Phylogenetic relationships among early-diverging eudicots based on four genes: were the eudicots ancestrally woody?

Based on analyses of combined data sets of three genes (18S rDNA, rbcL, and atpB), phylogenetic relationships among the early-diverging eudicot lineages (Ranunculales, Proteales, Trochodendraceae, Sabiaceae, and Buxaceae) remain unclear, as are relationships within Ranunculales, especially the placement of Eupteleaceae. To clarify relationships among these early-diverging eudicot lineages, we added entire sequences of 26S rDNA to the existing three-gene data set. In the combined analyses of four genes based on parsimony, ML, and Bayesian analysis, Ranunculales are strongly supported as a clade and are sister to other eudicots. Proteales appear as sister to the remaining eudicots, which are weakly (59%) supported as a clade. Relationships among Trochodendraceae, Buxaceae (including Didymeles), Sabiaceae, and Proteales remain unclear. Within Ranunculales, Eupteleaceae are sister to all other Ranunculales, with bootstrap support of 70% in parsimony analysis and with posterior probability of 1.00 in Bayesian analysis. Our character reconstructions indicate that the woody habit is ancestral, not only for the basal angiosperms, but also for the eudicots. Furthermore, Ranunculales may not be ancestrally herbaceous, as long maintained. The woody habit appears to have been ancestral for several major clades of eudicots, including Caryophyllales, and asterids.     

Multiple fruiting of the edible mushroomCoprinus cinereus from explants of the stipe

Summary Carpophores were developed on defined medium from explants of stipes produced in the dark using an expansionless mutant (no. 190) ofCoprinus cinereus. Explants taken from stipes within 24 h of formation developed multiple fruit within 3–12 days without formation of mycelia. Fruiting levels were affected by stipe age (24 h), medium composition, explant size, and polarity of the explant on fruiting medium. This method offers a new tool for developmental studies and may also be of use to commercial mushroom growers.     

5,7-Diphenyl-3-ureidohexahydroazepin-2-ones as Cholecystokinin-B receptor ligands

A series of 5,7-diphenyl-3-ureidohexahydroazepin-2-one cholecystokinin-B (CCK-B) receptor antagonists was synthesized using Beckmann ring expansion of a suitable 2,4-diphenylcyclohexanone as a key step. SAR studies revealed the importance of the 5-aryl group for high and selective CCK-B receptor affinity, as illustrated in compound (−)-10i (CCK-B IC₅₀ = 6.8 nM).     

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than na?ve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among na?ve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.     

Uncoupling ligand-dependent and -independent mechanisms for mitogen-activated protein kinase activation by the murine Ron receptor tyrosine kinase

Receptor tyrosine kinases (RTKs) activate downstream signaling through cognate growth factor receptor-induced dimerization and autophosphorylation. Overexpression of RTKs can lead to constitutive activation due to increased dimerization in the absence of ligand, and downstream signals are presumed to be the same as the ligand-induced signals. We have shown that the murine Ron (mRon) receptor tyrosine kinase exhibits constitutive activation of the MAP kinase pathway that is independent of the two docking site tyrosines, whereas activation of this pathway in response to ligand (macrophage-stimulating protein) is abolished in the absence of these tyrosines. Furthermore, we identified three tyrosines (Tyr-1175, Tyr-1265, and Tyr-1294) within the kinase domain that play critical but overlapping roles in controlling constitutive Erk activation by mRon. Phenylalanine mutations at these three tyrosines results in a receptor that fails to constitutively activate the Erk pathway but retains the ability to induce Erk phosphorylation in response to ligand stimulation. The ability of mRon to activate the MAP kinase pathway is dependent on c-Src activity, and we have shown that c-Src co-immunoprecipitates with mRon. c-Src fails to interact with mRon when the three tyrosines required for MAP kinase activation are mutated, whereas the presence of any one of these tyrosines alone restores Erk phosphorylation and recruitment of c-Src. Thus, the ligand-dependent and -independent activity of mRon can be uncoupled through the alteration of selective sets of tyrosines.     

Hypoxanthine-guanine phosphoribosyltransferase: Mosaicism in the peripheral erythrocytes of a heterozygote for a normal and a mutant enzyme

A mutant form of erythrocyte hypoxanthine-guanine phosphoribosyltransferase with an abnormal isoenzyme pattern was found in a patient with a partial enzyme deficiency and X-linked gout. This abnormal pattern was a marker for the mutant enzyme in hemolysate from the heterozygote for the enzyme deficiency.These studies were generously supported by the Medical Research Council of Canada (MRC # MA4758) and the Canadian Arthritis and Rheumatism Society.     

"Super p53" mice exhibit enhanced DNA damage response,are tumor resistant and age normally

The tumor suppressor p53 is critical in preventing cancer due to its ability to trigger proliferation arrest and cell death upon the occurrence of a variety of stresses, most notably, DNA damage and oncogenic stress. Here, we report the generation and characterization of mice carrying supernumerary copies of the p53 gene in the form of large genomic transgenes. Prior to this, we demonstrate that the p53 transgenic allele (p53-tg), when present in a p53-null genetic background, behaves as a functional replica of the endogenous gene. "Super p53" mice, carrying p53-tg alleles in addition to the two endogenous alleles, exhibit an enhanced response to DNA damage. Importantly, "super p53" mice are significantly protected from cancer when compared with normal mice. Finally, in contrast to previously reported mice with constitutively active p53, "super p53" mice do not show any indication of premature aging, probably reflecting the fact that p53 is under normal regulatory control. Together, our results prove that cancer resistance can be enhanced by a simple genetic modification and in the absence of undesirable effects.     

Variations in soil N cycling and trace gas emissions in wet tropical forests

We used a previously described precipitation gradient in a tropical montane ecosystem of Hawai’i to evaluate how changes in mean annual precipitation (MAP) affect the processes resulting in the loss of N via trace gases. We evaluated three Hawaiian forests ranging from 2200 to 4050 mm year⁻¹ MAP with constant temperature, parent material, ecosystem age, and vegetation. In situ fluxes of N₂O and NO, soil inorganic nitrogen pools (NH₄⁺ and NO₃⁻), net nitrification, and net mineralization were quantified four times over 2 years. In addition, we performed ¹⁵N-labeling experiments to partition sources of N₂O between nitrification and denitrification, along with assays of nitrification potential and denitrification enzyme activity (DEA). Mean NO and N₂O emissions were highest at the mesic end of the gradient (8.7±4.6 and 1.1±0.3 ng N cm⁻² h⁻¹, respectively) and total oxidized N emitted decreased with increased MAP. At the wettest site, mean trace gas fluxes were at or below detection limit (≤0.2 ng N cm⁻² h⁻¹). Isotopic labeling showed that with increasing MAP, the source of N₂O changed from predominately nitrification to predominately denitrification. There was an increase in extractible NH₄⁺ and decline in NO₃⁻, while mean net mineralization and nitrification did not change from the mesic to intermediate sites but decreased dramatically at the wettest site. Nitrification potential and DEA were highest at the mesic site and lowest at the wet site. MAP exerts strong control N cycling processes and the magnitude and source of N trace gas flux from soil through soil redox conditions and the supply of electron donors and acceptors.