Identification of serine proteases from Leishmania braziliensis

Leishmania (V) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nalpha-benzoyl-L-arginine ethyl ester hydrochloride and Nalpha-p-tosyl-L-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-L-phenyl-alanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.     

Long-Term Consequences of Grazing and Burning on Northern Peatland Carbon Dynamics

Abstract Using a 50-year-old field experiment, we investigated the effects of the long-term land management practices of repeated burning and grazing on peatland vegetation and carbon dynamics (C). Plant community composition, C stocks in soils and vegetation, and C fluxes of CO₂, CH₄ and DOC, were measured over an 18-month period. We found that both burning and grazing reduced aboveground C stocks, and that burning reduced C stocks in the surface peat. Both burning and grazing strongly affected vegetation community composition, causing an increase in graminoids and a decrease in ericoid subshrubs and bryophytes relative to unburned and ungrazed controls; this effect was especially pronounced in burned treatments. Soil microbial properties were unaffected by grazing and showed minor responses to burning, in that the C:N ratio of the microbial biomass increased in burned relative to unburned treatments. Increases in the gross ecosystem CO₂ fluxes of respiration and photosynthesis were observed in burned and grazed treatments relative to controls. Here, the greatest effects were seen in the burning treatment, where the mean increase in gross fluxes over the experimental period was greater than 40%. Increases in gross CO₂ fluxes were greatest during the summer months, suggesting an interactive effect of land use and climate on ecosystem C cycling. Collectively, our results indicate that long-term management of peatland has marked effects on ecosystem C dynamics and CO₂ flux, which are primarily related to changes in vegetation community structure.     

VSGdb: a database for trypanosome variant surface glycoproteins,a large and diverse family of coiled coil proteins

Background  

Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long α-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only ~20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for α helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome-specific questions, we have created VSGdb, a database of all known sequences.     

In vivo and in vitro activity and mechanism of action of the multidrug cytarabine-L-glycerylyl-fluorodeoxyuridine

Multidrugs have the potential to bypass resistance. We investigated the in vitro activity and resistance circumvention of the multidrug cytarabine-L-fluorodeoxyuridine (AraC-L-5FdU), linked via a glycerophospholipid linkage. Cytotoxicity was determined using sensitive (A2780, FM3A/0) and resistant (AG6000, AraC resistant, deoxycytidine kinase deficient; FM3A/TK-, 5FdU resistant, thymidine kinase deficient) cell lines. Circumvention of nucleoside transporter and activating enzymes was determined using specific inhibitors, HPLC analysis and standard radioactivity assays. AraC-L-5FdU was active (IC50: 0.03 microM in both A2780 and FM3A/0), had some activity in AG6000 (IC50: 0.28 microM), but no activity in FM3A/TK(-) (IC50: 18.3 microM). AraC-nucleotides were not detected in AG6000. 5FdU-nucleotides were detected in all cell lines. AraC-L-5FdU did not inhibit TS in FM3A/TK(-) (5%). Since phosphatase/nucleotidase-inhibition reduced cytotoxicity 7-70-fold, cleavage seems to be outside the cell, presumably to nucleotides, and then to nucleosides. The multidrug was orally active in the HT-29 colon carcinoma xenografts which are resistant toward the single drugs.     

Safeguarding marine life: conservation of biodiversity and ecosystems

Reviews in Fish Biology and Fisheries - Marine ecosystems and their associated biodiversity sustain life on Earth and hold intrinsic value. Critical marine ecosystem services include maintenance of...     

Auxin Resistant1 and PIN-FORMED2 Protect Lateral Root Formation in Arabidopsis under Iron Stress

A stunted root system is a significant symptom of iron (Fe) toxicity, yet little is known about the effects of excess Fe on lateral root (LR) development. In this work, we show that excess Fe has different effects on LR development in different portions of the Arabidopsis (Arabidopsis thaliana) root system and that inhibitory effects on the LR initiation are only seen in roots newly formed during excess Fe exposure. We show that root tip contact with Fe is both necessary and sufficient for LR inhibition and that the auxin, but not abscisic acid, pathway is engaged centrally in the initial stages of excess Fe exposure. Furthermore, Fe stress significantly reduced PIN-FORMED2 (PIN2)-green fluorescent protein (GFP) expression in root tips, and pin2-1 mutants exhibited significantly fewer LR initiation events under excess Fe than the wild type. Exogenous application of both Fe and glutathione together increased PIN2-GFP expression and the number of LR initiation events compared with Fe treatment alone. The ethylene inhibitor aminoethoxyvinyl-glycine intensified Fe-dependent inhibition of LR formation in the wild type, and this inhibition was significantly reduced in the ethylene overproduction mutant ethylene overproducer1-1. We show that Auxin Resistant1 (AUX1) is a critical component in the mediation of endogenous ethylene effects on LR formation under excess Fe stress. Our findings demonstrate the relationship between excess Fe-dependent PIN2 expression and LR formation and the potential role of AUX1 in ethylene-mediated LR tolerance and suggest that AUX1 and PIN2 protect LR formation in Arabidopsis during the early stages of Fe stress.Iron (Fe) is an essential trace element for plants (Pilon et al., 2009), and species differ greatly in how much Fe they require for optimal growth (Wheeler and Power, 1995; Batty and Younger, 2003). As Fe is frequently limiting, Fe deficiency is more commonly studied than toxicity arising from excess Fe exposure (Lei et al., 2014; Bashir et al., 2015; Briat et al., 2015). Fe is also a major focus for efforts in biofortification by targeting Fe transporters (Zhai et al., 2014; Pinto and Ferreira, 2015). However, the excessive presence of Fe in soils is equally common, in particular in soils characterized by low pH and hypoxic or anoxic conditions (Connolly and Guerinot, 2002). Toxicity arising from excess Fe exposure is recognized as one of the major plant diseases attributable to abiotic factors that impact the development and yield potential in the world’s leading cereal crops, rice (Oryza sativa) and wheat (Triticum aestivum; Becker and Asch, 2005; Khabaz-Saberi et al., 2012). Understanding the mechanisms underlying excess Fe toxicity is therefore essential.Plastic responses in the plant’s root system architecture are known to constitute a major mechanism by which plants cope with fluctuating environments. Lateral roots (LRs), which typically comprise the majority of the root system, contribute pivotally to nutrient acquisition from soil, and modulating LR development is a very important avoidance strategy for plants when confronted with unfavorable edaphic conditions, such as high salinity or heavy metals (Ivanov et al., 2003). In the case of excess exposure to Fe, stunting of the root system is among the chief symptoms of toxicity (Becker and Asch, 2005). However, while some information has been emerging on the primary root axis (Li et al., 2015), the specific role of the plant’s LR apparatus remains poorly studied. Yamauchi and Peng (1995) reported retardation of root growth and a reduction in LR length and number under excess Fe conditions. Recently, Reyt et al. (2015) showed that excess Fe had no significant effect on LR initiation in the LR branching zone and that ferritins play an important role in LR emergence under excess Fe in this portion of the root, although the authors had not investigated LR development in the root portions near the growing tip of the primary root. Because LR initiation is restricted to specific pericycle cell files adjacent to a xylem pole in the basal region of the meristem (De Smet et al., 2007; Fukaki and Tasaka, 2009), and LR formation in this new growing root portion may be more susceptible to stress stimuli, such as observed with exposure to high NH₄⁺ and salt (Duan et al., 2013; Li et al., 2013), it is reasonable to suggest that modulation of LR formation near the growing tip of the primary root is critical to the response to excess Fe stress.In Arabidopsis (Arabidopsis thaliana), the development of LRs proceeds through the following stages: lateral root primordia (LRP) initiation, establishment, emergence, activation into mature LRs, and final maintenance of LR elongation (Fukaki and Tasaka, 2009; Péret et al., 2009). The hormones abscisic acid (ABA) and auxin are important internal negative and positive regulators during LR development, respectively (Fukaki and Tasaka, 2009). ABA has been implicated in LRP emergence and meristem activation independent of auxin (De Smet et al., 2003). Auxin is an important internal positive regulator during LR development (Fukaki and Tasaka, 2009), and auxin transport is critical (Blilou et al., 2005). Mutants in auxin efflux carriers such as PIN-FORMED (PIN) and P-Glycoprotein show significant defects in LR formation (Fukaki and Tasaka, 2009; Péret et al., 2009). For example, LR initiation frequency was significantly reduced in pin2 and pin3 mutants (Dubrovsky et al., 2009), and PIN2 was also shown to be involved in exogenous and endogenous signal-mediated LR development (by brassinosteroid, jasmonate, and fungal challenge; Li et al., 2005; Felten et al., 2009; Sun et al., 2009). Similarly, Auxin Resistant1 (AUX1), an auxin influx carrier, also regulates LRP positioning and initiation (De Smet et al., 2007). While both AUX1 and PIN2 are required specifically for the basipetal transport of auxin through the outer root cell layers (Fukaki and Tasaka, 2009), PIN1 localized at the basal end of vascular cells is responsible for direct acropetal auxin flow in the root stele (Blilou et al., 2005). Recently, the roles of ethylene on LR development have also been highlighted, and the ethylene-mediated LR formation is dependent on the auxin pathway (Ivanchenko et al., 2008; Lewis et al., 2011). Ethylene treatment could mediate fluorescence of AUX1 and PIN2 fluorescent protein fusions at the root tip (Růzicka et al., 2007; Lewis et al., 2011). Although ABA, auxin, and ethylene signals have been implicated as important for LR development, it is not known whether and how the three hormones are involved in the response of LR formation to Fe stress.The previously described phenotypes and physiological processes related to Fe toxicity do not clarify the effect of excess Fe on LR formation. In this study, we employed the Arabidopsis wild type and ABA-, auxin-, and ethylene-related mutants to explore the LR formation response to Fe toxicity and to elucidate the roles of ABA, auxin, and ethylene. Potential mechanisms involved in the early stress response to Fe stress are discussed.     

Predator‐prey migration phenologies remain synchronised in a warming catchment

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Turbidity alters pre‐mating social interactions between native and invasive stream fishes

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