Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis

Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity—as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S₀) for each fraction; a unique correlation between S₀ and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how—and in which extent—the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Functional heterogeneity in a plant–frugivore assemblage enhances seed dispersal resilience to habitat loss

The ability of ecosystems to maintain their functions after disturbance (ecological resilience) depends on heterogeneity in the functional capabilities among species within assemblages. Functional heterogeneity may affect resilience by determining multiplicity between species in the provision of functions (redundancy) and complementarity between species in their ability to respond to disturbances (response diversity), but also by promoting the maintenance of biological information that enables ecosystems to reorganize themselves (ecological memory). Here, we assess the role of the components of the functional heterogeneity of a plant–frugivore assemblage on the resilience of seed dispersal to habitat loss. For three years, we quantified the distributions of fruits, frugivorous thrushes (Turdus spp.) and dispersed seeds, as well as frugivore diet and movement, along a gradient of forest cover in N Spain. The abundances and the spatial distributions of fruits and birds varied between years. The different thrushes showed similar diets but differed in spatial behavior and response to habitat loss, suggesting the occurrence of both functional redundancy and response diversity. Forest cover and fruit availability affected the spatial distribution of the whole frugivore assemblage. Fruit tracking was stronger in years when fruits were scarcer but more widespread across the whole fragmented landscape, entailing larger proportions of seeds dispersed to areas of low forest cover and open microhabitats. Rather than depending on redundancy and/or response diversity, seed dispersal resilience mostly emerged from the ecological memory conferred by the inter‐annual variability in fruit production and the ability of thrushes to track fruit resources across the fragmented landscape. Ecological memory also derived from the interaction of plants and frugivores as source organisms (trees in undisturbed forest), mobile links (birds able to disperse seeds into the disturbed habitat), and biological legacies (remnant trees and small forest patches offering scattered fruit resources across the landscape).     

Developmental Basis of Phallus Reduction during Bird Evolution

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Quantification of Anti‐Addictive Alkaloids Ibogaine and Voacangine in In Vivo‐ and In Vitro‐Grown Plants of Two Mexican Tabernaemontana Species

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti‐addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse‐grown plants, in vitro‐grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> extracellular adherence protein domain 4

The pathogenic bacterium Staphylococcus aureus has evolved to actively evade many aspects of the human innate immune system by expressing a series of secreted inhibitory proteins. Among these, the extracellular adherence protein (Eap) has been shown to inhibit the classical and lectin pathways of the complement system. By binding to complement component C4b, Eap is able to inhibit formation of the CP/LP C3 pro-convertase. Secreted full-length, mature Eap consists of four ~98 residue domains, all of which adopt a similar beta-grasp fold, and are connected through a short linker region. Through multiple biochemical approaches, it has been determined that the third and fourth domains of Eap are responsible for C4b binding. Here we report the backbone and side-chain resonance assignments of the 11.3 kDa fourth domain of Eap. The assignment data has been deposited in the BMRB database under the accession number 26726.     

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A₂, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.     

Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study

Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT) (EC 2.3.1.84) catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD) that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT) using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD) simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This mechanism is in agreement with previous results, obtained in our group, in alcohol acyltransferase from Vasconcellea pubescens (VpAAT1).     

Tracking Nutrient Routing in Avian Consumers in a Subtropical Desert

The nutrients animals ingest are allocated to serve different functions. We used contrasting C stable isotope signatures of dominant vegetation types in a North American subtropical desert to decipher how avian consumers allocate nutrients to fuel oxidative metabolism and to construct tissues. We conducted C stable isotope analysis of breath and feathers collected from nectarivores (hummingbirds) and of breath, plasma, and red blood cell samples collected from frugivores, granivores, and insectivores. Based on varying nutrient characteristics of food sources, we expected that for frugivores and granivores, CAM‐derived food (RC_CAM) would have similar importance for oxidative metabolism and for tissue building, that RC_CAM in nectarivores and insectivores would be more important for fueling metabolism than for generating tissues, and that (although low) RC_CAM in insectivores would be higher for sustaining metabolism than for building tissues. Our predictions held true for nectarivores and granivores, but RC_CAM use in tissue building was lower than expected in frugivores and higher than expected in insectivores. Our examination at the trophic guild, population, and individual levels showed that in general, nutrients used to sustain oxidative metabolism and tissue construction had a uniform isotopic origin. This finding suggests that the avian community under investigation does not route different food groups to fulfill different needs. However, we found some exceptions, indicating that birds can use different food sources for different functions, irrespective of trophic guild.