Chaperone-assisted expression,purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.     

Energy use by the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) for dispersal by flight

The mountain pine beetle Dendroctonus ponderosae Hopkins is a major native pest of Pinus Linnaeus (Pinaceae) in western North America. Host colonization by the mountain pine beetle is associated with an obligatory dispersal phase, during which beetles fly in search of a suitable host. Mountain pine beetles use stored energy from feeding in the natal habitat to power flight before host colonization and brood production. Lipids fuel mountain pine beetle flight, although it is not known whether other energy sources are also used during flight. In the present study, we compare the level of energy substrates, proteins, carbohydrates and lipids of individual mountain pine beetles flown on flight mills with unflown control beetles. We use a colorimetric method to measure the entire metabolite content of each individual beetle. The present study reveals that mountain pine beetles are composed of more protein and lipid than carbohydrate. Both female and male mountain pine beetles use lipids and carbohydrates as energy sources during flight. There is variation between sexes, however, in the energy substrates used for flight. Male mountain pine beetles use protein, in addition to lipids and carbohydrates, to fuel flight, whereas protein content is not different between flown and control females.     

Climate change alters plant biogeography in Mediterranean prairies along the West Coast,USA

Projected changes in climate are expected to have widespread effects on plant community composition and diversity in coming decades. However, multisite, multifactor climate manipulation studies that have examined whether observed responses are regionally consistent and whether multiple climate perturbations are interdependent are rare. Using such an experiment, we quantified how warming and increased precipitation intensity affect the relative dominance of plant functional groups and diversity across a broad climate gradient of Mediterranean prairies. We implemented a fully factorial climate manipulation of warming (+2.5–3.0 °C) and increased wet‐season precipitation (+20%) at three sites across a 520‐km latitudinal gradient in the Pacific Northwest, USA. After seeding with a nearly identical mix of native species at all sites, we measured plant community composition (i.e., cover, richness, and diversity), temperature, and soil moisture for 3 years. Warming and the resultant drying of soils altered plant community composition, decreased native diversity, and increased total cover, with warmed northern communities becoming more similar to communities further south. In particular, after two full years of warming, annual cover increased and forb cover decreased at all sites mirroring the natural biogeographic pattern. This suggests that the extant climate gradient of increasing heat and drought severity is responsible for a large part of the observed biogeographic pattern of increasing annual invasion in US West Coast prairies as one moves further south. Additional precipitation during the rainy season did little to relieve drought stress and had minimal effects on plant community composition. Our results suggest that the projected increase in drought severity (i.e., hotter, drier summers) in Pacific Northwest prairies may lead to increased invasion by annuals and a loss of forbs, similar to what has been observed in central and southern California, resulting in novel species assemblages and shifts in functional composition, which in turn may alter ecosystem functions.     

Variation in global codon usage bias among prokaryotic organisms is associated with their lifestyles

Background  

It is widely acknowledged that synonymous codons are used unevenly among genes in a genome. In organisms under translational selection, genes encoding highly expressed proteins are enriched with specific codons. This phenomenon, termed codon usage bias, is common to many organisms and has been recognized as influencing cellular fitness. This suggests that the global extent of codon usage bias of an organism might be associated with its phenotypic traits.     

Water-Soluble Ruthenium Complexes Bearing Activity Against Protozoan Parasites

Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl₂(HL)(HPTA)₂]Cl₂ with HL = bioactive 5-nitrofuryl containing thiosemicarbazones and PTA?=?1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl₂(HL4)(HPTA)₂]Cl₂ complex, with HL4?=?N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC₅₀)?=?5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI) >38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.     

Hematology and serum biochemistry in debilitated, free-ranging raccoon dogs (Nyctereutes procyonoides) infested with sarcoptic mange

Frequent outbreaks of Sarcoptes scabiei infestation in raccoon dogs (Nyctereutes procyonoides) have been reported in Japan. Although many raccoon dogs are brought to Kanazawa Zoological Garden (Yokohama, Kanagawa, Japan) because of S. scabiei infestation and debilitation, some of them die of asthenia. The clinical status of severely debilitated raccoon dogs must be determined to save their lives. In this study, we compared hematological and serum biochemical values between severely debilitated and nondebilitated raccoon dogs infested with S. scabiei. The total protein, albumin, glucose, and calcium values of debilitated raccoon dogs were significantly lower than those of nondebilitated raccoon dogs. On the other hand, debilitated raccoon dogs had significantly higher aspartate aminotransferase, total bilirubin, blood urea nitrogen, sodium, chloride, and phosphorus values than did nondebilitated raccoon dogs. The increase in the blood urea nitrogen value was particularly dramatic. The present study revealed that debilitated raccoon dogs infested with S. scabiei exhibited abnormal hematological values compared with nondebilitated raccoon dogs infested with S. scabiei. Clinically, the raccoon dogs developed malnutrition and sepsis if the mange infestation was untreated. Moreover, dehydration associated with appetite loss may have resulted in insufficient renal perfusion. These findings suggest that chronic S. scabiei infestations debilitated the raccoon dogs and resulted in physiological changes that were detected with hematological and serum biochemical tests.     

Structure of the Drosophila apoptosome at 6.9 å resolution

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ～6.9?? resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.     

Aurones serve as probes of beta-amyloid plaques in Alzheimer's disease

A novel series of aurone derivatives for in vivo imaging of beta-amyloid plaques in the brain of Alzheimer's disease (AD) were synthesized and characterized. When in vitro binding studies using Abeta(1-42) aggregates were carried out with aurone derivatives, they showed high binding affinities for Abeta(1-42) aggregates at the K(i) values ranging from 1.2 to 6.8 nM. When in vitro plaque labeling was carried out using double transgenic mice brain sections, the aurone derivatives intensely stained beta-amyiloid plaques. Biodistribution studies in normal mice after i.v. injection of the radioiodinated aurones displayed high brain uptake (1.9-4.6% ID/g at 2 min) and rapid clearance from the brain (0.11-0.26% ID/g at 60 min), which is highly desirable for amyloid imaging agents. The results in this study suggest that novel radiolabeled aurones may be useful amyloid imaging agents for detecting beta-amyloid plaques in the brain of AD.     

Small‐plot studies comparing pheromone and juice baits for mass‐trapping invasive Synanthedon myopaeformis in Canada

Recent introduction of Synanthedon myopaeformis (Borkhausen) (Lepidoptera: Sesiidae) into organic apple‐growing areas of Canada has stimulated research on semiochemical‐based management of this European pest. Replicated, small‐plot (0.16 ha) experiments were conducted to compare sex pheromone, 3Z,13Z‐octadecadienyl acetate (10 mg), Concord grape juice (300 ml), or their combination, as mass‐trapping lures at trap densities equivalent to 12.5, 25, 50, and 100 traps ha^?1. Total numbers of male and female moths removed from test plots increased significantly with trap density in all juice‐based mass‐trapping experiments. In pheromone mass‐trapping experiments, however, total catches of males did not increase significantly as trap densities were increased and catches appeared to plateau with 25–50 traps ha^?1. With pheromone‐based mass‐trapping, significantly fewer males were caught in pheromone‐baited assessment traps at the centre of each mass‐trapping plot than in identical traps in untreated plots. This reduction is indicative of significant trap interference or trap ‘shut‐down’. Increasing the density of juice‐based mass‐trapping had no effect on catches of male or female moths in juice‐baited assessment traps, indicating a short range of attraction and lack of interference between juice traps. Pheromone‐ and juice‐based mass trapping removed similar numbers of males at each trap density tested, respectively, but summed catches of males and females were greatest with juice baits. Combining pheromone and juice into a single mass‐trapping treatment (50 traps ha^?1) did not significantly increase catches of males or females relative to either treatment alone. If a practical bisexual mass‐trapping system is going to be developed for S. myopaeformis, then identification of volatile kairomones in Concord grape juice may be useful.