Structural and functional characterization of a novel phosphodiesterase from Methanococcus jannaschii

Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular (biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-A resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two beta-sheets flanked by alpha-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni(2+) or Mn(2+) produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni(2+) and Mn(2+) being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase.     

Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins

For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.     

Conservation value of timber quality versus associated non-timber quality stands for understory diversity in Nothofagus forests

Conservation strategies of forested landscapes must consider biodiversity of the included site types, i.e. timber-quality forests and associated non-timber-quality stands. The objectives were to characterize forest overstory structure in timber-quality versus associated non-timber-quality stands; and to compare their understory communities. Six forest types were sampled in Nothofagus forests of Tierra del Fuego (Argentina): two timber-quality N. pumilio forests, and four associated non-timber-quality stands (edge, N. antarctica, wetlands and streamside forests). Overstory structure and understory vegetation (species richness, frequencies, cover and biomass) were characterized during spring and summer seasons. Analysis of variance and multivariates were carried out. Overstory structure differed across the site types, with higher tree size, canopy closure and tree volume in timber-quality stands. Fifty-one understory plant species were observed, but understory variables varied with site types, especially wetlands (highest native and exotic richness, cover and biomass, and 25% of exclusive species). Site types were grouped in three: N. antarctica stands, streamside stands and the other N. pumilio forests according to multivariate analysis. Forty three percent of plants were distributed in all site types, and all timber-quality forest understory species were present in some associated non-timber-quality stands. Timber-quality N. pumilio forests have a marginal value for understory conservation compared to associated non-timber-quality stands, because these last include all the plants observed in timber-quality forests and also possess many exclusive species. Therefore, protection of associated non-timber-quality stands during forest management planning could increase understory conservation at landscape level, and these could be better reserves of understory diversity than retentions of timber-quality stands.     

Plasminogen activators and their inhibitors in a human mammary cell line (HBL-100). Modulation by glucocorticoids

Culture of human mammary HBL-100 cells in the presence of dexamethasone, a synthetic glucocorticoid, resulted in opposite effects on the production of the two plasminogen activators (PAs): a decrease in urokinase-type PA (u-PA) and a concomitant increase in tissue-type PA (t-PA). Two PA-specific inhibitors, one related to that produced by bovine aortic endothelial cells, and the other related to that isolated from human placenta, were also produced by these cells; dexamethasone did not affect the production of either of these inhibitors. The glucocorticoid effects observed on PA enzymatic activities were associated with changes in PA mRNA levels. Experiments using inhibitors of RNA and protein synthesis suggested that the glucocorticoid-induced decrease in u-PA mRNA was a secondary event, requiring synthesis of new regulatory proteins; in contrast, the increase in t-PA mRNA appeared to be a direct effect on t-PA gene expression.     

Adequacy of a systems structure in the modeling of training effects on performance.

A systems model of training effects on performance was applied to eight initially untrained subjects who were volunteers for an endurance training program for the purpose of verifying the statistical adequacy of the systems structure. In the model initially proposed by T. W. Calvert, E. W. Banister, M. V. Savage, and T. Bach (IEEE Trans. Syst. Man Cybern. 6: 94-102, 1976), the performance changes were related to the successive training loads by three first-order transfer functions. In the present study, the number of first-order components was statistically tested. A model including only one component, which had a positive effect on the performance, provided a significant fit with the performances in every subject. A second component significantly improved the fit in only two subjects. This further component, which had a negative effect on performance, was identified as fatigue. Nevertheless, a two-antagonistic component model is proposed to provide a good representation of the training responses. However, the low level of exercise demands and the inaccuracy of the fit could have impaired the evidencing of a fatiguing effect during the presently studied training protocol.     

Gout. Mechanisms of inflammation in gout

An acute attack of gout is a paradigm of acute sterile inflammation, as opposed to pyogenic inflammation. Recent studies suggest that the triggering of IL-1β release from leucocytes lies at the heart of a cascade of processes that involves multiple cytokines and mediators. The NLRP3 inflammasome appears to have a specific role in this regard, but the biochemical events leading to its activation are still not well understood. We review the known mechanisms that underlie the inflammatory process triggered by urate crystals and suggest areas that require further research.     

Root traits associated with nutrient exploitation following defoliation in three coexisting perennial grasses in a semi-arid savanna

Experiments were conducted to evaluate root traits associated with nutrient exploitation following defoliation in three coexisting perennial grasses in a semi‐arid savanna. Root length density was determined within soil cores directly beneath plants, nitrogen uptake was evaluated by excised‐root assay with (¹⁵NH₄)₂SO₄, and mycorrhizal root colonization was estimated by observation of root segments. Root length density was lowest for Bouteloua curtipendula, intermediate for Eriochloa sericea, and highest for Aristida purpurea indicating that root length density was a more important trait for the mid‐seral than the late‐seral species. Rates of ¹⁵N uptake were greatest in the least grazing tolerant late‐seral species, E. sericea, intermediate in the mid‐seral species, A. purpurea, and lowest in the most grazing tolerant late‐seral species, B. curtipendula. Two successive defoliations reduced ¹⁵N uptake 60% in the late‐seral species with the greatest uptake rate (E. sericea), but not in species with lowest uptake rates (B. curtipendula). Root length colonization was consistently high (33–61%) in all three species suggesting that these C₄ perennial grasses may function as obligate mycotrophs. Contrasting responses among the two late‐seral species indicate that the least grazing tolerant species, E. sericea, appears best adapted for nutrient exploitation while the most grazing tolerant species, B. curtipendula, appears best adapted for efficient nutrient retention. Contrasting responses of nitrogen uptake to short‐term defoliation parallel the population responses of these two coexisting late‐seral species to long‐term herbivory. These data indicate that herbivory may shift interspecific competitive interactions by mediating nutrient exploitation and that a trade‐off may exist between nutrient exploitation and herbivory tolerance in these species.     

Ubiquitination of human AP-endonuclease 1 (APE1) enhanced by T233E substitution and by CDK5

Apurinic/apyrimidinic endonuclease-1 (APE1) is a multifunctional DNA repair/gene regulatory protein in mammalian cells, and was recently reported to be phosphorylated at Thr233 by CDK5. We here report that ubiquitination of T233E APE1, a mimicry of phospho-T233 APE1, was markedly increased in multiple cell lines. Expression of CDK5 enhanced monoubiquitination of endogenous APE1. Polyubiquitinated APE1 was decreased when K48R ubiquitin was expressed, suggesting that polyubiquitination was mediated mainly through Lys48 of ubiquitin. The ubiquitination activity of MDM2, consistent in its role for APE1 ubiquitination, was increased for T233E APE1 compared to the wild-type APE1. In mouse embryonic fibroblasts lacking the MDM2 gene, ubiquitination of T233E APE1 was still observed probably because of the decreased degradation activity for monoubiquitinated APE1 and because of backup E3 ligases in the cells. Monoubiquitinated APE1 was present in the nucleus, and analyzing global gene expression profiles with or without induction of a ubiquitin-APE1 fusion gene suggested that monoubiquitination enhanced the gene suppression activity of APE1. These data reveal a delicate balance of ubiquitination and phosphorylation activities that alter the gene regulatory function of APE1.