Trypanosoma rangeli: An alkaline ecto-phosphatase activity is involved with survival and growth of the parasite

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca²⁺ present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (P_i). Levamisole at the IC₅₀ spared significantly parasite growth when β-GP was the sole source of P_i and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.     

Statistical Analysis of Peptide-Induced Graded and All-or-None Fluxes in?Giant Vesicles

Antimicrobial, cytolytic, and cell-penetrating peptides induce pores or perturbations in phospholipid membranes that result in fluxes of dyes into or out of lipid vesicles. Here we examine the fluxes induced by four of these membrane-active peptides in giant unilamellar vesicles. The type of flux is determined from the modality of the distributions of vesicles as a function of their dye content using the statistical Hartigan dip test. Graded and all-or-none fluxes correspond to unimodal and bimodal distributions, respectively. To understand how these distributions arise, we perform Monte Carlo simulations of peptide-induced dye flux into vesicles using a very simple model. The modality of the distributions depends on the rate constants of pore opening and closing, and dye flux. If the rate constants of pore opening and closing are both much smaller than that of dye flux through the pore, all-or-none influx occurs. However, if one of them, especially the rate constant for pore opening, increases significantly relative to the flux rate constant, the process becomes graded. In the experiments, we find that the flux type is the same in giant and large vesicles, for all peptides except one. But this one exception indicates that the flux type cannot be used to unambiguously predict the mechanism of membrane permeabilization by the peptides.     

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.     

Stoichiometric modeling of oxidation of reduced inorganic sulfur compounds (Riscs) in Acidithiobacillus thiooxidans

The prokaryotic oxidation of reduced inorganic sulfur compounds (RISCs) is a topic of utmost importance from a biogeochemical and industrial perspective. Despite sulfur oxidizing bacterial activity is largely known, no quantitative approaches to biological RISCs oxidation have been made, gathering all the complex abiotic and enzymatic stoichiometry involved. Even though in the case of neutrophilic bacteria such as Paracoccus and Beggiatoa species the RISCs oxidation systems are well described, there is a lack of knowledge for acidophilic microorganisms. Here, we present the first experimentally validated stoichiometric model able to assess RISCs oxidation quantitatively in Acidithiobacillus thiooxidans (strain DSM 17318), the archetype of the sulfur oxidizing acidophilic chemolithoautotrophs. This model was built based on literature and genomic analysis, considering a widespread mix of formerly proposed RISCs oxidation models combined and evaluated experimentally. Thiosulfate partial oxidation by the Sox system (SoxABXYZ) was placed as central step of sulfur oxidation model, along with abiotic reactions. This model was coupled with a detailed stoichiometry of biomass production, providing accurate bacterial growth predictions. In silico deletion/inactivation highlights the role of sulfur dioxygenase as the main catalyzer and a moderate function of tetrathionate hydrolase in elemental sulfur catabolism, demonstrating that this model constitutes an advanced instrument for the optimization of At. thiooxidans biomass production with potential use in biohydrometallurgical and environmental applications. Biotechnol. Bioeng. 2013; 110: 2242–2251. © 2013 Wiley Periodicals, Inc.     

Population Composition and Ectoparasite Prevalence on Bats (Sturnira ludovici; Phyllostomidae) in Forest Fragments and Coffee Plantations of Central Veracruz,Mexico

Studies comparing the abundance of frugivorous bats in shade‐coffee plantations and forest fragments report contradictory results, and have not taken into account the landscape context in which coffee plantations are immersed. Variables of population composition such as abundance, sex proportion, and reproductive condition, together with biological tags (i.e., bat fly prevalence), can provide information about spatiotemporal dynamics of habitats used by bats. In the central part of Veracruz, Mexico, we compared population variables and ectoparasite prevalence of the highland yellow‐shouldered bat (Sturnira ludovici) in two landscapes, one dominated by shade‐coffee plantations and another by forest fragments. Comparing these attributes between these two landscapes will increase our knowledge about the role of this agro‐ecosystem in the conservation of this species, which is an important seed disperser of cloud forest vegetation. Total abundance and proportion of females was greater in forest fragments than in coffee plantations, whereas the percentage of reproductive females and bat fly prevalence was similar between landscapes. Our results show that landscapes with forest fragments harbor the greatest abundance of S. ludovici, but shade‐coffee plantations also are utilized by S. ludovici and likely adjacent forest remnants provide enough food resources for this species and other frugivores. Moreover, this study provides more evidence documenting the importance of preserving the last cloud forest fragments in the central region of Veracruz, Mexico, and suggests that using shade‐coffee plantations to connect forest fragments may be an effective way of maintaining populations of S. ludovici and likely other volant frugivores.     

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1 mg CLC/120 × 10⁶ sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.     

Bacterial community composition of anthropogenic biochar and Amazonian anthrosols assessed by 16S rRNA gene 454 pyrosequencing

Biochar (BC) is a common minor constituent of soils and is usually derived from the burning of wood materials. In the case of Amazonian dark earth (ADE) soils, the increased amount of this material is believed to be due to anthropogenic action by ancient indigenous populations. In this study, we use 16S rRNA gene pyrosequencing to assess the bacterial diversity observed in the BC found in ADEs as well as in the dark earth itself and the adjacent Acrisol. Samples were taken from two sites, one cultivated with manioc and one with secondary forest cover. Analyses revealed that the community structure found in each sample had unique features. At a coarse phylogenetic resolution, the most abundant phyla in all sequence libraries were Actinobacteria, Acidobacteria, Verrucomicrobia and Proteobacteria that were present in similar relative abundance across all samples. However, the class composition varied between them highlighting the difference between the Acrisol and the remaining samples. This result was also corroborated by the comparison of the OTU composition (at 97 % identity). Also, soil coverage has shown an effect over the community structure observed in all samples. This pattern was found to be significant through unweighted UniFrac as well as P tests. These results indicate that, although the ADEs are found in patches within the Acrisols, the contrasting characteristics found between them led to the development of significantly different communities.     

Differential sensitivity to detergents of actin cytoskeleton from nerve endings

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.