Genome sequencing and analysis of Aspergillus oryzae

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.     

SWAP-70 is a guanine-nucleotide-exchange factor that mediates signalling of membrane ruffling

Phosphoinositide-3-OH kinase (PI(3)K), activated through growth factor stimulation, generates a lipid second messenger, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 is instrumental in signalling pathways that trigger cell activation, cytoskeletal rearrangement, survival and other reactions. However, some targets of PtdIns(3,4,5)P3 are yet to be discovered. We demonstrate that SWAP-70, a unique signalling protein, specifically binds PtdIns(3,4,5)P3. On stimulation by growth factors, cytoplasmic SWAP-70, which is dependent on PI(3)K but independent of Ras, moved to cell membrane rearrangements known as ruffles. However, mutant SWAP-70 lacking the ability to bind PtdIns(3,4,5)P3 blocked membrane ruffling induced by epidermal growth factor or platelet-derived growth factor. SWAP-70 shows low homology with Rac-guanine nucleotide exchange factors (GEFs), and catalyses PtdIns(3,4,5)P3-dependent guanine nucleotide exchange to Rac. SWAP-70-deficient fibroblasts showed impaired membrane ruffling after stimulation with epidermal growth factor, and failed to activate Rac fully. We conclude that SWAP-70 is a new type of Rac-GEF which, independently of Ras, transduces signals from tyrosine kinase receptors to Rac.     

Tristable nematic liquid-crystal device using micropatterned surface alignment

It has long been appreciated that liquid-crystal (LC) devices in which the LC molecules adopt multiple stable orientations could drastically reduce the power consumption required for high-information-content displays. But for the commonly used nematic LCs, which are intrinsically uniaxial in symmetry, no industrially feasible multi-stable LC device has been realized. Recently we demonstrated how bistability can be robustly engineered into a nematic LC device, by patterning a substrate with an orientational chequerboard pattern that enforces orthogonal LC alignment in neighbouring square domains. As a result of the four-fold symmetry of the pattern, the two diagonal axes of the chequerboard become equally stable macroscopic orientations. Here we extend this symmetry approach to obtain a tristable surface-aligned nematic LC. A microscopic pattern exhibiting six-fold symmetry is inscribed on a polyimide surface using the stylus of an atomic force microscope. The hexagonal symmetry of the microscopic orientational domains in turn gives rise to three stable macroscopic LC orientations, which are mutually switchable by an in-plane electric field. The resulting switching mode is surface driven, and hence should be compatible with demanding flexible display applications.     

An origin of the radio jet in M87 at the location of the central black hole

Powerful radio jets from active galactic nuclei are thought to be powered by the accretion of material onto the supermassive black hole (the 'central engine'). M87 is one of the closest examples of this phenomenon, and the structure of its jet has been probed on a scale of about 100 Schwarzschild radii (R(s), the radius of the event horizon). However, the location of the central black hole relative to the jet base (a bright compact radio 'core') remains elusive. Observations of other jets indicate that the central engines are located about 10(4)-10(6)R(s) upstream from the radio core. Here we report radio observations of M87 at six frequencies that allow us to achieve a positional accuracy of about 20?microarcseconds. As the jet base becomes more transparent at higher frequencies, the multifrequency position measurements of the radio core enable us to determine the upstream end of the jet. The data reveal that the central engine of M87 is located within 14-23R(s) of the radio core at 43?GHz. This implies that the site of material infall onto the black hole and the eventual origin of the jet reside in the bright compact region seen on the image at 43?GHz.     

Structural basis of HutP-mediated anti-termination and roles of the Mg2+ ion and L-histidine ligand

HutP regulates the expression of the hut structural genes of Bacillus subtilis by an anti-termination mechanism and requires two components, Mg2+ ions and L-histidine. HutP recognizes three UAG triplet units, separated by four non-conserved nucleotides on the terminator region. Here we report the 1.60-A resolution crystal structure of the quaternary complex (HutP-L-histidine-Mg2+-21-base single-stranded RNA). In the complex, the RNA adopts a novel triangular fold on the hexameric surface of HutP, without any base-pairing, and binds to the protein mostly by specific protein-base interactions. The structure explains how the HutP and RNA interactions are regulated critically by the l-histidine and Mg2+ ion through the structural rearrangement. To gain insights into these structural rearrangements, we solved two additional crystal structures (uncomplexed HutP and HutP-L-histidine-Mg2+) that revealed the intermediate structures of HutP (before forming an active structure) and the importance of the Mg2+ ion interactions in the complexes.     

p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.     

Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting

Catalytic processes on surfaces have long been studied by probing model reactions on single-crystal metal surfaces under high vacuum conditions. Yet the vast majority of industrial heterogeneous catalysis occurs at ambient or elevated pressures using complex materials with crystal faces, edges and defects differing in their catalytic activity. Clearly, if new or improved catalysts are to be rationally designed, we require quantitative correlations between surface features and catalytic activity--ideally obtained under realistic reaction conditions. Transmission electron microscopy and scanning tunnelling microscopy have allowed in situ characterization of catalyst surfaces with atomic resolution, but are limited by the need for low-pressure conditions and conductive surfaces, respectively. Sum frequency generation spectroscopy can identify vibrations of adsorbed reactants and products in both gaseous and condensed phases, but so far lacks sensitivity down to the single molecule level. Here we adapt real-time monitoring of the chemical transformation of individual organic molecules by fluorescence microscopy to monitor reactions catalysed by crystals of a layered double hydroxide immersed in reagent solution. By using a wide field microscope, we are able to map the spatial distribution of catalytic activity over the entire crystal by counting single turnover events. We find that ester hydrolysis proceeds on the lateral {1010} crystal faces, while transesterification occurs on the entire outer crystal surface. Because the method operates at ambient temperature and pressure and in a condensed phase, it can be applied to the growing number of liquid-phase industrial organic transformations to localize catalytic activity on and in inorganic solids. An exciting opportunity is the use of probe molecules with different size and functionality, which should provide insight into shape-selective or structure-sensitive catalysis and thus help with the rational design of new or more productive heterogeneous catalysts.     

Remote triggering of deep earthquakes in the 2002 Tonga sequences

It is well established that an earthquake in the Earth's crust can trigger subsequent earthquakes, but such triggering has not been documented for deeper earthquakes. Models for shallow fault interactions suggest that static (permanent) stress changes can trigger nearby earthquakes, within a few fault lengths from the causative earthquake, whereas dynamic (transient) stresses carried by seismic waves may trigger earthquakes both nearby and at remote distances. Here we present a detailed analysis of the 19 August 2002 Tonga deep earthquake sequences and show evidence for both static and dynamic triggering. Seven minutes after a magnitude 7.6 earthquake occurred at a depth of 598 km, a magnitude 7.7 earthquake (664 km depth) occurred 300 km away, in a previously aseismic region. We found that nearby aftershocks of the first mainshock are preferentially located in regions where static stresses are predicted to have been enhanced by the mainshock. But the second mainshock and other triggered events are located at larger distances where static stress increases should be negligible, thus suggesting dynamic triggering. The origin times of the triggered events do not correspond to arrival times of the main seismic waves from the mainshocks and the dynamically triggered earthquakes frequently occur in aseismic regions below or adjacent to the seismic zone. We propose that these events are triggered by transient effects in regions near criticality, but where earthquakes have difficulty nucleating without external influences.     

Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.     

HAR1 mediates systemic regulation of symbiotic organ development

Symbiotic root nodules are beneficial to leguminous host plants; however, excessive nodulation damages the host because it interferes with the distribution of nutrients in the plant. To keep a steady balance, the nodulation programme is regulated systemically in leguminous hosts. Leguminous mutants that have lost this ability display a hypernodulating phenotype. Through the use of reciprocal and self-grafting studies using Lotus japonicus hypernodulating mutants, har1 (also known as sym78), we show that the shoot genotype is responsible for the negative regulation of nodule development. A map-based cloning strategy revealed that HAR1 encodes a protein with a relative molecular mass of 108,000, which contains 21 leucine-rich repeats, a single transmembrane domain and serine/threonine kinase domains. The har1 mutant phenotype was rescued by transfection of the HAR1 gene. In a comparison of Arabidopsis receptor-like kinases, HAR1 showed the highest level of similarity with CLAVATA1 (CLV1). CLV1 negatively regulates formation of the shoot and floral meristems through cell-cell communication involving the CLV3 peptide. Identification of hypernodulation genes thus indicates that genes in leguminous plants bearing a close resemblance to CLV1 regulate nodule development systemically, by means of organ-organ communication.