Atomic mutagenesis of the ribosomal Sarcin-Ricin-Loop to study EF-G GTPase activation

Translocation factor EF-G, possesses a low basal GTPase activity, which is stimulated by the ribosome. One potential region of the ribosome that triggers GTPase activity of EF-G is the Sarcin-Ricin-Loop (SRL) (helix 95) in domain VI of the 23S rRNA. Structural data showed that the tip of the SRL closely approaches GTP in the active center of EF-G, structural probing data confirmed that EF-G interacts with nucleotides G2655, A2660, G2661 and A2662.1-3 The exocyclic group of adenine at A2660 is required for stimulation of EF-G GTPase activity by the ribosome as demonstrated using atomic mutagenesis.4 Recent crystal structures of EF-G on the ribosome, gave more insights into the molecular mechanism of EF-G GTPase activity.5 Based on the structure of EF-Tu on the ribosome1, the following mechanism of GTPase activation was proposed: upon binding of EF-G to the ribosome, the conserved His92 (E.coli) changes its position, pointing to the γ-phosphate of GTP. In this activated state, the phosphate of residue A2662 of the SRL positions the catalytic His in its active conformation. It was further proposed that the phosphate oxygen of A2662 is involved in a charge-relay system, enabling GTP hydrolysis. In order to test this mechanism, we use the atomic mutagenesis approach, which allows introducing non-natural modifications in the SRL, in the context of the complete 70S ribosome. Therefore, we replaced one of the non-bridging oxygens of A2662 by a methyl group. A methylphosphonat is not able to position or activate a histidine, as it has no free electrons and therefore no proton acceptor function. These modified ribosomes were then tested for stimulation of EF-G GTPase activity. First experiments show that one of the two stereoisomers incorporated into ribosomes does not stimulate GTPase activity of EF-G, whereas the other is active. From this we conclude that indeed the non-bridging phosphate oxygen of A2662 is involved in EF-G GTPase activation by the ribosome. Ongoing experiments aim at revealing the contribution of this non-bridging oxygen at A2662 to the mechanism of EF-G GTPase activation at the atomic level.

Miocene radiolarians of the Sulu Sea

Radiolarians are sporadic in sediments collected in the Sulu Sea during ODP Leg 124. Due to the generally poor preservation and low abundance of radiolarians in Sulu Sea sediments, no biostratigraphic datums are well defined, although three radiolarian zones are identified. Most samples containing radiolarians are pelagic or hemipelagic clays with varying proportions of volcanic ash. Detailed analysis of Sulu Sea radiolarians was limited to Miocene successions. Pliocene and Quaternary occurrences of radiolarians were noted but have not been zoned. The late middle Miocene of Sites 769 and 771 is represented by an assemblage of radiolarians (Diartus petterssoni Zone) that is entirely replaced by massive pyrite. This type of preservation develops only under anoxic conditions. The development of widespread anoxia in Sulu Sea waters in the late middle Miocene was probably the result of hydrologic isolation of basin waters, and may be associated with eustatic sea level fall over the silled basin. Upper lower Miocene pelagic and hemipelagic sediments that overlie pyroclastics and basalt flows in the Sulu Sea sites contain moderately to very poorly preserved radiolarians of the Calocycletta costata Zone. A thin unit of marine claystone was recovered from between the thick pyroclastics and basement rocks at Site 768. Radiolarians present in these claystones are rare and very poorly preserved. This radiolarian assemblage probably represents the C. costata Zone, although very poor preservation and low abundance make this interpretation equivocal. The radiolarian zones identified constrain the age of basin formation to late early Miocene or earlier.

Radiolarians of the Celebes Sea

Two sites were drilled in the Celebes Sea as part of Ocean Drilling Program Leg 124; Site 767 and Site 770. Radiolarians are preserved in Paleogene pelagic claystones with minor occurrences in certain Neogene successions. The brown clays that immediately overlie basalt at both sites contain radiolarians of the late middle Eocene Podocyrtis chalara Zone. Late Eocene radiolarians are not found, due to dissolution and probable hiatus. The Oligocene is represented by the Theocyrtis tuberosa and Dorcadospyris ateuchus Zones. Oligocene sediments are strongly dominated by abundant and diverse radiolarians of the TristylospyrislDorcadospyris lineage. Preservation of Paleogene radiolarian assemblages ranges from good to very poor. Late Miocene radiolarians of the Didymocyrtis antepenultima Zone are found only in Site 770. Other Neogene sediments are barren of radiolarian remains, with the exception of latest Pleistocene and Holocene sediments.

Multi Domain-Specific Modeling of the Security Concerns of Service-Oriented Architectures

Service-Oriented Architectures (SOA), and Web Services (WS), the technology generally used to implement them, achieve the integration of heterogeneous technologies, providing interoperability, and yielding the reutilization of pre-existent systems. Model-driven development methodologies provide inherent benefits such as increased productivity, greater reuse, and better maintainability, to name a few. Efforts on achieving model-driven development of SOAs already exist, but there is currently no standard solution that addresses non-functional aspects of these services as well. This paper presents an approach to integrate these non-functional aspects in the development of web services, with an emphasis on security.

Role of maize stover incorporation on nitrogen oxide emissions in a non-irrigated Mediterranean barley field.

Aims Agricultural soils in semiarid Mediterranean areas are characterized by low organic matter contents and low fertility levels. Application of crop residues and/or manures as amendments is a cost-effective and sustainable alternative to overcome this problem. However, these management practices may induce important changes in the nitrogen oxide emissions from these agroecosystems, with additional impacts on carbon dioxide emissions. In this context, a field experiment was carried out with a barley (Hordeum vulgare L.) crop under Mediterranean conditions to evaluate the effect of combining maize (Zea mays L.) residues and N fertilizer inputs (organic and/or mineral) on these emissions. Methods Crop yield and N uptake, soil mineral N concentrations, dissolved organic carbon (DOC), denitrification capacity, N2O, NO and CO2 fluxes were measured during the growing season. Results The incorporation of maize stover increased N2O emissions during the experimental period by c. 105 %. Conversely, NO emissions were significantly reduced in the plots amended with crop residues. The partial substitution of urea by pig slurry reduced net N2O emissions by 46 and 39 %, with and without the incorporation of crop residues respectively. Net emissions of NO were reduced 38 and 17 % for the same treatments. Molar DOC:NO 3 − ratio was found to be a robust predictor of N2O and NO fluxes. Conclusions The main effect of the interaction between crop residue and N fertilizer application occurred in the medium term (4–6 month after application), enhancing N2O emissions and decreasing NO emissions as consequence of residue incorporation. The substitution of urea by pig slurry can be considered a good management strategy since N2O and NO emissions were reduced by the use of the organic residue.

Structural basis of an embryonically lethal single Ala → Thr mutation in the vnd/NK-2 homeodomain

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to −5°C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25°C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix–turn–helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency1

The mobility of elements within plants contributes to a plant species' tolerance of nutrient deficiencies in the soil. The genetic manipulation of within-plant nutrient movement may therefore provide a means to enhance plant growth under conditions of variable soil nutrient availability. In these experiments tobacco (Nicotiana tabacum) was engineered to synthesize sorbitol, and the resultant effect on phloem mobility of boron (B) was determined. In contrast to wild-type tobacco, transgenic tobacco plants containing sorbitol exhibit a marked increase in within-plant B mobility and a resultant increase in plant growth and yield when grown with limited or interrupted soil B supply. Growth of transgenic tobacco could be maintained by reutilization of B present in mature tissues or from B supplied as a foliar application to mature leaves. In contrast, B present in mature leaves of control tobacco lines could not be used to provide the B requirements for new plant growth. 10B-labeling experiments verified that B is phloem mobile in transgenic tobacco but is immobile in control lines. These results demonstrate that the transgenic enhancement of within-plant nutrient mobility is a viable approach to improve plant tolerance of nutrient stress.

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Analysis of the relation between the sequence and secondary and three-dimensional structures of immunoglobulin molecules.

Methods of structural and statistical analysis of the relation between the sequence and secondary and three-dimensional structures are developed. About 5000 secondary structures of immunoglobulin molecules from the Kabat data base were predicted. Two statistical analyses of amino acids reveal 47 universal positions in strands and loops. Eight universally conservative positions out of the 47 are singled out because they contain the same amino acid in > 90% of all chains. The remaining 39 positions, which we term universally alternative positions, were divided into five groups: hydrophobic, charged and polar, aromatic, hydrophilic, and Gly-Ala, corresponding to the residues that occupied them in almost all chains. The analysis of residue-residue contacts shows that the 47 universal positions can be distinguished by the number and types of contacts. The calculations of contact maps in the 29 antibody structures revealed that residues in 24 of these 47 positions have contacts only with residues of antiparallel beta-strands in the same beta-sheet and residues in the remaining 23 positions always have far-away contacts with residues from other beta-sheets as well. In addition, residues in 6 of the 47 universal positions are also involved in interactions with residues of the other variable or constant domains.

Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by site-directed mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of the [Fe4S4]3+ core.

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.

Microwave-Assisted Extraction of Phenolic Compounds from Almond Skin Byproducts (Prunus amygdalus): A Multivariate Analysis Approach

A microwave-assisted extraction (MAE) procedure to isolate phenolic compounds from almond skin byproducts was optimized. A three-level, three-factor Box–Behnken design was used to evaluate the effect of almond skin weight, microwave power, and irradiation time on total phenolic content (TPC) and antioxidant activity (DPPH). Almond skin weight was the most important parameter in the studied responses. The best extraction was achieved using 4 g, 60 s, 100 W, and 60 mL of 70% (v/v) ethanol. TPC, antioxidant activity (DPPH, FRAP), and chemical composition (HPLC-DAD-ESI-MS/MS) were determined by using the optimized method from seven different almond cultivars. Successful discrimination was obtained for all cultivars by using multivariate linear discriminant analysis (LDA), suggesting the influence of cultivar type on polyphenol content and antioxidant activity. The results show the potential of almond skin as a natural source of phenolics and the effectiveness of MAE for the reutilization of these byproducts.

Decomposition of nitrogen-15 labeled hoop pine harvest residues in subtropical Australia

Information on decomposition of harvest residues may assist in the maintenance of soil fertility in second rotation (2R) hoop pine plantations (Araucaria cunninghamii Aiton ex A. Cunn.) of subtropical Australia. The experiment was undertaken to determine the dynamics of residue decomposition and fate of residue-derived N. We used N-15-labeled hoop pine foliage, branch, and stem material in microplots, over a 30-mo period following harvesting. We examined the decomposition of each component both singly and combined, and used C-13 cross-polarization and magic-angle spinning nuclear magnetic resonance (C-13 CPMAS NMR) to chart C transformations in decomposing foliage. Residue-derived N-15 was immobilized in the 0- to 5-cm soil layer, with approximately 40% N-15 recovery in the soil from the combined residues by the end of the 30-mo period. Total recovery of N-15 in residues and soil varied between 60 and 80% for the combined-residue microplots, with 20 to 40% of the residue N-15 apparently lost. When residues were combined within microplots the rate of foliage decomposition decreased by 30% while the rate of branch and stem decomposition increased by 50 and 40% compared with rates for these components when decomposed separately. Residue decomposition studies should include a combined-residue treatment. Based on C-15 CPMAS NMR spectra for decomposing foliage, we obtained good correlations for methoxyl C, aryl C, carbohydrate C and phenolic C with residue mass, N-15 enrichment, and total N. The ratio of carbohydrate C to methoxyl C may be useful as an indicator of harvest residue decomposition in hoop pine plantations.

Organic Matter Dynamics in the Wet Tropics Under Different Sugarcane Residue Management Regimes

Retention of sugarcane leaves and tops on the soil surface after harvesting has almost completely replaced pre- and post-harvest burning of crop residues in the Australian sugar industry. Since its introduction around 25 years ago, residue retention has increased soil organic matter to improve soil fertility as well as improve harvest flexibility and reduce erosion. However, in the wet tropics residue retention also poses potential problems of prolonged waterlogging, and late-season release of nitrogen which can reduce sugar content of the crop. The objective of this project is to examine the management of sugarcane residues in the wet tropics using a systems approach. Subsidiary objectives are (a) to improve understanding of nitrogen cycling in Australian sugarcane soils in the wet tropics, and (b) to identify ways to manage crop residues to retain their advantages and limit their disadvantages. Project objectives will be addressed using several approaches. Historic farm production data recorded by sugar mills in the wet tropics will be analysed to determine the effect of residue burning or retention on crop yield and sugar content. The impact of climate on soil processes will be highlighed by development of an index of nitrogen mineralisation using the Agricultural Production Systems Simulator (APSIM) model. Increased understanding of nitrogen cycling in Australian sugarcane soils and management of crop residues will be gained through a field experiment recently established in the Australian wet tropics. From this experiment the decomposition and nitrogen dynamics of residues placed on the soil surface and incorporated will be compared. The effect of differences in temperature, soil water content and pH will be further examined on these soils under glasshouse conditions. Preliminary results show a high ammonium to nitrate ratio in tropics soils, which may be due to low rates of nitrification that increase the retention of nitrogen in a form (ammonium) that is less subject to leaching. Further results will be presented at Congress.