Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H(2)O(2)) in the airways through its ability to oxidize thiocyanate (SCN(-)) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 μM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O(2), Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN(-). On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.     

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin-A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat-storing lipocyte phenotype. Circulating retinol is associated with plasma retinol-binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [(3)H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high-affinity membrane receptor for RBP-retinol complex (KD = 4.93 nM), and both cell types expressed a low-affinity one (KD = 234 nM). The RBP-retinol complex, but not the BSA-delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein-protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane-bound retinol. RBP-delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin-delivered retinol was mainly associated with membranes. Disturbing the clathrin-mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo-RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.     

��-Amyloid Causes Depletion of Synaptic Vesicles Leading to Neurotransmission Failure

Alzheimer disease is a progressive neurodegenerative brain disorder that leads to major debilitating cognitive deficits. It is believed that the alterations capable of causing brain circuitry dysfunctions have a slow onset and that the full blown disease may take several years to develop. Therefore, it is important to understand the early, asymptomatic, and possible reversible states of the disease with the aim of proposing preventive and disease-modifying therapeutic strategies. It is largely unknown how amyloid β-peptide (Aβ), a principal agent in Alzheimer disease, affects synapses in brain neurons. In this study, we found that similar to other pore-forming neurotoxins, Aβ induced a rapid increase in intracellular calcium and miniature currents, indicating an enhancement in vesicular transmitter release. Significantly, blockade of these effects by low extracellular calcium and a peptide known to act as an inhibitor of the Aβ-induced pore prevented the delayed failure, indicating that Aβ blocks neurotransmission by causing vesicular depletion. This new mechanism for Aβ synaptic toxicity should provide an alternative pathway to search for small molecules that can antagonize these effects of Aβ.     

Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.     

Hypolipidemic and antiatherosclerotic potential of Pleurotus ostreatus, cultived by submerged fermentation in the high-fat diet fed rats

The ability of Pleurotus ostreatus biomass, cultived by submerged fermentation, to produce beneficial effect on lipid profile and macrophages activity during a high-fat diet (HFD) for a long-term intake was investigated. Blood samples were collected through cardiac puncture to measure the plasma cholesterol, triglycerides, low-density protein (LDL), high-density protein (HDL), aspartate aminotransferase (AST) activity, urea-blood urea nitrogen (BUN)/creatinine ratio of rats fed on an HFD for 4 months. Dosage of lipid hydroperoxides was carried out on methanolic extract of liver tissue. Peritoneal macrophages activity was evaluated in relation to the superoxide anion, hydrogen peroxide and nitric oxide production, phagocytosis and lysosomal volume. The administration of P. ostreatus significantly altered the lipid profile and oxidative stress as related to the LDL and triglycerides decrease and inhibitory effects on superoxide anion and hydrogen peroxide production. All findings of this study lead us to suggest that the P. ostreatus maybe a beneficial agent in the hyperlipidemia and atherosclerosis treatments.     

Putative local adaptations modulate the interactions of the carnivorous plant Drosera uniflora Willd (1809) (Droseraceae) with cushion and shrub nurse plants

Cushion and shrub plants are typical high mountain nurse plants. In Magellanic tundras of the Cordillera del Sarao on the coast of Chile, the carnivorous plant Drosera uniflora grows in association with cushions of Donatia fascicularis and the shrubs Chusquea montana var. nigricans and Lepidothamnus fonkii. The different microhabitats for recruitment, in addition to the limited gene flow of D. uniflora, enable us to hypothesise that this plant manifests putative local adaptations, expressed in seed germination and population abundance. Our aim was to evaluate the local adaptation of D. uniflora to cushion and shrub microhabitats by estimating germination and abundance. Local adaptation of seed germination was determined by means of reciprocal transplant experiments. Abundance was determined in small plots located inside, on the edge and outside the nurse plants. Seed origin and growth substrate play a decisive role in D. uniflora germination. Seeds that originate from cushions plant habitat and germinate in the same substrate do so in greater numbers than when those which originate from shrubby habitats. Conversely, seeds that originate from shrubby habitat and germinate in the same substrate do not exhibit significant differences. Abundance was always greater inside than outside of the nurse plants. We concluded that availability and quality of different microhabitats for seed germination and recruitment, together with a limited gene flow, would trigger putative local adaptations in D. uniflora, modulating a long history of interactions with the plants that act as nurse plants.     

Advantages of granivory in seasonal environments: feeding ecology of an arboreal seed predator in Amazonian forests

Specialized seed predators are uncommon in arboreal vertebrate assemblages, and the hypothesis that consuming seeds of immature fruits – which may be available for relatively long periods compared to mature fruit – could reduce seasonal food scarcity experienced by generalist frugivores remains largely untested. To test this hypothesis, we examined the diet and feeding ecology of bald‐faced saki monkeys Pithecia irrorata in a largely intact forest mosaic of southeastern Peru based on systematic monitoring of five habituated groups over a three‐year period and compared the relative availability of ripe and unripe fruits in their diet. Plant phenology data from individual tree crowns showed that, compared to ripe fruits, immature fruits were available in more tree species, in greater quantities, and for longer periods. Despite pronounced community‐wide seasonal changes in fruit production at our study area, feeding patterns of bald‐faced saki remained largely invariant: fruits comprised approximately 95% of the species’ monthly diet, with seeds alone accounting for 75%, with no major monthly dietary shifts. The flexible exploitation by this species of a consistently available food supply for which it faces little competition likely reduces foraging effort and consumption of less desirable foods, even during prolonged periods of overall fruit scarcity. The relative rarity of immature fruit specialists in tropical forests may reflect the fact that processing the hard pericarps and neutralizing the toxicities of immature seeds present substantial evolutionary hurdles that few arboreal vertebrate species have overcome.     

Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain

Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.     

The hypothesis of sympatric speciation as the dominant generator of endemism in a global hotspot of biodiversity

Allopatric or sympatric speciation influence the degree to which closely related species coexist in different manners, altering the patterns of phylogenetic structure and turnover among and between communities. The objective of this study was to examine whether phylogenetic community structure and turnover in the Brazilian Atlantic Forest permit conclusions about the dominant process for the formation of extant angiosperm richness of tree species. Therefore, we analyzed phylogenetic community structure (MPD, MNTD) as well as taxonomic (Jaccard similarity) and phylogenetic turnover (betaMPD, betaMNTD) among and between 49 tree communities distributed among three different habitat types. Mean annual precipitation and mean annual temperature in each survey area were estimated. Phylogenetic community structure does not differ between habitat types, although MPD reduces with mean annual temperature. Jaccard similarity decreases and betaMNTD increases with spatial distance and environmental differences between study sites. Spatial distance explains the largest portions of variance in the data, indicating dispersal limitation and the spatial aggregation of recently formed taxa, as betaMNTD is related to more recent evolutionary events. betaMPD, that is related to deep evolutionary splits, shows no spatial or environmental pattern, indicating that older clades are equally distributed across the Brazilian Atlantic Forest. While similarity pattern indicates dispersal limitations, the spatial turnover of betaMNTD is consistent with a high degree of sympatric speciation generating extant diversity and endemism in the Brazilian Atlantic Forest. More comprehensive approaches are necessary to reduce spatial sampling bias, uncertainties regarding angiosperm diversification patterns and confirm sympatric speciation as the dominant generator for the formation of extant species diversity in the Brazilian Atlantic Forest.