The moss genes <Emphasis Type="Italic">PpSKI1</Emphasis> and <Emphasis Type="Italic">PpSKI2</Emphasis> encode nuclear SnRK1 interacting proteins with homologues in vascular plants

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant’s ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.     

Synthesis physicochemical profile and PAMPA study of new NO-donor edaravone co-drugs

A new class of co-drugs were synthesised by joining antioxidant edaravone with a vasodilating substructure containing NO-donor nitrooxy functions, and characterised for their stability in different media, lipophilicity and permeability profile. The products display good stability in water/co-solvent at different pH. Conversely, they are rapidly metabolised into edaravone and NO-donor moieties when incubated in human serum or rat-liver homogenates. In the latter conditions time dependent production of nitrite/nitrate (NO(x)) occurs. The compounds display wide-ranging lipophilicity. PAMPA studies predict good gastrointestinal absorption for a number of these compounds. The title products are potentially useful for treating ROS-related conditions accompanied by decreased NO availability.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.     

A native nitrogen-fixing shrub facilitates weed invasion

Invasions by exotic weedy plants frequently occur in highly disturbed or otherwise anthropogenically altered habitats. Here we present evidence that, within California coastal prairie, invasion also can be facilitated by a native nitrogen-fixing shrub, bush lupine (Lupinus arboreus). Bush lupines fix nitrogen and grow rapidly, fertilizing the sandy soil with nitrogen-rich litter. The dense lupine canopy blocks light, restricting vegetative growth under bushes. Heavy insect herbivory kills lupines, opening exposed nitrogen-rich sites within the plant community. Eventual re-establishment of lupine occurs because of an abundant and long-lived seed bank. Lupine germination, rapid growth, shading and fertilization of sites, and then death after only a few years, results in a mosaic of nutrient-rich sites that are available to invading species. To determine the role of bush lupine death and nitrogen enrichment in community composition, we examined nutrient dynamics and plant community characteristics within a site only recently colonized by lupine, comparing patches where lupines had recently died or were experimentally killed with adjacent areas lacking lupine. In experimentally killed patches, instantaneous pool sizes of exchangeable ammonium and nitrate nitrogen were higher than in adjacent sites free of lupine. Seedlings of the introduced grass Bromus diandrus accumulated 48% greater root biomass and 93% more shoot biomass when grown in a greenhouse in soil collected under experimentally killed lupines compared to B. diandrus seedlings grown in soil collected at least 1 m away from lupines. At the end of the spring growing season, total above-ground live plant biomass was more than twice as great in dead lupine patches as in the adjacent lupine-free grassland, but dead lupine patches contained 47% fewer plant species and 57% fewer native species. Sites where lupines have repeatedly died and reestablished during recent decades support an interstitial grassland community high in productivity but low in diversity, composed of mostly weedy introduced annual plants. In contrast, at a site only recently colonized by bush lupines, the interstitial grassland consists of a less productive but more diverse set of native and introduced species. We suggest that repeated bouts of lupine germination, establishment, and death can convert a rich native plant community into a less diverse collection of introduced weeds.     

Question 9: Prospects for the Construction of Artificial Cells or Protocells

The construction of artificial cells or protocells that are a simplified version of contemporary cells will have implications for both the understanding of the origins of cellular Life and the design of “cell-like” chemical factories. In this short communication, we discuss the progress and remaining issues related to the construction of protocells from metabolic products. We further outline the de novo design of a simple chemical system that mimics the functional properties of a living cell without being composed of molecules of biological origin, thereby addressing issues related to Life’s origins. Presented at: International School of Complexity—4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.