Insights into the origin and distribution of biodiversity in the Brazilian Atlantic forest hot spot: a statistical phylogeographic study using a low-dispersal organism

The relative importance of the processes that generate and maintain biodiversity is a major and controversial topic in evolutionary biology with large implications for conservation management. The Atlantic Forest of Brazil, one of the world''s richest biodiversity hot spots, is severely damaged by human activities. To formulate an efficient conservation policy, a good understanding of spatial and temporal biodiversity patterns and their underlying evolutionary mechanisms is required. With this aim, we performed a comprehensive phylogeographic study using a low-dispersal organism, the land planarian species Cephaloflexa bergi (Platyhelminthes, Tricladida). Analysing multi-locus DNA sequence variation under the Approximate Bayesian Computation framework, we evaluated two scenarios proposed to explain the diversity of Southern Atlantic Forest (SAF) region. We found that most sampled localities harbour high levels of genetic diversity, with lineages sharing common ancestors that predate the Pleistocene. Remarkably, we detected the molecular hallmark of the isolation-by-distance effect and little evidence of a recent colonization of SAF localities; nevertheless, some populations might result from very recent secondary contacts. We conclude that extant SAF biodiversity originated and has been shaped by complex interactions between ancient geological events and more recent evolutionary processes, whereas Pleistocene climate changes had a minor influence in generating present-day diversity. We also demonstrate that land planarians are an advantageous biological model for making phylogeographic and, particularly, fine-scale evolutionary inferences, and propose appropriate conservation policies.     

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.     

Actividad del sistema neuronal LHRH en un modelo animal de retraso del crecimiento

ObjectiveMild and chronic energy restriction results in growth retardation with puberal delay, a nutritional disease known as nutritional dwarfing (ND). The aim of the present study was to assess the profile of hypothalamic luteinizing hormone-releasing hormone (LHRH) release, at baseline and under glutamate stimulation, in ND rats to elucidate gonadotrophic dysfunction. Reproductive ability during refeeding was also studied.Material and methodsAt weaning, 60 male rats were assigned to two groups of 30 animals each: a control and an experimental group. Control rats were fed ad libitum with a balanced rodent diet. The experimental group received 80% of the diet consumed by the control group for 4 weeks. After 4 weeks of food restriction, the ND group was fed freely for 8 weeks. Ten rats from each group were sacrificed every 4 weeks for assays.ResultsAt week 4, body weight and length were significantly diminished in the experimental group vs. the control group (p<0.001). No changes were observed in LHRH baseline release, pulse frequency or amplitude in the experimental group compared with the control group at any time. However, under glutamate stimulation, LHRH release was significantly higher in ND rats than in control rats at week 4 (p<0.05). Refeeding the ND group allowed the rats to reach overall growth and reproductive ability.ConclusionsThe results of the present study suggest that the response to the facilitatory effect of glutamate on LHRH release in post-restricted ND rats is probably related to a lesser central nervous system maturation in relation to their chronological age. The adequate somatic growth and normal reproductive ability attained with refeeding suggest the reversibility of the two energetically costly processes compromised by global, mild and chronic food restriction.     

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase,Emerging from Nonconventional Codon Usage and Directed Mutation

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k_cat/K_m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes'' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.β-Fructofuranosidases (EC 3.2.1.26) are enzymes of biotechnological interest that catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. In general, they exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH), along with invertases, inulinases, and fructosyltransferases (http://www.cazy.org). The GH32 family has been studied intensely, and some three-dimensional structures are now available, such as that of inulinase from Aspergillus awamorii (26), fructan-exohydrolase from Cichorium intybus (CiFEH) (34, 36), or invertase from Thermotoga maritima (2, 3) and Arabidopsis thaliana (35). These proteins contain a five-blade β-propeller N-terminal catalytic module and a C-terminal β-sandwich domain (19). Multiple-sequence alignment of GH32 proteins, which are included in the GH-J clan together with the GH68 proteins of the inulosucrase family, reveals the presence of three conserved motifs, each containing a key acidic residue (in boldface) implicated in substrate binding and hydrolysis: Asn-Asp-Pro-Asn-Gly (NDPNG), Arg-Asp-Pro (RDP), and Glu-Cys (EC) (28). These conserved residues are implicated in a double-displacement reaction in which a covalent glycosyl-enzyme intermediate is formed. Thus, the catalytic mechanism proposed for the Saccharomyces cerevisiae invertase implies that Asp23 (NDPNG) acts as a nucleophile and Glu204 (EC) acts as the acid/base catalyst (29), whereas Asp309 (RDP) of Acetobacter diazotropicus levansucrase influences the efficiency of sucrose hydrolysis (7) and Arg188 and Asp189 of the latter motif define the substrate binding and specificity of exoinulinase from A. awamorii toward fructopyranosyl residues (26).As well as hydrolyzing sucrose, β-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds, depending on the source of the enzyme (12, 21, 31). FOS act as prebiotics, and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis (16). Currently, FOS are mainly produced by Aspergillus fructosyltransferase in industry (10, 31), providing a mixture of FOS with an inulin-type structure that contains β-(2→1)-linked fructose oligomers (¹F-FOS: 1-kestose or nystose). Curiously, when the link between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose) involves a β-(2→6) link, the prebiotic properties of the FOS may be enhanced beyond that of commercial FOS (23).The yeast Schwanniomyces occidentalis (also called Debaryomyces occidentalis) produces a number of extracellular enzymes that make it of interest in biotechnology. Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1, 9), as well as an invertase (17). In addition, we also characterized an extracellular β-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5). This enzyme exhibited a transfructosylating activity that efficiently produces the trisaccharides 6-kestose and 1-kestose in the ratio 3:1, generating the highest 6-kestose yield yet reported, as far as we know. The Ffase three-dimensional structure has recently been solved (6) and represented as a homodimer, each modular subunit arranged like other GH32 enzymes. The Asp50 (NDPNG) and Glu230 (EC) located at the center of the propeller are the catalytic residues implicated in substrate binding and hydrolysis, whereas Arg178 and Asp179 form the RDP motif (6).The genetic codes of some yeasts incorporate certain variations. For example, while CUG was believed to be a universal codon for leucine, in the cytoplasm of certain species of the genus Candida (15) it encodes a serine, as in Pichia farinosa (33). The reassignment of this codon is mediated by a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (25).Here, we show that deviation from the standard use of the CUG leucine codon to encode serine was correlated with the transferase capacity and specificity of the Ffase enzyme. Indeed, the S196L substitution enhanced the transferase activity of the enzyme 3-fold. Several site-directed mutants were generated and characterized to study their transferase capacities. These results are considered on the basis of the enzymes'' three-dimensional structure, which enables a novel putative binding site of sucrose that serves as a water substitute donor in the hydrolytic reaction yielding the tranglycosylation product 6-kestose to be identified.     

A peptide based on the pore-forming domain of pro-apoptotic poliovirus 2B viroporin targets mitochondria

Non-structural poliovirus 2B protein induces plasma membrane permeabilization and has been recently implicated in triggering apoptosis via the mitochondrial pathway. Here we describe that the pore-forming P3 peptide, based on the 2B amphipathic domain, translocates through the plasma membrane of culture cells and targets mitochondria. Cell permeabilization by P3 versions of different lengths, together with peptide uptake analyses supported an internalization mechanism dependent on P3 capacity to interact physically with lipid bilayers and establish permeating pores therein. Internalized P3 was found associated with mitochondria, but contrary to the parental 2B protein, the short peptide did not affect the morphology or cell distribution of these organelles, nor induced apoptosis. We conclude that P3 constitutes a mitochondriotropic sequence, which is however devoid of 2B pro-apoptotic activity.     

Melatonin prevents glutamate-induced oxytosis in the HT22 mouse hippocampal cell line through an antioxidant effect specifically targeting mitochondria

The pineal hormone melatonin has neuroprotective effects in a large number of models of neurodegeneration. Melatonin crosses the blood-brain barrier, shows a decrease in its nocturnal peaks in blood with age that has been associated with the development of neurodegenerative disorders, and has been shown to be harmless at high concentrations. These properties make melatonin a potential therapeutic agent against neurodegenerative disorders but the pathways involved in such neuroprotective effects remain unknown. In the present report we study the intracellular pathways implicated in the complete neuroprotection provided by melatonin against glutamate-induced oxytosis in the HT22 mouse hippocampal cell line. Our results strongly suggest that melatonin prevents oxytosis through a direct antioxidant effect specifically targeted at the mitochondria. Firstly, none of the described transducers of melatonin signalling seems to be implicated in the neuroprotection provided by this indole. Secondly, melatonin does not prevent cytosolic GSH depletion-dependent increase in reactive oxygen species (ROS), but it totally prevents mitochondrial ROS production despite the fact that the latter is much higher than the former. And finally, there is a high correlation between the concentration at which melatonin and closely related indoles exert a direct antioxidant effect in vitro and a neuroprotective effect against glutamate-induced oxytosis.     

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA^Thr synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA^Thr. Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs.     

A Single Mutation in the Gene Responsible for the Mucoid Phenotype of Bifidobacterium animalis subsp. lactis Confers Surface and Functional Characteristics

Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.