational changes in various proteins involved in the regulation of cell adhesion and motility.
Characterization of the survival effect of tumour necrosis factor-α in human neutrophils
Authors: Walmsley S.R. , Cowburn A.S. , Sobolewski A. , Murray J. , Farahi N. , Sabroe I. , Chilvers E.R.
Published: June 2004

Granulocyte apoptosis has been proposed as a fundamental, injury-limiting granulocyte-clearance mechanism. As such, inhibition of this process may prevent the resolution of inflammation. Our previous studies have shown that TNFα (tumour necrosis factor-α) has a bi-modal influence on the rate of constitutive neutrophil apoptosis in vitro, causing early acceleration and late inhibition of this process. The pro-apoptotic effect is uniquely TNFR1 (TNF receptor 1) and TNFR2-dependent and the latter survival process is mediated via phosphoinositide 3-kinase and NF-κB (nuclear factor-κB) activation. In the present study, we show that, in contrast with GM-CSF (granulocyte/macrophage colony-stimulating factor), the delayed addition (i.e. at 6 h) of TNFα increases its survival effect despite substantial loss of neutrophil TNFR1 and TNFR2 at that time. This paradox was resolved using PBMC (peripheral blood mononuclear cell)-deplete and 5% PBMC-replete neutrophil cultures, where the enhanced survival effect observed after delayed TNFα addition was shown to be PBMC-dependent. TNFR2-blocking antibodies had no effect on the late survival effect of TNFα, implying a TNFR1-dependent process. Finally, I-κBα (inhibitory κB-α) and NF-κB time-course studies demonstrated that the survival effects of both GM-CSF and TNFα could be explained by maintenance of functional NF-κB.
What can we learn from highly purified neutrophils?
Authors: Sabroe I. , Prince L.R. , Dower S.K. , Walmsley S.R. , Chilvers E.R , Whyte M.K.B.
Published: June 2004

Neutrophil purification has traditionally been performed by centrifugation of leucocytes through density gradients. These reliable methods produce populations that are typically >95% pure neutrophils, and have allowed the widespread study of the function of these cells. Our recent work has suggested that residual monocytes may play a more important role than has been previously realized, and suggest that for some functional experiments, further purification of cells is required to understand fully the neutrophil phenotype.
Gene profiling of in vitro and in vivo models of delayed neutrophil apoptosis: a common pathway?
Authors: O'Neill A. , Greenan M.C. , Doyle B. , Fitzpatrick J.M. , Watson R.W.G.
Published: June 2004

Mechanisms responsible for the termination of an inflammatory response include the activation of a genetic programme of cellular suicide termed apoptosis, which leads to the elimination of the cellular effectors of acute inflammation, particularly the neutrophil. However, delays in this response result in the persistence of inflammation and the development of inflammatory disorders. Understanding the mechanism that inhibits the process of cell death may be helpful in the treatment of inflammatory disorders. Inflammatory cytokines have been shown to inhibit apoptosis through stabilization of the mitochondria and inhibition of the caspase cascade. To date, how these processes are inhibited remains the central question. We hypothesize that the decision for the delay in neutrophil apoptosis is made through signals delivered on the cell surface, which activate combinations of specific genes that inhibit the cell death pathway. Gene chip microarray experiments were performed in in vivo and in vitro models of delayed neutrophil apoptosis. Analysis has yielded changes in a large number of genes involved in inflammation, metabolism, signalling, mitochondrial function and apoptosis. A number of genes have been identified as suitable targets responsible for the regulation of neutrophil apoptosis and their expression was confirmed by real-time PCR and explored at the level of the protein. Their functional role in the apoptotic response is now being determined. One significant finding is that the gene patterns of delay in vitro and in vivo appear to be different, indicating the possibility for different pathways regulating the delay in neutrophil apoptosis.
Early membrane events in polymorphonuclear cell (PMN) apoptosis: membrane blebbing and vesicle release, CD43 and CD16 down-regulation and phosphatidylserine externalization
Authors: Nusbaum P. , Lainé C. , Seveau S. , Lesavre P. , Halbwachs-Mecarelli L.
Published: June 2004

CD43 down-regulation during the apoptosis of PMN (polymorphonuclear cells) is not caused by proteolysis or internalization. Could it be released with bleb-derived membrane vesicles? Membrane blebbing was followed by microscopy on PMN ‘synchronized’ by an overnight incubation at 15°C before their spontaneous apoptosis at 37°C. Released vesicles were quantified by flow cytometry. Membrane blebbing, release of bleb-derived membrane vesicles, decrease of CD43/CD16 expression and phosphatidylserine externalization occurred simultaneously. However, caspase and PKC inhibition prevented annexin binding but not blebbing, vesicle release or CD43 expression decrease; myosin light chain kinase inhibition prevented cell blebbing and vesicle release but had no effect on CD43/CD16 down-regulation or annexin V binding. By electron microscopy, CD43 appeared poorly expressed on membrane blebs and concentrated at bleb ‘necks’. In conclusion, CD43 down-regulation is not caused by cell blebbing. Cell blebbing, phospholipid ‘flip-flop’ and CD43/CD16 down-regulation are independent membrane events.
Regulation of neutrophil apoptosis by Mcl-1
Authors: Edwards S.W. , Derouet M. , Howse M. , Moots R.J.
Published: June 2004

Neutrophils rapidly undergo spontaneous apoptosis, but this process can be considerably delayed by exposure to a variety of agents such as pro-inflammatory cytokines. The anti-apoptotic protein of the Bcl-2 family, Mcl-1, plays a key role in the regulation of neutrophil apoptosis. The protein has some unusual properties compared with other family members, including an extremely high turnover rate. Many factors, such as cytokines and local oxygen concentrations, can regulate cellular levels of Mcl-1 via transcription and post-transcriptional modification, control the survival time of neutrophils within tissues and thereby influence the inflammatory response.
The importance of resolution of inflammation in the pathogenesis of ANCA-associated vasculitis
Authors: Harper L. , Williams J.M. , Savage C.O.
Published: June 2004

The primary small-vessel systemic vasculitides are disorders that target small blood vessels, inducing vessel wall inflammation, and are associated with the development of anti-neutrophil cytoplasmic antibodies. Multiple organs are attacked, including the lungs and kidneys. Increasing knowledge of pathogenesis suggests that the antibodies activate neutrophils inappropriately, leading to endothelial and vascular damage. Cytokines, such as tumour necrosis factor, can facilitate damage by priming the neutrophils and activating endothelial cells. Apoptosis of infiltrating neutrophils is also disrupted by anti-neutrophil cytoplasmic antibody activation, and removal of these effete cells occurs in a pro-inflammatory manner, promoting persistent inflammation. The autoimmune response may be promoted by aberrant phagocytosis of apoptotic neutrophils by dendritic cells. Understanding the pathogenesis can help to rationalize existing therapies and indicate new approaches to therapy.
Annexin 1 and neutrophil apoptosis
Authors: Perretti M. , Solito E.
Published: June 2004

ANXA1 (annexin 1), a member of the ‘annexin’ family of calcium- and phospholipid-binding proteins, was originally identified as an endogenous mediator of the anti-inflammatory actions of glucocorticoids. However, this protein exerts multiple inhibitory effects on the host inflammatory response, including a preferential regulation of the adhesion step of blood-borne neutrophil within the microenvironment of an inflamed vasculature. It is now emerging that ANXA1 is endowed with other roles, since the protein is abundant in inflammatory exudates as it is produced and released by the extravasated neutrophil. In the present paper, we review the novel proapoptotic effect of ANXA1 and discuss its potential with respect to the pathophysiology of inflammation and leucocyte recruitment.
The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance
Authors: Peters J.A. , Kelley S.P. , Dunlop J.I. , Kirkness E.F. , Hales T.G. , Lambert J.J.
Published: June 2004

5-HT3 (5-hydroxytryptamine type 3) receptors are cation-selective ion channels of the Cys-loop transmitter-gated ion channel superfamily. Two 5-HT3 receptor subunits, 5-HT3A and 5-HT3B, have been characterized in detail, although additional putative 5-HT3 subunit genes (HTR3C, HTR3D and HTR3E) have recently been reported. 5-HT3 receptors function as homopentameric assemblies of the 5-HT3 subunit, or heteropentamers of 5-HT3A and 5-HT3B subunits of unknown stoichiometry. The single-channel conductances of human recombinant homomeric and heteromeric 5-HT3 receptors are markedly different, being <1 and approx. 16 pS respectively. Paradoxically, from the results of studies performed on the closely related nicotinic acetylcholine receptor, the channel-lining M2 domain of the 5-HT3A subunit is predicted to enhance cation conduction, whereas that of the 5-HT3B subunit would not. The present study describes a novel determinant of single-channel conductance, outwith the M2 domain, which accounts for this anomaly. Utilizing a panel of chimaeric 5-HT3A and 5-HT3B subunits, a profound determinant of single-channel conductance was traced to a putative amphipathic helix (the ‘HA stretch’) within the large cytoplasmic loop of the receptor. Replacement of three arginine residues (R432, R436 and R440) unique to the HA stretch of the 5-HT3A subunit with the aligned residues (Q395, D399 and A403) of the 5-HT3B subunit increased the single-channel conductance 28-fold. Significantly, from ultrastructural studies of the Torpedo nicotinic acetylcholine receptor, the key residues may be components of narrow openings within the inner vestibule of the channel, located in the cytoplasm, which contribute to the permeation pathway. Our findings indicate an important and hitherto unappreciated function for the HA stretch in the Cys-loop family of transmitter-gated ion channels.
Electronic conduction in shock-compressed water
Authors: Celliers P. M. , Collins G. W. , Hicks D. G. , Koenig M. , Henry E. , Benuzzi-Mounaix A. , Batani D. , Bradley D. K. , Da Silva L. B. , Wallace R. J. , Moon S. J. , Eggert J. H. , Lee K. K. M. , Benedetti L. R. , Jeanloz R. , Masclet I. , Dague N. , Marchet B. , Rabec Le Gloahec M. , Reverdin Ch. , Pasley J. , Willi O. , Neely D. , Danson C.
Published: June 2004

The optical reflectance of a strong shock front in water increases continuously with pressure above 100 GPa and saturates at ∼45% reflectance above 250 GPa. This is the first evidence of electronic conduction in high pressure water. In addition, the water Hugoniot equation of state up to 790 GPa (7.9 Mbar) is determined from shock velocity measurements made by detecting the Doppler shift of reflected light. From a fit to the reflectance data we find that an electronic mobility gap ∼2.5 eV controls thermal activation of electronic carriers at pressures in the range of 100–150 GPa. This suggests that electronic conduction contributes significantly to the total conductivity along the Neptune isentrope above 150 GPa.
Nonlinear effects associated with interactions of intense photons with a photon gas
Authors: Shukla Padma K. , Marklund Mattias , Tskhakaya Davy D. , Eliasson Bengt
Published: June 2004

The derivative correction to the Heisenberg–Euler Lagrangian has been introduced. A general dispersion relation for a photon traveling on a slowly varying background electromagnetic field has been presented. A set of equations describing the nonlinear propagation of an electromagnetic pulse on a radiation fluid background is then derived. Novel modulational and filamentational instabilities are found, and using numerical methods, it has been shown that electromagnetic pulses may collapse and split into pulse trains. Also presented are analytical results concerning the collapse, split, and Mach cone formation. The implications of the results are discussed.
Turbulent vortex dipoles in a shallow water layer
Authors: Sous Damien , Bonneton Natalie , Sommeria Joël
Published: June 2004

This paper describes an experimental study on turbulent dipolar vortices in a shallow water layer. Dipoles are generated by an impulsive horizontal jet, by which a localized three-dimensional turbulent flow region is created. Dipole emergence is only controlled by the confinement number C=Q/H2tinj whereas the jet Reynolds number Re=Q/v has no influence in the studied range 50 000<Re<75 000 (H is the water depth, v is the kinematical viscosity, Q the injected momentum flux and tinj the injection duration). When C>2, the flow becomes quasi-two-dimensional and a single vortex dipole emerges in most cases. By qualitative observations and application of particle image velocimetry, the main dipole features have been determined. The shallow water dipoles are characterized by the simultaneous presence of several scales of turbulence: A quasi-two-dimensional main flow at large scale and three-dimensional turbulent motions at small scale. A vertical circulation takes place in the dipole front. A theoretical model is presented and compared to experimental results. The three-dimensional turbulence production occurs mainly in the frontal circulation. A good agreement has been found between the model prediction and the measurements for the velocity evolution.
Uphill transport and the probabilistic transport model
Authors: van Milligen B. Ph. , Carreras B. A. , Sánchez R.
Published: June 2004

Profile consistency is a long-standing mystery of transport in thermonuclear fusion plasmas. This phenomenon is critically tested by studying the system response to off-axis fueling or heating. The present paper investigates the potential of a recently proposed probabilistic transport model to simulate this phenomenon, and it is found that most of the observed phenomenology can be reproduced, at least qualitatively. In particular, the observed differences between tokamaks and stellarators under off-axis heating may find an explanation in this framework.
Power deposition by neutral beam injected fast ions in field-reversed configurations
Authors: Takahashi Toshiki , Kato Takayuki , Kondoh Yoshiomi , Iwasawa Naotaka
Published: June 2004

The effects of Coulomb collisions on neutral beam (NB) injected fast ions into field-reversed configuration (FRC) plasmas are investigated by calculating the single particle orbits, where the ions are subject to the slowing-down and pitch-angle collisions. The Monte Carlo method is used for the pitch-angle scattering, and the friction term is added to the equation of motion to show the effects of the slowing-down collision, such as the deposited power profile. The calculation parameters used are relevant to the NB injection on the FRC injection experiment device [T. Asai, Y. Suzuki, T. Yoneda, F. Kodera, M. Okubo, and S. Goto, Phys. Plasmas 7, 2294 (2000)]. It is found that the dominant local power deposition occurs in the open field region between the X point and the mirror point because of a concentration of fast ions and a longer duration travel at the mirror reflection point. In the present calculation, the maximum deposited power to the FRC plasma is about 10% of the injected power. Although the pitch-angle scattering by Coulomb collision destroys the mirror confinement of NB injected fast ions, this effect is found to be negligible. The loss mechanism due to nonadiabatic fast ion motion, which is intrinsic in nonuniform FRC plasmas, has a much greater effect than the pitch-angle scattering by Coulomb collision.
Time development of a current-free double-layer
Authors: Charles C. , Boswell R. W.
Published: June 2004

The time development of a supersonic (∼2.3cs) argon ion beam generated by a current-free double-layer (DL) is obtained by pulsing a “helicon” discharge (13.56 MHz) and measuring the total ion current and the ion beam current during the first few milliseconds using a retarding field energy analyzer. The ion beam current is detected during the plasma breakdown phase (60–250 μs) and is stable thereafter (⩾250 μs). Temporal measurements of the floating potential upstream and downstream of the DL show evidence of wall charging in the plasma source during the first 250 μs of the discharge which appear to be related to the appearance of the double-layer. A comparison between the DL case and a somewhat higher pressure “non DL” case suggests that the double-layer is formed at ∼100 μs.
Multiple isoforms of starch branching enzyme-I in wheat: lack of the major SBE-I isoform does not alter starch phenotype
Authors: Regina Ahmed , Kosar-Hashemi Behjat , Li Zhongyi , Rampling Lynette , Cmiel Mark , Gianibelli Maria C. , Konik-Rose Christine , Larroque Oscar , Rahman Sadequr , Morell Matthew K.
Published: June 2004

The role of starch branching enzyme-I (SBE-I) in determining starch structure in the endosperm has been investigated. Null mutations of SBE-I at the A, B and D genomes of wheat were identified in Australian wheat varieties by immunoblotting. By combining individual null mutations at the B and D genomes through hybridisation, a double-null mutant wheat, which lacks the B and D isoforms of SBE-I, was developed. Wheat mutants lacking all the three isoforms of SBE-I were generated from a doubled haploid progeny of a cross between the BD double-null mutant line and a Chinese Spring (CS) deletion line lacking the A genome isoform. Comparison of starch from this mutant wheat to that from wild type revealed no substantial alteration in any of the structural or functional properties analysed. Further analysis of this triple-null mutant line revealed the presence of another residual peak of SBE-I activity, referred to as SBE-Ir, in wheat endosperm representing < 3% of the activity of SBE-I in wild type endosperm.
The effect of different height reducing genes on the early growth of wheat
Authors: Ellis Marc H. , Rebetzke Greg J. , Chandler Peter , Bonnett David , Spielmeyer Wolfgang , Richards Richard A.
Published: June 2004

Genes that reduce height without compromising seedling vigour or coleoptile length have great potential for wheat improvement. We therefore investigated the effects of various reduced height (Rht) genes on the early stages of plant development, using a combination of near isogenic, recombinant, mutant and wild type comparisons. Gibberellin (GA) insensitivity caused by Rht-B1b or Rht-D1b was associated with reduced leaf elongation rate and coleoptile length. Similar results were found for two other sources of dwarfing, Rht11 and Rht17. We found one class of Rht genes (e.g. Rht8) which had no effect on coleoptile length, leaf elongation rate or responsiveness to GA, indicating that these dwarfing genes may act later in wheat development to reduce height and increase harvest index, without affecting early growth. A third class of Rht genes was found in three durum backgrounds. These had reduced coleoptile lengths and leaf elongation rates, but had a greater response to GA than the corresponding tall varieties. We discuss these results in relation to the possible mechanisms underlying the reduction in height and the suitability of the different Rht genes for wheat improvement.
A validation, comparison and error analysis of two heat-pulse methods for measuring sap flow in Eucalyptus marginata saplings
Authors: Bleby Timothy M. , Burgess Stephen S. O. , Adams Mark A.
Published: June 2004

We validated and compared two heat-pulse methods for measuring sap flow in potted Eucalyptus
marginata Donn ex. Smith (jarrah) saplings. During daylight hours and under well-watered conditions, rates of sap flow (0.1–0.5 kg h–1) measured by the established compensation heat-pulse method (CHPM) and the newly developed heat-ratio method (HRM) were similar to rates measured with a weighing lysimeter, and most of the time there was no significant difference (P<0.001) between methods. The HRM accurately described sap flow at night when rates of flow were low (< 0.1 kg h–1) or near zero, but the CHPM was unable to measure low rates of sap flow due to its inability to distinguish heat-pulse velocities below a threshold velocity of 0.1 kg h–1 (3–4 cm h–1). The greatest potential for error in the calculation of daily sap flow was associated with the misalignment of temperature sensors, the estimation of sapwood area and the method used to acquire total sap flow from point measurements of sap velocity. A direct comparison of the two heat-pulse methods (applied synchronously) revealed that the HRM had a more convincing mechanism for correcting spacing errors and was more resistant to random fluctuation in measurements than the CHPM. While we view the HRM more favourably than the CHPM in some key areas, both methods are valid and useful, within their constraints, for measuring transpiration in jarrah and other woody species.
Accounting for the effect of temperature on squid growth in nature: from hypothesis to practice
Authors: Forsythe J. W.
Published: June 2004

The impact of temperature on cephalopod growth has become a productive area of study. Current knowledge of squid growth owes much to earlier laboratory studies on octopuses and cuttlefishes that revealed rapid temperature-sensitive growth. Advances in laboratory culture of squids eventually revealed the dramatic extent to which rising water temperature accelerates growth rates. This led to proposal and testing of a working hypothesis, the Forsythe Hypothesis, that during periods of gradually warming temperature, monthly cohorts of squids experience warmer conditions and grow faster, and perhaps larger, than older, earlier-hatched cohorts. The advent of statolith increment analysis for determining age in field-caught squids has provided a powerful tool in ground-truthing this hypothesis in nature. This hypothesis, now termed here the Forsythe Effect, has been laboratory- and field-tested over the past 10 years and been strongly supported. Food availability and inherent species-specific physiological limits must also be accounted for in predicting growth. Gaps still exist in our precise understanding of the temperature-induced changes in form and duration of squid growth. Acute and chronic ocean-scale seawater-temperature change events will compel us to look to squids as ‘bioindicators’ of environmental condition and to statoliths as the ‘archives’ of this information.