Mechanisms of Convergent Egg Provisioning in Poison Frogs

Download : Download video (29MB)     

Optimization of N-benzyl-5-nitrofuran-2-carboxamide as an antitubercular agent

The optimization campaign for a nitrofuran antitubercular hit (N-benzyl-5-nitrofuran-2-carboxamide; JSF-3449) led to the design, synthesis, and biological profiling of a family of analogs. These compounds exhibited potent in vitro antitubercular activity (MIC?=?0.019–0.20?μM) against the Mycobacterium tuberculosis H37Rv strain and low in vitro cytotoxicity (CC₅₀?=?40–>120?μM) towards Vero cells. Significant improvements in mouse liver microsomal stability and mouse pharmacokinetic profile were realized by introduction of an α, α-dimethylbenzyl moiety. Among these compounds, JSF-4088 is highlighted due to its in vitro antitubercular potency (MIC?=?0.019?μM) and Vero cell cytotoxicity (CC₅₀?>?120?μM). The findings suggest a rationale for the continued evolution of this promising series of antitubercular small molecules.     

Asymmetric synthesis of d-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol

Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.     

Separating the anti-apoptotic and mitotic roles of survivin

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and has an essential role in mitosis. To date whether these two functions can be divorced has not been addressed. Here we show that the linker region between the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin and COOH-terminal alpha helix may be the key to separating its roles. When overexpressed survivin is present in interphase cells and shuttles between the cytoplasm and nucleus. Here we identify a rev-like nuclear exportation signal (NES) in the central domain of survivin and demonstrate that point mutations within this region cause accumulation of survivin in the nucleus. Interestingly cells expressing NES mutants exhibit reduced survival after X-irradiation. Moreover, cells expressing survivin(L98A)-green fluorescent protein (GFP) showed increased poly(ADP-ribose) polymerase-cleavage and caspase-3 activity after tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment compared with cells expressing full-length survivin-green fluorescent protein. These data suggest a direct link between the interphase localization of survivin and cellular responsiveness to apoptotic stimuli. Using a cell proliferation assay, we also found that ectopic expression of NES mutants can complement for depletion of endogenous survivin, indicating that they can execute the mitotic duties of survivin. Thus we demonstrate for the first time that 1) survivin has a functional NES; 2) nuclear accumulation of overexpressed survivin correlates with increased sensitivity of cells to ionising radiation; and 3) the anti-apoptotic and mitotic roles of survivin can be separated through mutation of its NES. Separating these two functions of survivin could open up new possibilities for therapeutic strategies aimed at eliminating cancer cells yet preserving normal cell viability.     

Arbuscular mycorrhizal fungi in the tree seedlings of two Australian rain forests: occurrence, colonization, and relationships with plant performance

The roots of rain forest plants are frequently colonized by arbuscular mycorrhizal fungi (AMF) that can promote plant growth in the nutrient poor soils characteristic of these forests. However, recent studies suggest that both the occurrence of AMF on rain forest plants and the dependence of rain forest plants on AMF can be highly variable. We examined the occurrence and levels of AMF colonization of some common seedling species in a tropical and a subtropical rain forest site in Queensland, Australia. We also used a long-term database to compare the growth and mortality rates of seedling species that rarely formed AMF with those that regularly formed AMF. In both forests, more than one-third of the seedling species rarely formed AMF associations, while 40% of species consistently formed AMF in the tropical site compared to 27% in the subtropical site. Consistent patterns of AMF occurrence were observed among plant families at the two sites. Variation among seedling species in AMF occurrence or colonization was not associated with differences in seed mass among species, variation in seedling size and putative age within a species, or lack of AMF inoculum in the soil. Comparisons of four seedling species growing both in the shaded understory and in small canopy gaps revealed an increase in AMF colonization in two of the four species in gaps, suggesting that light limitation partially explains the low occurrence of AMF. Seedling survival was significantly positively associated with seed biomass but not with AMF colonization. Furthermore, seedling species that regularly formed AMF and those that did not had similar rates of growth and survival, suggesting that mycorrhizal and nonmycorrhizal strategies were equivalent in these forests. Furthermore, the high numbers of seedlings that lacked AMF and the overall low rate of seedling growth (the average seedling required 6 years to double its height) suggest that most seedlings did not receive significant indirect benefits from AMF through connection to canopy trees via a common mycorrhizal network.     

A locus conferring resistance to Colletotrichum higginsianum is shared by four geographically distinct Arabidopsis accessions

Colletotrichum higginsianum is a hemibiotrophic fungal pathogen that causes anthracnose disease on Arabidopsis and other crucifer hosts. By exploiting natural variation in Arabidopsis we identified a resistance locus that is shared by four geographically distinct accessions (Ws‐0, Kondara, Gifu‐2 and Can‐0). A combination of quantitative trait loci (QTL) and Mendelian mapping positioned this locus within the major recognition gene complex MRC‐J on chromosome 5 containing the Toll‐interleukin‐1 receptor/nucleotide‐binding site/leucine‐rich repeat (TIR‐NB‐LRR) genes RPS4 and RRS1 that confer dual resistance to C. higginsianum in Ws‐0 ( Narusaka et al., 2009 ). We find that the resistance shared by these diverse Arabidopsis accessions is expressed at an early stage of fungal invasion, at the level of appressorial penetration and establishment of intracellular biotrophic hyphae, and that this determines disease progression. Resistance is not associated with host hypersensitive cell death, an oxidative burst or callose deposition in epidermal cells but requires the defense regulator EDS1, highlighting new functions of TIR‐NB‐LRR genes and EDS1 in limiting early establishment of fungal biotrophy. While the Arabidopsis accession Ler‐0 is fully susceptible to C. higginsianum infection, Col‐0 displays intermediate resistance that also maps to MRC‐J. By analysis of null mutants of RPS4 and RRS1 in Col‐0 we show that these genes, individually, do not contribute strongly to C. higginsianum resistance but are both required for resistance to Pseudomonas syringae bacteria expressing the Type III effector, AvrRps4. We conclude that distinct allelic forms of RPS4 and RRS1 probably cooperate to confer resistance to different pathogens.