bluuebunny/crossref_metadata_2025 huggingface dataset:
Finding extraterrestrial sites for thermophiles
Authors: Naylor T.
Published: June 2004

Virtually our entire knowledge of the universe comes from two sorts of measurement of the electromagnetic radiation from the stars and galaxies within it; either their flux through relatively wide bandpasses (photometry), or measurements of the shape and wavelength of relatively narrow lines via spectroscopy. These techniques are now being used to discover planets outside our solar system, and perhaps in the next 10 years will begin to characterize them. If a serious search is to be made for extraterrestrial thermophiles, we need predictions for the effects of thermophiles on their host planets that are observable with these techniques. In this paper I shall outline what sorts of observation are likely to be used in the next 15 years for extra-solar planet work. All of the journal articles quoted here can be found through http://adsabs.harvard.edu/abstract_service.html, and often also accessed as preprints at http://uk.arxiv.org/form/astro%20ph?MULTI=form%20±%20interface.
The hyperthermophilic origin of life revisited
Authors: Schwartzman D.W. , Lineweaver C.H.
Published: June 2004

We revisit the case for the hyperthermophilic scenario for the origin of life and the last common ancestor. Evidence includes studies of phylogenetic trees, rRNA, G and C content, hyperthermophilic proteins, correlations between maximal temperature tolerances and genetic distances, saline stabilization of DNA/RNA, and the inferred climatic temperatures of the early Earth. Although some doubts remain, the case for hot biogenesis and the last common ancestor has gotten stronger.
Sulphate metabolism among thermophiles and hyperthermophiles in natural aquatic systems
Authors: Roychoudhury A.N.
Published: June 2004

Although controversial, the idea that hydrothermal systems may have been the site for prebiotic synthesis of organic molecules and origin of life is widely supported. For the nascent life to survive, it must have had some sort of metabolic mechanism for generating energy. However, little is known of the specific metabolic pathways utilized by the early life forms or the effect of high temperatures on their activity. Recent research on natural high temperature aquatic environments, though limited because of difficult field logistics and experimental problems, is revolutionizing our understanding of possible energy-generating redox pathways, such as sulphate reduction. An abridged review of research on thermophilic sulphate reduction is presented here. Because of a complex interplay between microbiological and geochemical entities involved, and the uncertainties that modern hydrothermal systems are proxy for biogeochemical conditions on early Earth, great caution is required for interpretation and extrapolation of data from these studies to primordial times. Furthermore, a general lack of integrated geological and microbiological studies towards a common understanding of origin and sustenance of life on Earth is starkly evident from this review.
Phylogenomics of hyperthermophilic Archaea and Bacteria
Authors: Klenk H.-P. , Spitzer M. , Ochsenreiter T. , Fuellen G.
Published: June 2004

The location of hyperthermophilic organisms in the tree of life has been the source of many exciting discussions during the last two decades. It inspired not only novel hypotheses for the early evolution of the organisms, but also the isolation of many new species of Archaea and Bacteria from hot environments, as well as microbial genome sequencing and phylogenomic analyses. In view of the new wealth of genetic information generated from several analysed genomes of the hyperthermophiles, we can only conclude that the question of their exact phylogenetic location and evolutionary origin is presently as open as ever before.
Shuffling of Sulfolobus genomes by autonomous and non-autonomous mobile elements
Authors: Brügger K. , Torarinsson E. , Redder P. , Chen L. , Garrett R.A.
Published: June 2004

Each of the sequenced Sulfolobus genomes contains large numbers of putatively mobile elements, both IS elements (insertion sequence elements) and MITEs (miniature inverted-repeat transposable elements). There are 344 in the 3.0 Mb genome of Sulfolobus solfataricus P2 and 95 in the 2.7 Mb genome of Sulfolobus tokodaii. In the former they constitute more than 10% of the genome. Experimental data suggest that transposition of IS elements occurs frequently. Moreover, the gene order between the two organisms differs greatly, indicating that multiple rearrangements have occurred. This has also led to considerable speculation as to how the cells are viable. Recently, a third Sulfolobus genome was completed which contains no IS elements or MITEs. This enabled us to compare the gene orders of the three genomes and provide evidence for mobile element-induced rearrangements of sections of the genomes.
Genetic elements of Thermococcales
Authors: Prieur D. , Erauso G. , Geslin C. , Lucas S. , Gaillard M. , Bidault A. , Mattenet A.-C. , Rouault K. , Flament D. , Forterre P. , Le Romancer M.
Published: June 2004

This minireview summarizes our current knowledge about archaeal genetic elements in the hyperthermophilic order Thermococcales in the phylum Euryarchaeota. This includes recent work on the first virus of Pyrococcus, PAV1, the discovery of novel unique virus morphotypes in hot deep-sea environments, and preliminary observations on novel cryptic plasmids. We also review the work accomplished over the last 5 years in the development of genetic tools for members of the Pyrococcus and Thermococcus genera, mainly in our laboratories.
Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms
Authors: Johnson M.R. , Montero C.I. , Conners S.B. , Shockley K.R. , Pysz M.A. , Kelly R.M.
Published: June 2004

Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures. Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats. Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.
Alkalithermophiles
Authors: Wiegel J. , Kevbrin V.V.
Published: June 2004

Alkalithermophiles are an exciting subset of extremophilic organisms and represent extremophiles that are adapted to two extreme conditions, i.e. to a combination of alkaline and thermobiotic growth conditions. Among the anaerobic alkalithermophiles are representatives of both Bacteria and Archaea within a wide variety of physiological types and systematic groups, although a great majority belongs to the Firmicutes. Alkaliphiles have been isolated from a variety of niches including mesobiotic and neutrophilic soils and sediments. Interestingly anaerobic isolates from mesobiotic and neutrophilic niches exhibit shorter doubling times than isolates from thermobiotic niches; some anaerobic alkalithermophiles exhibit extremely fast growth rates, i.e. doubling times as short as 10 min. Their adaptation to both high pH and high temperature draws our attention not only because they are potential sources of industrially valuable enzymes but also because of their adaptive mechanisms to external environmental parameters. They could thus function as model organisms for extraterrestrial life in some environments and for theories on the origin of life. Alkalithermophiles, as far we know, do not represent the most thermophilic nor the most alkaliphilic of micro-organisms but represent the most alkaliphilic ones among the thermophiles and vice versa. We believe that the presently known species are only the tip of the iceberg and therefore that they do not represent the true boundaries under which life can thrive in respect to high temperature in alkaline environments.
The outer membrane of the hyperthermophilic archaeon Ignicoccus: dynamics, ultrastructure and composition
Authors: Näther D.J. , Rachel R.
Published: June 2004

Ignicoccus is the only archaeal genus known today whose cells possess an outer membrane. According to freeze-etch experiments, it is composed of two leaflets which become separated in the fracture process. Here we show by transmission electron microscopy that the two leaflets can also be visualized in ultrathin sections; they exhibit highly different staining intensities. Biochemical analysis proves the presence of lipids as well as membrane proteins. Various derivatives of the archaeal lipid ‘archaeol’ could be identified, many of which were glycosylated. The protein set is dominated by four membrane proteins, one or several of which may form pores. The outer membrane itself is a dynamic structure: periplasmic vesicles can be visualized in various stages of a fusion process, and, although rarely, vesicles are seen on the outer cell surface, either in a release or a fusion process. Future studies will focus on the outer membrane proteins in order to understand their role in outer membrane permeability, e.g. what kinds of transport processes they facilitate.
Archaeal integrases and mechanisms of gene capture
Authors: She Q. , Shen B. , Chen L.
Published: June 2004

Archaeal integrases facilitate the formation of two distinctive types of integrated element within archaeal chromosomes: the SSV type and pNOB8 type. The former carries a smaller N-terminal and a larger C-terminal integrase gene fragment, and the latter an intact integrase gene. All integrated elements overlap tRNA genes that were target sites for integration. It has been demonstrated that SSV (Sulfolobus spindle virus) viruses, carrying an SSV-type integrase gene, and conjugative plasmids, carrying a pNOB8-type integrase, are integrative elements. Two mechanisms have been proposed for stably maintaining an integrated element within archaeal chromosomes. There is also evidence for changes having occurred in the captured integrated elements present in archaeal genomes. Thus we infer that site-specific integration constitutes an important mechanism for horizontal gene transfer and genome evolution.
Archaeal histones: structures, stability and DNA binding
Authors: Reeve J.N. , Bailey K.A. , Li W-t. , Marc F. , Sandman K. , Soares D.J.
Published: June 2004

Structures, stability and DNA-binding properties have been established for archaeal histones from mesophiles, thermophiles and hyperthermophiles. Most archaeal histones are simply histone folds that are stabilized by dimer formation. Archaeal histones and the histone folds of the eukaryotic nucleosome core histones share a common ancestry and bind and wrap DNA similarly using conserved residues. The histone-fold residues that stabilize dimer–dimer interactions within an archaeal histone core contribute to determining archaeal histone–DNA affinity.
DNA replication in thermophiles
Authors: Majerník A.I. , Jenkinson E.R. , Chong J.P.J.
Published: June 2004

DNA replication enzymes in the thermophilic Archaea have previously attracted attention due to their obvious use in methods such as PCR. The proofreading ability of the Pyrococcus furiosus DNA polymerase has resulted in a commercially successful product (Pfu polymerase). One of the many notable features of the Archaea is the fact that their DNA processing enzymes appear on the whole to be more like those found in eukaryotes than bacteria. These proteins also appear to be simpler versions of those found in eukaryotes. For these reasons, archaeal organisms make potentially interesting model systems to explore the molecular mechanisms of processes such as DNA replication, repair and recombination. Why archaeal DNA-manipulation systems were adopted over bacterial systems by eukaryotic cells remains a most interesting question that we suggest may be linked to thermophily.
The replication protein of the Sulfolobus islandicus plasmid pRN1
Authors: Lipps G.
Published: June 2004

The thermoacidophile crenarchaeote Sulfolobus ssp. is one of the best-studied Archaea. Cryptic and conjugative plasmids as well as viruses have been described for this genus. For the majority of the genetic elements only the genome sequence and the basic characteristics were determined. In contrast the fusellovirus SSV1 and the cryptic plasmid pRN1, which is the smallest known genetic element of the crenarchaeota, have been studied in more detail. The three gene products of the plasmid pRN1 have been characterized biochemically. The replication protein of the plasmid, a multifunctional enzyme, has a novel domain, termed prim/pol domain. This domain constitutes the first member of the DNA polymerase family E. Based on the biochemical characterization of the gene products a model of how pRN1 is replicated in vivo is proposed.
Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii
Authors: Shen Y. , Tang X.-F. , Matsui E. , Matsui I.
Published: June 2004

Family D DNA polymerase (PolD) has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. We successfully cloned, expressed, and purified the family D DNA polymerase from Pyrococcus horikoshii (PolDPho). By site-directed mutagenesis, we identified amino acid residues Asp-1122 and Asp-1124 of a large subunit as the essential residues responsible for DNA-polymerizing activity. We analysed the domain structure using proteins truncated at the N- and C-termini of both small and large subunits (DP1Pho and DP2Pho), and identified putative regions responsible for subunit interaction, oligomerization and regulation of the 3´-5´ exonuclease activity in PolDPho. It was also found that the internal region of the putative zinc finger motif (cysteine cluster II) at the C-terminal of DP2Pho is involved in the 3´-5´ exonuclease activity. Using gel filtration analysis, we determined the molecular masses of the recombinant PolDPho and the N-terminal putative dimerization domain of the large subunit, and proposed that PolD from P. horikoshii probably forms a heterotetrameric structure in solution. Based on these results, a model regarding the subunit interaction and regulation of activity of PolDPho is proposed.
Rescue of protein splicing activity from a Magnetospirillum magnetotacticum intein-like element
Authors: Southworth M.W. , Yin J. , Perler F.B.
Published: June 2004

The self-catalytic protein splicing mechanism is mediated by the intein plus the first amino acid following the intein C-terminus (termed the +1 residue). Although polymorphisms of conserved residues elsewhere in inteins have been widely reported, no splicing-competent intein has been observed without a Ser, Thr or Cys in this functionally essential +1 position. This residue is the nucleophile in two steps of the protein splicing pathway: ligation of the extein fragments during transesterification and formation of a peptide bond between the exteins by an acyl rearrangement. An intein-like element in a hypothetical protein (gene Magn8951) from Magnetospirillum magnetotacticum has all intein signature sequences except the +1 residue, where it has a Tyr. Although the Tyr side-chain hydroxyl can potentially mediate the transesterification reaction, an acyl shift has never been observed with this residue. When the activities of this bacterial intein-like element were studied, protein splicing was not observed and N-terminal cleavage predominated. Mutation of Tyr+1 to Phe or Ala indicated that the Tyr side-chain hydroxyl was not necessary for N-terminal cleavage. Protein splicing activity could be rescued by ‘reversion’ of Tyr+1 to Cys.
Hyperthermophilic dehydrogenase enzymes
Authors: Littlechild J.A. , Guy J.E. , Isupov M.N.
Published: June 2004

Archaeal dehydrogenases are often found to be of a specific class of dehydrogenase which has low sequence identity to the equivalent bacterial and eukaryotic counterparts. This paper focuses on two different types of hyperthermophilic dehydrogenase enzyme that have been cloned and over-expressed in Escherichia coli. The crystallographic structures of the apo form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from Sulfolobus solfataricus and the related holo form of GAPDH from Methanothermus fervidus have been solved to high resolution. The zinc-containing structure of ADH (alcohol dehydrogenase) from Aeropyrum pernix has also been solved as a quaternary complex with the cofactor NADH and the inhibitor octanoic acid. The results show that despite the low sequence identity to the related enzymes found in other organisms the fold of the protein chain is similar. The archaeal GAPDH enzymes show a relocation of the active site which is a feature of evolutionary interest. The high thermostability of these three archaeal dehydrogenases can be attributed to a combination of factors including an increase in the number of salt bridges and hydrophobic interactions, a higher percentage of secondary structure and the presence of disulphide bonds.
Hyperthermophilic and salt-dependent formyltransferase from Methanopyrus kandleri
Authors: Shima S. , Thauer R.K. , Ermler U.
Published: June 2004

Methanopyrus kandleri is a hyperthermophilic methanogenic archaeon, which grows on H2 and CO2 as its sole energy source. Its growth temperature optimum is 98°C. One of the interesting characteristics of this archaeon is its high intracellular salt content. The organism has been reported to contain the trianionic cDPG (cyclic 2,3-diphosphoglycerate) and K+ at concentrations of 1.1 and 3 M, respectively. Reflecting the high cellular salt concentration, the enzymes in this organism are adapted not only to high temperature but also to high salt concentrations. The formyltransferase from M. kandleri was characterized extensively with respect to thermo- and halophilicity. The crystal structure of the formyltransferase at 1.73 Å shows the enzyme to be composed of four identical subunits of molecular mass 32 kDa. The formyltransferase is thermostable and active only at relatively high concentrations of potassium phosphate (1 M) or other salts with strongly hydrated anions (strong salting-out salts). Potassium phosphate and potassium cDPG were found to be equivalent in activating and stabilizing the enzyme. At low concentrations of these salts, the enzyme is inactive and thermolabile. It was shown by equilibrium sedimentation analysis that the enzyme is in a monomer/dimer/tetramer equilibrium, the equilibrium constant being dependent on the concentration of salts: the higher oligomeric species increase with increasing salt concentrations. Evidence was provided that the monomer is both inactive and thermolabile. Experiments using a mutation which is directed to break surface ion pairs between two dimers indicated that dimerization is required for activity and tetramerization leads to thermostability.
Oxygen and carbon dioxide kinetic challenges for thermophilic mineral bioleaching processes
Authors: de Kock S.H. , Barnard P. , du Plessis C.A.
Published: June 2004

Agitated bacterial tank bioleaching reactors are currently sparged with air to satisfy both oxygen and CO2 requirements of microbial cells. Under high-sulphide loading conditions, as is the case with high-grade metal sulphide concentrates, the microbial and chemical demand for oxygen is significantly increased during the bioleaching process. Sparging with enriched oxygen gas may offer an alternative process option to increased agitation and sparged aeration, to overcome the mass transfer difficulties at elevated temperatures where thermophilic Archaea, rather than Bacteria, are used. In the case of air sparging, the DO (dissolved oxygen) concentration in tank reactors could not be increased to a point where it would become inhibitory due to the limited oxygen content of air (20.9% O2). The use of enriched oxygen in such reactors at large scale does, however, pose its own set of process risks. The first aim of this investigation was, therefore, to determine the effects of various DO concentrations, in both the limiting and inhibitory ranges, on the microbial activity of Sulfolobus sp. U40813, a typical thermophilic mineral-leaching archaeon. Secondly, the effect of CO2 concentration on the rate of ferrous iron oxidation was investigated. Both the oxygen and CO2 kinetics were examined in controlled batch cultures at 78°C, using ferrous sulphate and potassium tetrathionate as energy sources. The optimal DO concentration for iron oxidation was found to be between 1.5 and 4.1 mg·l−1. The use of elevated DO concentrations (above 4.1 mg·l−1) inhibited the ferrous oxidation rates. The optimal gas CO2 concentration for ferrous iron oxidation was found to be in the range 7–17% (v/v). The iron oxidation rates were, however, severely limited at CO2 concentrations less than 7%, indicating that the CO2 supply was limiting in this range and inhibited the microbial growth rate.
Development of an ideal starch saccharification process using amylolytic enzymes from thermophiles
Authors: Satyanarayana T. , Noorwez S.M. , Kumar S. , Rao J.L.U.M. , Ezhilvannan M. , Kaur P.
Published: June 2004

The extensive efforts to screen thermophilic fungi and bacteria, isolated from various environmental samples, have resulted in the selection of Thermomucor indicae-seudaticae, Geobacillus thermoleovorans NP33 and G. thermoleovorans NP54 for the production of glucoamylase, amylopullulanase and α-amylase, respectively. Submerged and solid-state fermentation processes were optimized for maximizing the secretion of glucoamylase by T. indicae-seudaticae. The production of amylopullulanase and α-amylase by NP33 and NP54 in submerged fermentation was also optimized. Glucoamylase was optimally active at pH 7.0 and 60°C and was shown to saccharify soluble as well as raw starches. Amylopullulanase and α-amylase exhibited optima at pH 7.0 and 100°C and saccharified starch efficiently. Differential inhibition and action on mixed substrates clearly suggested that there are two separate active sites for α-amylase and pullulanase activities of amylopullulanase. Both α-amylase and amylopullulanase are high maltose-forming and Ca2+-independent. These amylolytic enzymes have been shown to be useful in starch saccharification alone and in combination.
Metagenomics, gene discovery and the ideal biocatalyst
Authors: Cowan D.A. , Arslanoglu A. , Burton S.G. , Baker G.C. , Cameron R.A. , Smith J.J. , Meyer Q.
Published: June 2004

With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the ‘ideal biocatalyst’ for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of ‘sequence space’ that is not addressed by traditional screening.
Embden–Meyerhof–Parnas and Entner–Doudoroff pathways in Thermoproteus tenax: metabolic parallelism or specific adaptation?
Authors: Ahmed H. , Tjaden B. , Hensel R. , Siebers B.
Published: June 2004

Genome data as well as biochemical studies have indicated that – as a peculiarity within hyperthermophilic Archaea – Thermoproteus tenax uses three different pathways for glucose metabolism, a variant of the reversible EMP (Embden–Meyerhof–Parnas) pathway and two different modifications of the ED (Entner–Doudoroff) pathway, a non-phosphorylative and a semi-phosphorylative version. An overview of the three different pathways is presented and the physiological function of the variants is discussed.
Metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus: dynamic enzyme–enzyme interactions involved in the formation of the channelling complex
Authors: Massant J. , Glansdorff N.
Published: June 2004

Protection of thermolabile metabolites and coenzymes is a somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles. Detailed information about the mechanisms used by thermophiles to protect these thermolabile metabolites and coenzymes is still scarce. A case in point is CP (carbamoyl phosphate), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. Recently we obtained the first evidence for a physical interaction between two hyperthermophilic enzymes for which kinetic evidence had suggested that these enzymes channel a highly thermolabile and potentially toxic intermediate. By physically interacting with each other, CKase (carbamate kinase) and OTCase (ornithine carbamoyltransferase) prevent thermodenaturation of CP in the aqueous cytoplasmic environment. The CP channelling complex involving CKase and OTCase or ATCase (aspartate carbamoyltransferase), identified in hyperthermophilic archaea, provides a good model system to investigate the mechanism of metabolic channelling and the molecular basis of protein–protein interactions in the physiology of extreme thermophiles.
Arginine regulation in Thermotoga neapolitana and Thermotoga maritima
Authors: Charlier D.
Published: June 2004

Experimental data and in silico analyses of sequenced bacterial genomes indicate that arginine repressor (ArgR) proteins and their respective target sites are surprisingly well conserved in very diverse bacteria. Arginine regulation therefore constitutes an interesting model system from the study of evolutionary aspects of bacterial regulation. Moreover, arginine repressor molecules are multifunctional, they repress the arginine biosynthetic genes and are involved in the activation of the various arginine catabolic pathways. Studies on the arginine repressor from the hyperthermophiles Thermotoga neapolitana and Thermotoga maritima have reinforced the uniform view of the bacterial ArgR–operator interaction, but have also revealed that the Thermotoga repressor exhibits unique features. Thus, its DNA-binding activity is nearly arginine-independent and exhibits poor sequence specificity. ArgRTn has a remarkable capacity to bind heterologous arginine operators and half-site targets.
Towards an understanding of isoform specificity in phosphoinositide 3-kinase signalling in lymphocytes
Authors: Fruman D.A.
Published: June 2004

The PI3K (phosphoinositide 3-kinase) signalling pathway promotes proliferation and transformation in many cell types. This is particularly well illustrated by studies of primary lymphocytes and their leukaemic counterparts. PI3K activation is required for proliferation of T cells and B cells, and certain oncogenes cause leukaemia in part by promoting PI3K signalling. Genetic manipulation of this pathway, together with biochemical studies in primary lymphocytes, has begun to shed light on the molecular mechanisms by which PI3K contributes to the proliferation of normal and transformed lymphocytes. In particular, targeted gene disruption in mice has allowed the identification of specific isoforms of PI3K that are required for distinct cellular responses. Continued investigation of isoform specificity in PI3K signalling, as well as the characterization of critical downstream targets of PI3K signalling, may reveal strategies for the therapeutic control of immune responses and leukaemia.
Btk-dependent regulation of phosphoinositide synthesis
Authors: Carpenter C.L.
Published: June 2004

Activation of the BCR (B cell antigen receptor) stimulates the production of both PtdIns(3,4,5)P3 and Ins(1,4,5)P3. PtdIns(3,4,5)P3 and Ins(1,4,5)P3 are generated from a common substrate, PtdIns(4,5)P2. In some systems, continuous PtdIns(4,5)P2 synthesis is necessary for maximal Ins(1,4,5)P3 production, but whether this is true for the BCR, and whether PtdIns(4,5)P2 synthesis is regulated following BCR activation, are not known. We found that Btk (Bruton's tyrosine kinase), a member of the Tec family of cytoplasmic protein tyrosine kinases, is constitutively associated with PIP5Ks (phosphatidylinositol 4-phosphate 5-kinases), the enzymes that synthesize PtdIns(4,5)P2. Btk functions as a shuttle to bring PIP5K to the plasma membrane as a means of stimulating PtdIns(4,5)P2 synthesis. The Btk–PIP5K complex appears to localize to lipid rafts. This complex provides a novel shuttling mechanism that allows Btk to regulate the production of the substrate required by both its upstream activator phosphoinositide 3-kinase and its downstream target phospholipase Cγ2.
The tumour suppressor PTEN: involvement of a tumour suppressor candidate protein in PTEN turnover
Authors: Maehama T. , Okahara F. , Kanaho Y.
Published: June 2004

The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) plays essential roles in regulating signalling pathways involved in cell growth and apoptosis, and is inactivated in a wide variety of tumours. The role of PTEN as a tumour suppressor has been firmly established; however, the mechanism(s) by which its function and activity are regulated remains elusive. Here, we summarize recent progress in research directed towards trying to understand the molecular basis of regulatory mechanisms for PTEN. We also describe our novel finding that a tumour suppressor candidate protein binds to extreme C-terminal region of PTEN and regulates PTEN protein turnover.
Identification of novel effectors of invasive cell growth downstream of phosphoinositide 3-kinase
Authors: Kaufmann J. , Pronk G. , Giese K. , Klippel A.
Published: June 2004

Conventional approaches to identifying cancer targets are complicated by the chromosomal instability of tumour cells, and typically result in a large number of differentially expressed candidate genes with uncertain disease relevance. Here we present a novel approach which aims to elucidate the molecular changes that are induced after loss of tumour suppressor function. Using gene silencing tools, we mimic the loss of tumour suppressor function to identify key regulators of tumour initiation and progression. Loss of function of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) correlates with increased invasive cell growth due to the resulting chronic activation of the PI 3-kinase (phosphoinositide 3-kinase) pathway. Induced activation of PI 3-kinase either by inhibiting PTEN expression or by using p110*, a constitutively active PI 3-kinase, increased signalling and the invasive growth potential of cells. Using this unbiased approach we have identified novel downstream effectors of PI 3-kinase/PTEN signalling that mediate the behaviour of cells with a hyperactive PI 3-kinase pathway. These molecules represent candidate targets for therapeutic intervention in patients with PTEN-deficient tumours.
Phosphoinositide 3-kinase and Forkhead, a switch for cell division
Authors: Martínez-Gac L. , Álvarez B. , García Z. , Marqués M. , Arrizabalaga M. , Carrera A.C.
Published: June 2004

Cell cycle progression is a tightly controlled process. To initiate cell division, mitogens trigger a number of early signals that promote the G0–G1 transition by inducing cell growth and the activation of G1 cyclins. Activation of cyclin E/cdk2 (cyclin-dependent kinase 2) at the end of G1 is then required to trigger DNA synthesis (S phase entry). Among the early signals induced by mitogens, activation of PI3K (phosphoinositide 3-kinase) appears essential to induce cell cycle entry, as it regulates cell growth signalling pathways, which in turn determine the rate of cell cycle progression. Another mechanisms by which PI3K and its downstream effector protein kinase B regulate cell cycle entry is by inactivation of the FOXO (Forkhead Box, subgroup O) transcription factors, which induce expression of quiescence genes such as those encoding p27kip, p130 and cyclin G2. PI3K/FOXO then work as a complementary switch: when PI3K is active, FOXO transcription factors are inactive. The switch is turned on and off at different phases of the cell cycle, thus regulating cell cycle progression.
Chemokine inhibition – why, when, where, which and how?
Authors: Johnson Z. , Power C.A. , Weiss C. , Rintelen F. , Ji H. , Ruckle T. , Camps M. , Wells T.N.C. , Schwarz M.K. , Proudfoot A.E.I. , Rommel C.
Published: June 2004

Chemokines are small chemoattractant cytokines that control a wide variety of biological and pathological processes, ranging from immunosurveillance to inflammation, and from viral infection to cancer. Genetic and pharmacological studies have shown that chemokines are responsible for the excessive recruitment of leucocytes to inflammatory sites and damaged tissue. In the present paper, we discuss the rationale behind interfering with the chemokine system and introduce various points for therapeutic intervention using either protein-based or small-molecule inhibitors. Unlike other cytokines, chemokines signal via seven-transmembrane GPCRs (G-protein-coupled receptors), which are favoured targets by the pharmaceutical industry, and, as such, they are the first cytokines for which small-molecule-receptor antagonists have been developed. In addition to the high-affinity receptor interaction, chemokines have an in vivo requirement to bind to GAGs (glycosaminoglycans) in order to mediate directional cell migration. Prevention of the GAG interaction has been shown to be a viable therapeutic strategy. Targeting chemokine intracellular signalling pathways offers an alternative small-molecule approach. One of the key signalling targets downstream of a variety of chemokine receptors identified to date is PI3Kγ (phosphoinositide 3-kinase γ), a member of the class I PI3K family. Thus the chemokine system offers many potential entry points for innovative anti-inflammatory therapies for autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis and allergic contact dermatitis.
Phosphoinositide 3-kinase γ: kinase-dependent and -independent activities in cardiovascular function and disease
Authors: Alloatti G. , Montrucchio G. , Lembo G. , Hirsch E.
Published: June 2004

Cardiac function is controlled by GPCRs (G-protein-coupled receptors) which exert their function by triggering numerous signalling pathways, including the activation of PI3K (phosphoinositide 3-kinase). The GPCR-activated PI3Kγ is weakly expressed in the heart, but the deletion of its expression in mice causes remarkable phenotypes. Indeed, the lack of PI3Kγ does not modify heart rate and blood pressure, but does increase contractility, particularly in response to stimuli that enhance cardiac contractile force, such as catecholamines. Consistently, treatment of mutant cardiomyocytes with β-adrenergic agonists causes an abnormal increase in the elevation of cAMP production. On the other hand, PI3Kγ appears to play a role in mediating the contractile depression exerted by other GPCR agonists, such as PAF (platelet-activating factor), that are released in pathological conditions, such as after an ischaemic insult. The receptor for PAF coupled to Gi activates PI3Kγ, which, in turn, is essential to promote Akt phosphorylation, NOSIII (nitric oxide synthase isoform III) activation and the production of nitric oxide, a well characterized cardiodepressing agent. As a whole, PI3Kγ appears to negatively control cardiac contractility through different signalling mechanisms, thus becoming a possible drug target for the treatment of critical human cardiac pathologies, such as infarction or heart failure.
Inhibiting the phosphoinositide 3-kinase pathway for cancer treatment
Authors: Workman P.
Published: June 2004

There is extensive evidence from the molecular and genomic analysis of human cancers that the PI 3-kinase (phosphoinositide 3-kinase)–Akt/PKB (protein kinase B) pathway is deregulated in malignant progression. Furthermore, the causal involvement of PI 3-kinase is supported by gene-knockout mouse models. Prototype inhibitors show evidence of anticancer activity in vitro and in vivo animal models. The recent development of isoform-selective inhibitors shows considerable promise for cancer treatment.
Structural basis for ligand recognition by RGD (Arg-Gly-Asp)-dependent integrins
Authors: Takagi J.
Published: June 2004

Since the discovery of the RGD sequence motif as the essential cell attachment site in Fn (fibronectin), RGD-dependent ligand recognition by integrins has been the major focus of many integrin researches. Although many integrins recognize RGD-containing ligands, it is believed that residues outside the RGD motif provide specificity as well as high affinity for each integrin–ligand pair. These ‘secondary’ sites are generally assumed to interact directly with the α subunit of integrin, whereas the RGD motif binds primarily to the β subunit. This necessitates that the integrin–ligand interface comprises a relatively large, or even scattered, area. Molecular electron microscopy and single-particle analysis were performed on a headpiece fragment of integrin α5β1 in the presence and absence of bound ligand (Fn fragment), and revealed a marked shape change of the β subunit hybrid and I-like domains that is linked with the ligand docking. Furthermore, electron microscopy images revealed a focal rather than a large contact area at the α5β1–Fn interface, raising a question about ‘2-site docking model’. Kinetic analysis of real-time binding data showed that the synergy site greatly enhances kon but has little effect on the stability or koff of the complex, suggesting that the synergy site exerts its positive effect on α5β1 binding by facilitating the initial encounter, rather than by contributing to the protein–protein interaction surface. Thus the ligand recognition mechanism by integrins needs further refinement through more structural analyses of the complexes as well as kinetic analysis of binding data.
Monoclonal antibodies as probes of integrin priming and activation
Authors: Humphries M.J.
Published: June 2004

Integrins are a family of heterodimeric, transmembrane receptors that mediate a range of cell–cell and cell–extracellular matrix interactions in an array of physiological and pathophysiological situations. Integrin-mediated cell adhesion is dynamically regulated in vivo to facilitate cell anchorage and movement, but prevents aberrant trafficking and aggregation. Following ligand engagement, integrin signalling imposes a spatial constraint on the assembly of signalling complexes and controls the transduction of mechanical force to the cytoskeleton. This transmembrane passage of signals via integrins is achieved both by clustering of receptors, which makes the ligand and effector engagement more favourable kinetically, and by induction of conformational changes, that theoretically creates ligand and effector binding sites de novo. Clustering and conformational changes can be triggered both from the inside of the cell (resulting in acquisition of ligand-competent conformers) and from the outside (ligand-induced signalling). In this paper, these processes will be described and distinguished by the terms priming and activation, respectively. Although both clustering and conformation are important for integrin function, the latter will be the main focus of this article; in particular, the importance of monoclonal antibodies for the study of integrin shape changes.
Structural basis for integrin αIIbβ3 clustering
Authors: Li R. , Bennett J.S. , DeGrado W.F.
Published: June 2004

We have expressed two proteins that correspond to the transmembrane and cytoplasmic domains of integrin αIIb and β3 subunits. Characterization of these proteins, dispersed in anionic and zwitterionic micelles, revealed that, rather than interacting with each other, the two proteins associated into homodimers and homotrimers respectively. Moreover, studies using the TOXCAT assay system confirmed that the αIIb and β3 transmembrane domains can self-associate in biological cell membranes. Transmembrane domain-mediated homo-oligomerization provides a plausible structural basis for integrin clustering and could promote integrin activation as well. Indeed, replacing specific residues in the transmembrane helix of either αIIb or β3 with an asparagine residue resulted in a facilitated homo-oligomerization of the mutated transmembrane helix, promoted the formation of integrin clusters on the cell surface and shifted αIIbβ3 to its activated state. Thus these studies support the hypothesis that the transmembrane domains play a vital role in the function and regulation of αIIbβ3.
Hierarchical assembly of cell–matrix adhesion complexes
Authors: Zaidel-Bar R. , Cohen M. , Addadi L. , Geiger B.
Published: June 2004

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 μm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.
Talin controls integrin activation
Authors: Calderwood D.A.
Published: June 2004

Tight, dynamic control of the affinity of integrin adhesion receptors for their extracellular ligands (integrin activation) is essential for the development and functioning of multicellular organisms. Integrin activation is controlled by intracellular signals that, through their action on integrin cytoplasmic domains, induce conformational changes in integrin extracellular domains, resulting in increased affinity for the ligand. Recent results indicate that the binding of talin, a major actin-binding protein, to integrin β tails represents a final common step in integrin activation pathways. The major integrin-binding site lies within the talin FERM (four-point-one, ezrin, radixin, moesin) domain, and binding occurs via a variant of the classical PTB domain (phosphotyrosine-binding domain)–NPxY interaction. Formation of this talin–integrin complex plays a critical role in integrin activation, since mutations, in either talin or integrin β tails, which disrupt complex formation, inhibit integrin activation. Furthermore, use of RNA interference to knockdown talin expression selectively reveals that talin is essential for integrin activation in response to physiological agonists. Thus the association of the cytoskeletal protein talin with integrin β cytoplasmic domains is a critical step during integrin activation, and regulation of this step may be a final common element in the signalling pathways that control integrin activation.
Integrin regulation of cell signalling and motility
Authors: Juliano R.L. , Reddig P. , Alahari S. , Edin M. , Howe A. , Aplin A.
Published: June 2004

Integrins clearly play a key role in regulating both mitogenic signalling and cell migration. Thus integrins modulate the efficiency of the Erk (extracellular-signal-regulated kinase)/MAP kinase (mitogen-activated protein kinase) pathway, acting at several distinct levels. We have shown that both cAMP-dependent protein kinase and PAKs (p21-activated kinases) play a role in integrin regulation of the Erk pathway, acting primarily at the level of Raf-1. Integrins and PAKs also play a role in the control of cell migration. Thus we have discovered a novel protein that links the α5β1 integrin to migration controlled by Rho-family GTPases. This protein, termed Nischarin, is a large cytosolic macromolecule that is not related to well-known protein families. The N-terminus of Nischarin interacts with a short segment of the cytoplasmic domain of the α5 integrin subunit. Overexpression of Nischarin alters actin organization and inhibits Rac-driven cell migration and tumour cell invasion. Use of effector domain mutants of Rac suggest that Nischarin acts downstream of Rac, probably at the level of PAK-family kinases. These studies emphasize the intricate connection between integrins and Rho-family GTPases and their effectors in controlling both mitogenesis and migration.
Granulocyte apoptosis: who would work with a ‘real’ inflammatory cell?
Authors: Dransfield I. , Rossi A.G.
Published: June 2004

The neutrophil granulocyte is a key factor in cellular innate defence mechanisms against infection or tissue damage. Granulocyte apoptosis is now acknowledged to have a critical role in progression of inflammatory responses. Granulocytes are preprogrammed to die with important physiological mechanisms for non-inflammatory clearance. Shutdown of secretory capacity represents an important aspect of the programme of biochemical events that accompany neutrophil apoptosis together with surface molecular changes that serve to identify apoptotic cells as targets for phagocytic removal. Defining the underlying regulatory mechanisms together with the changes in patterns of gene/protein expression associated with granulocyte death remains a challenge. Use of novel strategies for inducing cell death will allow biochemical approaches to dissect the underlying pathways. Although study of granulocyte cell death has especial difficulties when compared with other cell types, there are clearly potential benefits for new therapeutic approaches to treat inflammatory diseases.
Early events in spontaneous neutrophil apoptosis
Authors: Scheel-Toellner D. , Wang K.-Q. , Webb P.R. , Wong S.H. , Craddock R. , Assi L.K. , Salmon M. , Lord J.M.
Published: June 2004

Neutrophils are very abundant, short-lived leucocytes and their death by apoptosis is central to homoeostasis and the resolution of inflammation, yet the trigger for apoptosis is still a topic of debate. Depolarization of the mitochondrial membrane has been supposed to initiate neutrophil spontaneous apoptosis, as neutrophils gradually lose the anti-apoptotic protein Mcl-1 and Bax translocates and inserts into the mitochondrial membrane. However, other reports show that caspase 8 is required for neutrophil apoptosis, suggesting the involvement of DR (death receptor) signalling. As DR ligation is not required for neutrophil apoptosis, this raises the intriguing possibility that activation of caspase 8 during neutrophil apoptosis occurs via a novel mechanism. In the present paper, we discuss the current evidence for mechanisms occurring in neutrophil apoptosis, which could trigger DR signalling in the absence of DR ligation.