Insights into assembly from structural analysis of bacteriophage PRD1

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.     

Contrasting origins of the upper mantle revealed by hafnium and lead isotopes from the Southeast Indian Ridge

The origin of the isotopic signature of Indian mid-ocean ridge basalts has remained enigmatic, because the geochemical composition of these basalts is consistent either with pollution from recycled, ancient altered oceanic crust and sediments, or with ancient continental crust or lithosphere. The radiogenic isotopic signature may therefore be the result of contamination of the upper mantle by plumes containing recycled altered ancient oceanic crust and sediments, detachment and dispersal of continental material into the shallow mantle during rifting and breakup of Gondwana, or contamination of the upper mantle by ancient subduction processes. The identification of a process operating on a scale large enough to affect major portions of the Indian mid-ocean ridge basalt source region has been a long-standing problem. Here we present hafnium and lead isotope data from across the Indian-Pacific mantle boundary at the Australian-Antarctic discordance region of the Southeast Indian Ridge, which demonstrate that the Pacific and Indian upper mantle basalt source domains were each affected by different mechanisms. We infer that the Indian upper-mantle isotope signature in this region is affected mainly by lower continental crust entrained during Gondwana rifting, whereas the isotope signature of the Pacific upper mantle is influenced predominantly by ocean floor subduction-related processes.     

Tungsten isotope evidence that mantle plumes contain no contribution from the Earth's core

Osmium isotope ratios provide important constraints on the sources of ocean-island basalts, but two very different models have been put forward to explain such data. One model interprets (187)Os-enrichments in terms of a component of recycled oceanic crust within the source material. The other model infers that interaction of the mantle with the Earth's outer core produces the isotope anomalies and, as a result of coupled (186)Os-(187)Os anomalies, put time constraints on inner-core formation. Like osmium, tungsten is a siderophile ('iron-loving') element that preferentially partitioned into the Earth's core during core formation but is also 'incompatible' during mantle melting (it preferentially enters the melt phase), which makes it further depleted in the mantle. Tungsten should therefore be a sensitive tracer of core contributions in the source of mantle melts. Here we present high-precision tungsten isotope data from the same set of Hawaiian rocks used to establish the previously interpreted (186)Os-(187)Os anomalies and on selected South African rocks, which have also been proposed to contain a core contribution. None of the samples that we have analysed have a negative tungsten isotope value, as predicted from the core-contribution model. This rules out a simple core-mantle mixing scenario and suggests that the radiogenic osmium in ocean-island basalts can better be explained by the source of such basalts containing a component of recycled crust.     

Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity

Elucidating the signalling mechanisms by which obesity leads to impaired insulin action is critical in the development of therapeutic strategies for the treatment of diabetes. Recently, mice deficient for S6 Kinase 1 (S6K1), an effector of the mammalian target of rapamycin (mTOR) that acts to integrate nutrient and insulin signals, were shown to be hypoinsulinaemic, glucose intolerant and have reduced beta-cell mass. However, S6K1-deficient mice maintain normal glucose levels during fasting, suggesting hypersensitivity to insulin, raising the question of their metabolic fate as a function of age and diet. Here, we report that S6K1-deficient mice are protected against obesity owing to enhanced beta-oxidation. However on a high fat diet, levels of glucose and free fatty acids still rise in S6K1-deficient mice, resulting in insulin receptor desensitization. Nevertheless, S6K1-deficient mice remain sensitive to insulin owing to the apparent loss of a negative feedback loop from S6K1 to insulin receptor substrate 1 (IRS1), which blunts S307 and S636/S639 phosphorylation; sites involved in insulin resistance. Moreover, wild-type mice on a high fat diet as well as K/K A(y) and ob/ob (also known as Lep/Lep) mice-two genetic models of obesity-have markedly elevated S6K1 activity and, unlike S6K1-deficient mice, increased phosphorylation of IRS1 S307 and S636/S639. Thus under conditions of nutrient satiation S6K1 negatively regulates insulin signalling.     

Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism

The molybdenum cofactor is part of the active site of all molybdenum-dependent enzymes, except nitrogenase. The molybdenum cofactor consists of molybdopterin, a phosphorylated pyranopterin, with an ene-dithiolate coordinating molybdenum. The same pyranopterin-based cofactor is involved in metal coordination of the homologous tungsten-containing enzymes found in archea. The molybdenum cofactor is synthesized by a highly conserved biosynthetic pathway. In plants, the multidomain protein Cnx1 catalyses the insertion of molybdenum into molybdopterin. The Cnx1 G domain (Cnx1G), whose crystal structure has been determined in its apo form, binds molybdopterin with high affinity and participates in the catalysis of molybdenum insertion. Here we present two high-resolution crystal structures of Cnx1G in complex with molybdopterin and with adenylated molybdopterin (molybdopterin-AMP), a mechanistically important intermediate. Molybdopterin-AMP is the reaction product of Cnx1G and is subsequently processed in a magnesium-dependent reaction by the amino-terminal E domain of Cnx1 to yield active molybdenum cofactor. The unexpected identification of copper bound to the molybdopterin dithiolate sulphurs in both structures, coupled with the observed copper inhibition of Cnx1G activity, provides a molecular link between molybdenum and copper metabolism.     

Membrane structure and interactions with protein and DNA in bacteriophage PRD1

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.     

The Vision Conference: Facilitating Creative Processes

This paper presents the principles behind the design and management of the Vision Conference: a one-day workshop in which a large group of participants meet to create ideas, projects, and visions for the future activities of a local community or an organization. A case study from a local community in Denmark is also presented to illustrate the organization, planning, and management of a Vision Conference. The paper focuses on the three central social processes of the conference: group work, problem solving, and facilitation. The paper ends with a discussion of creativity and creativity techniques suitable for Vision Conferences.     

Acylphosphatase overexpression triggers SH-SY5Y differentiation towards neuronal phenotype

An acylphosphatase (AcPase) overexpression study was carried out on SH-SY5Y neuroblastoma cells, using a green fluorescent fusion protein (AcP-GFP), with GFP acting as a reporter protein. The cellular proliferation rate was significantly reduced by overexpression of AcPase by a factor of ten. In contrast, clones transfected with two inactive AcPase mutants showed a growth rate comparable to control cells. This suggests that AcPase catalyzes the proliferative down-regulation. AcPase-overexpressing clones showed a physiological mortality rate as assessed by an MTT reduction test and by evaluation of necrotic markers. DNA fragmentation analysis and assays of caspase-3 and poly (ADP-ribose) polymerase (PARP)-active fragments showed no evidence of any apoptotic pattern. AcPase overexpression led to a marked increase in PARP activity as well as Bcl-2 content; these are commonly up-regulated during differentiative processes in neuronal cells. In fact, the typical differentiation marker, growth-associated-protein 43, was significantly up-regulated. Microscopic observations also showed a clear increase in the differentiative phenotype in AcPase-overexpressing cells. Our results clearly show that AcPase plays a primary causative role in neuronal differentiation.Received 3 May 2004; accepted 25 May 2004     

Caffeine as a psychomotor stimulant: mechanism of action

The popularity of caffeine as a psychoactive drug is due to its stimulant properties, which depend on its ability to reduce adenosine transmission in the brain. Adenosine A₁ and A_2A receptors are expressed in the basal ganglia, a group of structures involved in various aspects of motor control. Caffeine acts as an antagonist to both types of receptors. Increasing evidence indicates that the psychomotor stimulant effect of caffeine is generated by affecting a particular group of projection neurons located in the striatum, the main receiving area of the basal ganglia. These cells express high levels of adenosine A_2A receptors, which are involved in various intracellular processes, including the expression of immediate early genes and regulation of the dopamine- and cyclic AMP-regulated 32-kDa phosphoprotein DARPP-32. The present review focuses on the effects of caffeine on striatal signal transduction and on their involvement in caffeine-mediated motor stimulation.Received 8 July 2003; received after revision 7 September 2003; accepted 6 October 2003     

Horizontal gene transfer from Eukarya to Bacteria and domain shuffling: the α-amylase model

-Amylases are present in all kingdoms of the living world. Despite strong conservation of the tertiary structure, only a few amino acids are conserved in interkingdom comparisons. Animal -amylases are characterized by several typical motifs and biochemical properties. A few cases of such -amylases have been previously reported in some eubacterial species. We screened the bacterial genomes available in the sequence databases for new occurrences of animal-like -amylases. Three novel cases were found, which belong to unrelated bacterial phyla: Chloroflexus aurantiacus, Microbulbifer degradans, and Thermobifida fusca. All the animal-like -amylases in Bacteria probably result from repeated horizontal gene transfer from animals. The M. degradans genome also contains bacterial-type and plant-type -amylases in addition to the animal-type one. Thus, this species exhibits -amylases of animal, plant, and bacterial origins. Moreover, the similarities in the extra C-terminal domains (different from both the -amylase domain C and the starch-binding domain), when present, also suggest interkingdom as well as intragenomic shuffling.Received 17 October 2003; accepted 6 November 2003