Characterization and determination of titratable groups of proteins by linearization of titration curves. II. Application to lysozyme

The potentiometric acid-base titration curve of fully protonated lysozyme at ionic strengths of 0.10 and 1.0 m has been performed. The stoichiometry and the pK_a values of each titratable group have been determined through the linearization of titration curves. Two types of carboxylic groups with pK_a values of 3.76 and 5.02, the imidazole group with pK_a 7.37 and the amine group with pK_a 9.63, have been identified at an ionic strength of 0.10 m at 25.0°C. The number of titratable groups found per mole of protein has been 5.12 and 5.60 for the two types of carboxylic groups, 1.13 for the imidazole group, and 3.19 for the amino groups. The endpoint of the titration of the protein obtained by this method accords quite well with the endpoint obtained by the use of Gran function applied to the excess of strong base.     

Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis

Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

Ascorbate oxidase from Cucurbita maxima

Ascorbate oxidase is present in homogenates of the flesh of Cucurbita maxima fruits. Its activity is independent of ascorbate concentration over th     

Micro-buffy coats of whole blood: a method for the electron microscopic study of mononuclear cells

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.     

Effects of pair migratory behavior on breeding phenology and success in a partially migratory shorebird population

In migratory systems, variation in individual phenology can arise through differences in individual migratory behaviors, and this may be particularly apparent in partial migrant systems, where migrant and resident individuals are present within the same population. Links between breeding phenology and migratory behavior or success are generally investigated at the individual level. However, for breeding phenology in particular, the migratory behaviors of each member of the pair may need to be considered simultaneously, as breeding phenology will likely be constrained by timing of the pair member that arrives last, and carryover effects on breeding success may vary depending on whether pair members share the same migratory behavior or not. We used tracking of marked individuals and monitoring of breeding success from a partially migrant population of Eurasian oystercatchers (Haematopus ostralegus) breeding in Iceland to test whether (a) breeding phenology varied with pair migratory behavior; (b) within‐pair consistency in timing of laying differed among pair migratory behaviors; and (c) reproductive performance varied with pair migratory behavior, timing of laying, and year. We found that annual variation in timing of laying differed among pair migratory behaviors, with resident pairs being more consistent than migrant and mixed pairs, and migrant/mixed pairs breeding earlier than residents in most years but later in one (unusually cold) year. Pairs that laid early were more likely to replace their clutch after nest loss, had higher productivity and higher fledging success, independent of pair migratory behavior. Our study suggests that the links between individual migratory behavior and reproductive success can vary over time and, to a much lesser extent, with mate migratory behavior and can be mediated by differences in laying dates. Understanding these cascading effects of pair phenology on breeding success is likely to be key to predicting the impact of changing environmental conditions on migratory species.     

The bacterial yjdF riboswitch regulates translation through its tRNA-like fold

Unlike most riboswitches, which have one cognate effector, the bacterial yjdF riboswitch binds to diverse azaaromatic compounds, only a subset of which cause it to activate translation. We examined the yjdF aptamer domain by small-angle X-ray scattering and found that in the presence of activating ligands, the RNA adopts an overall shape similar to that of tRNA. Sequence analyses suggested that the yjdF aptamer is a homolog of tRNA^Lys, and that two of the conserved loops of the riboswitch are equivalent to the D-loop and T-loop of tRNA, associating to form an elbow-like tertiary interaction. Chemical probing indicated that this association is promoted by activating ligands such as chelerythrine and harmine. In its native mRNA context, activator ligands stabilize the tRNA-like fold of the yjdF aptamer, outcompeting the attenuated state in which its T-loop base pairs to the Shine–Dalgarno element of the mRNA. Moreover, we demonstrate that the liganded aptamer itself activates translation, as authentic tRNAs, when grafted into mRNA, can potently activate translation. Taken together, our data demonstrate the ability of tRNA to function as a small-molecule responsive cis regulatory element.     

Leishmania braziliensis causing human disease in Northeast Brazil presents loci with genotypes in long-term equilibrium

BackgroundLeishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil.Methodology/Principal findingsTwo temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008–2011) primary sample, N = 120; (1999–2001) validation sample, N = 35. Parasites were genotyped by Sanger’s sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward’s comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software.Conclusions/SignificanceThese findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite’s lifecycle.     

Climate and land use changes will degrade the configuration of the landscape for titi monkeys in eastern Brazil

Land use changes have profound effects on populations of Neotropical primates, and ongoing climate change is expected to aggravate this scenario. The titi monkeys from eastern Brazil (Callicebus personatus group) have been particularly affected by this process, with four of the five species now allocated to threatened conservation status categories. Here, we estimate the changes in the distribution of these titi monkeys caused by changes in both climate and land use. We also use demographic‐based, functional landscape metrics to assess the magnitude of the change in landscape conditions for the distribution predicted for each species. We built species distribution models (SDMs) based on maximum entropy for current and future conditions (2070), allowing for different global circulation models and contrasting scenarios of glasshouse gas concentrations. We refined the SDMs using a high‐resolution map of habitat remnants. We then calculated habitat availability and connectivity based on home‐range size and the dispersal limitations of the individual, in the context of a predicted loss of 10% of forest cover in the future. The landscape configuration is predicted to be degraded for all species, regardless of the climatic settings. This include reductions in the total cover of forest remnants, patch size and functional connectivity. As the landscape configuration should deteriorate severely in the future for all species, the prevention of further loss of populations will only be achieved through habitat restoration and reconnection to counteract the negative effects for these and several other co‐occurring species.     

Activity of Combined Antifungal Agents Against Multidrug-Resistant <Emphasis Type="Italic">Candida glabrata</Emphasis> Strains

In this study, we evaluated the in vitro activity of echinocandins, azoles, and amphotericin B alone and in combination against echinocandin/azole-sensitive and echinocandin/azole-resistant Candida glabrata isolates. Susceptibility tests were performed using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute document M27-A3. The checkerboard method was used to evaluate the fractional inhibitory concentration index of the interactions. Cross-resistance was observed among echinocandins; 15% of the isolates resistant to caspofungin were also resistant to anidulafungin and micafungin. Synergistic activity was observed in 70% of resistant C. glabrata when anidulafungin was combined with voriconazole or posaconazole. Higher (85%) synergism was found in the combination of caspofungin and voriconazole. The combinations of caspofungin with fluconazole, posaconazole and amphotericin B, micafungin with fluconazole, posaconazole and voriconazole, and anidulafungin with amphotericin B showed indifferent activities for the majority of the isolates. Anidulafungin combined with fluconazole showed the same percentage of synergism and indifference (45%). Antagonism was detected in 50% of isolates when micafungin was combined with amphotericin B. Combinations of echinocandins and antifungal azoles have great potential for in vivo assays which are required to evaluate the efficacy of these combinations against multidrug-resistant C. glabrata strains.