Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Sulfide oxidation coupled to arsenate reduction by a diverse microbial community in a soda lake

We characterized the arsenate-reducing, sulfide-oxidizing population of Mono Lake, California, by analyzing the distribution and diversity of rrnA, cbbL, and dissimilatory arsenate reductase (arrA) genes in environmental DNA, arsenate-plus sulfide-amended lake water, mixed cultures, and isolates. The arsenate-reducing community was diverse. An organism represented by an rrnA sequence previously retrieved from Mono Lake and affiliated with the Desulfobulbaceae (Deltaproteobacteria) appears to be an important member of the arsenate-reducing, sulfide-oxidizing community. Sulfide oxidation coupled with arsenate reduction appears to proceed via a two-electron transfer, resulting in the production of arsenite and an intermediate S compound that is subsequently disproportionated. A realgar-like As/S mineral was formed in some experiments.     

Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53

The white grub, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), is a major pest of turf and ornamental plants in Belgium. Previously, the combination of lethal concentration of the entomopathogenic nematodes Heterorhabditis megidis or Steinernema glaseri with the entomopathogenic fungus Metarhizium anisopliae (strain CLO 53) caused additive or synergistic mortality to third-instar H. philanthus in the laboratory and greenhouse. In this present study, we examined this interaction under field conditions and compared a combination of a commercial formulation of Heterorhabditis bacteriophora (Nema-green^®) and M. anisopliae. Controls were M. anisopliae, chlorpyrifos (Dursban 5 Granules) and H. bacteriophora. Field applications (surface or subsurface) were made against a mixed population of second/third-instar H. philanthus at a sport field and lawn infested in the province of West-Flanders. In both trials, the combination of M. anisopliae with H. bacteriophora at 5 × 10¹² conidia/ha +2.5 × 10⁹ infective juveniles/ha resulted in additive or synergistic effects, causing more than 95% grub mortality when the nematodes was applied 4 weeks after the application of fungus. However, application of nematode, chlorpyrifos or fungus alone provided 39–66%, 42–60% (surface) and 33–76%, 82–100% or 37–65%, (subsurface) control of H. philanthus. We concluded that the pathogen combinations we tested are compatible elements of integrated pest management and are likely to improve control of H. philanthus larvae and perhaps other insect pests beyond what is expected from single application of the pathogen.     

Involvement of p53 in gemcitabine mediated cytotoxicity and radiosensitivity in breast cancer cell lines

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) is one of the anti-metabolites drugs that target DNA replication. We evaluated dFdCyd cytotoxicity and its radiosensitizing ability in human breast cancer cell lines, MCF-7 (wild-type p53) and MDA-MB-231 (mutant-type p53) along with normal mammary epithelial cell line (MCF-12) for comparison. Radiosensitivity and cytotoxicity were measured by the clonogenic survival assays. DNA DSBs was studied by Pulse Field Gel Electrophoresis (PFGE) and cell cycle distribution was analyzed by flow cytometry. MDA-MB-231 cells were the most sensitive to the cytotoxicity of dFdCyd (IC(50) 5 nM) then MCF-7 (IC(50) 10nM), whereas MCF-12 cells were the most resistant to the cytotoxicity of dFdCyd (IC(50) 70 nM). MCF-12 and MCF-7 cell lines did not show any radiosensitization to dFdCyd, whereas the MDA-MB-231 cells showed significantly increased radioresistant to dFdCyd at equimolar concentration (p=0.002) and at IC(50) concentration (p<0.001). The DNA double strand breaks (DSBs) repair showed that dFdCyd neither increases DNA DSBs nor decreases the rate of their repair in MCF-12 and MCF-7 cell lines, while the same treatment in MDA-MB-231 cell line led to decrease the rate of DSBs or increase the rate of DNA repair (p=0.034). Therefore, dFdCyd is a cytotoxic agent, especially in the cancer cells irrespective of having wild-type or mutated p53 protein, but it is not effective as radiosensitizer in the cell lines used in this study. dFdCyd combined with radiation reduces the efficacy of chemo-radiotherapy in p53 mutated cells. Therefore, p53-mutated cancer could be a counter-indication for radiation-gemcitabine combined treatment.     

An experiment on comb orientation by honey bees (Hymenoptera: Apidae) in traditional hives

The orientation of combs in traditional beehives is extremely important for obtaining a marketable honey product. However, the factors that could determine comb orientation in traditional hives and the possibilities of inducing honey bees, Apis mellifera (L.), to construct more desirable combs have not been investigated. The goal of this experiment was to determine whether guide marks in traditional hives can induce bees to build combs of a desired orientation. Thirty-two traditional hives of uniform dimensions were used in the experiment. In 24 hives, ridges were formed on the inner surfaces of the hives with fermented mud to obtain different orientations, circular, horizontal, and spiral, with eight replicates of each treatment. In the remaining eight control hives, the inner surface was left smooth. Thirty-two well-established honey bee colonies from other traditional hives were transferred to the prepared hives. The colonies were randomly assigned to the four treatment groups. The manner of comb construction in the donor and experimental hives was recorded. The results showed that 22 (91.66%) of the 24 colonies in the treated groups built combs along the ridges provided, whereas only 2 (8.33%) did not. Comb orientation was strongly associated with the type of guide marks provided. Moreover, of the 18 colonies that randomly fell to patterns different from those of their previous nests, 17 (94.4%) followed the guide marks provided, irrespective of the comb orientation type in their previous nest. Thus, comb orientation appears to be governed by the inner surface pattern of the nest cavity. The results suggest that even in fixed-comb hives, honey bees can be guided to build combs with orientations suitable to honey harvesting, without affecting the colonies.     

A kinetic zipper model with intrachain interactions applied to nucleic acid hairpin folding kinetics

Single-stranded DNA and RNA hairpin structures with 4-10 nucleotides (nt) in the loop and 5-8 basepairs (bp) in the stem fold on 10-100 μs timescale. In contrast, theoretical estimate of first contact time of two ends of an ideal semiflexible polymer of similar lengths (with persistence length ~2-nt) is 10-100 ns. We propose that this three-orders-of-magnitude difference between these two timescales is a result of roughness in the folding free energy surface arising from intrachain interactions. We present a statistical mechanical model that explicitly includes all misfolded microstates with nonnative Watson-Crick (WC) and non-WC contacts. Rates of interconversion between different microstates are described in terms of two adjustable parameters: the strength of the non-WC interactions (ΔG(nWC)) and the rate at which a basepair is formed adjacent to an existing basepair (k(bp)(+)). The model accurately reproduces the temperature and loop-length dependence of the measured relaxation rates in temperature-jump studies of a 7-bp stem, single-stranded DNA hairpin with 4-20-nt-long poly(dT) loops, with ΔG(nWC) ≈ -2.4 kcal/mol and k(bp)(+) ≥ (1 ns)(-1), in 100 mM NaCl. Thus, our model provides a microscopic interpretation of the slow hairpin folding times as well as an estimate of the strength of intrachain interactions.     

Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.