Observation of covalent intermediates in an enzyme mechanism at atomic resolution

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.     

Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida

Many Gram-negative bacteria export proteins to the exterior. Some of these proteins are first secreted into the periplasm and then cross the outer membrane in a separate step. The source of energy required for the translocation is unknown. Export of the extracellular protein proaerolysin from the periplasm through the outer membrane of Aeromonas salmonicida is inhibited by a proton ionophore and by low extracellular pH. One possible explanation of these results is that a proton gradient across the outer membrane is required for export.     

Hierarchical self-assembly of F-actin and cationic lipid complexes: stacked three-layer tubule networks

We describe a distinct type of spontaneous hierarchical self-assembly of cytoskeletal filamentous actin (F-actin), a highly charged polyelectrolyte, and cationic lipid membranes. On the mesoscopic length scale, confocal microscopy reveals ribbonlike tubule structures that connect to form a network of tubules on the macroscopic scale (more than 100 micrometers). Within the tubules, on the 0.5- to 50-nanometer length scale, x-ray diffraction reveals an unusual structure consisting of osmotically swollen stacks of composite membranes with no direct analog in simple amphiphilic systems. The composite membrane is composed of three layers, a lipid bilayer sandwiched between two layers of actin, and is reminiscent of multilayered bacterial cell walls that exist far from equilibrium. Electron microscopy reveals that the actin layer consists of laterally locked F-actin filaments forming an anisotropic two-dimensional tethered crystal that appears to be the origin of the tubule formation.     

Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins

Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.     

Gene amplification of c-myc and N-myc in small cell carcinoma of the lung

The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.     

X-ray Absorption Spectroscopic Investigation of Trace Vanadium Sites in Coal

X-ray absorption spectroscopy was used to probe the chemical and structural environment of vanadium in coal. It was found that vanadium exists in at least two environments, in both of which it was coordinated to oxygens. There was no evidence of vanadium in nitrogen (porphyrin) or sulfide environments. It was also found that the vanadium environments in the raw coal did not survive unchanged in a liquefaction process. These findings have implications for coal cleaning processes and for trace element release into the liquefaction process stream.     

Aflatoxicol: major aflatoxin B1 metabolite in rat plasma

Aflatoxicol, a carcinogenic metabolite of the foodborne carcinogen aflatoxin B1 previously known only as a bioreduction product in vitro, was identified as the major aflatoxin metabolite in the plasma of Sprague-Dawley rats, a susceptible species, that had been doses orally or intravenously with aflatoxin B1 labeled with carbon-14. Alfatoxicol, however, was not detected in the plasma of similarly dosed mice and monkeys, which are both resistant to aflatoxin B1-induced cardinogenesis. The formation of aflatoxicol both in vitro and in vivo may be an indicatory of species sensitivity to aflatoxin-induced carcinogenesis and may be useful in the prediction of human susceptibility.     

Gravity field of jupiter from pioneer 11 tracking data

Significantly improved values of the zonal gravity harmonic coefficients J(39) J(4), and J(6) of Jupiter have been obtained from a preliminary analysis of Pioneer 11 spacecraft Doppler data taken while the spacecraft was near Jupiter. The new results, which will have an important application as boundary conditions for theoretical models of Jupiter's interior. are consistent with a planet in hydrostatic equilibrium.     

A flexible approach to managing variability in grain yield and nitrate leaching at within-field to farm scales

We up-scaled the APSIM simulation model of crop growth, water and nitrogen dynamics to interpret and respond to spatial and temporal variations in soil, season and crop performance and improve yield and decrease nitrate leaching. Grain yields, drainage below the maximum root depth and nitrate leaching are strongly governed by interaction of plant available soil water storage capacity (PAWC), seasonal rainfall and nitrogen supply in the water-limited Mediterranean-type environment of Western Australia (WA). APSIM simulates the interaction of these key system parameters and the robustness of its simulations has been rigorously tested with the results of several field experiments covering a range of soil types and seasonal conditions in WA. We used yield maps, soil and weather data for farms at two locations in WA to determine spatial and temporal patterns of grain yield, drainage below the maximum root depth and nitrate leaching under a range of weather, soil and nitrogen management scenarios. On one farm, we up-scaled APSIM simulations across the whole farm using local weather and fertiliser use data and the average PAWC values of soil type polygons. On a 70 ha field on another farm, we used a linear regression of apparent soil electrical conductivity (EC_a) measured by EM38 against PAWC to transform an EC_a map of the field into a high resolution (5 m grid) PAWC map. We then used regressions of simulated yields, drainage below the maximum root depth and nitrate leaching on PAWC to upscale the APSIM simulations for a range of weather and fertiliser management scenarios. This continuous mapping approach overcame the weakness of the soil polygons approach, which assumed uniformity in soil properties and processes within soil type polygons. It identified areas at greatest financial and environmental risks across the field, which required focused management and simulated their response to management interventions. Splitting nitrogen applications increased simulated wheat yields at all sites across the field and decreased nitrate leaching particularly where the water storage capacity of the soil was small. Low water storage capacity resulted in both low wheat yields and large leaching loss. Another management option to decrease leaching may be to grow perennial vegetation that uses more water and loses less by drainage.Paper from the 5th European Conference on Precision Agriculture (5ECPA), Uppsala, Sweden, 2005     

Phonon dispersions of fcc delta-plutonium-gallium by inelastic x-ray scattering

We report an experimental determination of the phonon dispersion curves in a face-centered cubic (fcc) delta-plutonium-0.6 weight % gallium alloy. Several unusual features, including a large elastic anisotropy, a small-shear elastic modulus C', a Kohn-like anomaly in the T1[011] branch, and a pronounced softening of the [111] transverse modes, are found. These features can be related to the phase transitions of plutonium and to strong coupling between the lattice structure and the 5f valence instabilities. Our results also provide a critical test for theoretical treatments of highly correlated 5f electron systems as exemplified by recent dynamical mean field theory calculations for delta-plutonium.