Mobilization of seed reserves during germination of four tropical species of the Amazon Rainforest

Seed reserve mobilization during germination of the Amazonian species Myciaria dubia (camu-camu), Eugenia stipitata(araca-boi), Dipteryx odorata (cumaru) and Hymenaea courbaril (jatoba) was evaluated. Seeds were placed in germination chambers at 30 degrees C with it 12 h photoperiod. Analysis of primary metabolites (carbohydrates, lipids and proteins) and fatty acid composition were carried out in quiescent seeds and at four germination stages after radicle protrusion. Germination was high in all species but there were statistically significant differences between species. Differences were also observed with regard to the duration of the germination period. The seeds showed variation in the content and composition of the analyzed compounds. indicating that the mobilization rates of these compounds may affect germination velocity.

The modularity of seed dispersal: differences in structure and robustness between bat- and bird-fruit networks

In networks of plant-animal mutualisms, different animal groups interact preferentially with different plants, thus forming distinct modules responsible for different parts of the service. However, what we currently know about seed dispersal networks is based only on birds. Therefore, we wished to fill this gap by studying bat-fruit networks and testing how they differ from bird-fruit networks. As dietary overlap of Neotropical bats and birds is low, they should form distinct mutualistic modules within local networks. Furthermore, since frugivory evolved only once among Neotropical bats, but several times independently among Neotropical birds, greater dietary overlap is expected among bats, and thus connectance and nestedness should be higher in bat-fruit networks. If bat-fruit networks have higher nestedness and connectance, they should be more robust to extinctions. We analyzed 1 mixed network of both bats and birds and 20 networks that consisted exclusively of either bats (11) or birds (9). As expected, the structure of the mixed network was both modular (M = 0.45) and nested (NODF = 0.31); one module contained only birds and two only bats. In 20 datasets with only one disperser group, bat-fruit networks (NODF = 0.53 +/- A 0.09, C = 0.30 +/- A 0.11) were more nested and had a higher connectance than bird-fruit networks (NODF = 0.42 +/- A 0.07, C = 0.22 +/- A 0.09). Unexpectedly, robustness to extinction of animal species was higher in bird-fruit networks (R = 0.60 +/- A 0.13) than in bat-fruit networks (R = 0.54 +/- A 0.09), and differences were explained mainly by species richness. These findings suggest that a modular structure also occurs in seed dispersal networks, similar to pollination networks. The higher nestedness and connectance observed in bat-fruit networks compared with bird-fruit networks may be explained by the monophyletic evolution of frugivory in Neotropical bats, among which the diets of specialists seem to have evolved from the pool of fruits consumed by generalists.

Seed dispersal and predation in the endemic Atlantic rainforest palm Astrocaryum aculeatissimum across a gradient of seed disperser abundance

Tropical forests have been subject to intense hunting of medium and large frugivores that are important in dispersing large-seeded species. It has been hypothesized that in areas with extinction or low abundance of medium and large-bodied animals the density of small rodents may increase. Therefore, this increment in the density of small rodents may compensate for the absence or low abundance of medium and large frugivores on seed removal and seed dispersal. Here, we fill up this gap in the literature by determining if seed removal, seed dispersal, and seed predation by small rodents (spiny rats, Trinomys inheringi and squirrels, Sciurus ingrami) are maintained in defaunated areas. We accessed seed removal, seed dispersal, seed predation, and seedling recruitment of an endemic Atlantic rainforest palm, Astrocaryum aculeatissimum, in a gradient of abundance of agoutis. We found that seed removal, scatter hoarding, and seed predation increase with the abundance of agoutis. In contrast, the proportion of dispersed but non-cached seeds decreased with the abundance of agoutis. We did not find any effect of the abundance of agoutis on seed dispersal distance, but we did find a positive trend on the density of seedlings. We concluded that small rodents do not compensate the low abundance of agoutis on seed removal, scatter hoarding, and seed predation of this palm tree. Moreover, areas in which agoutis are already extinct did not present any seed removal or scatter hoarding, not even by small rodents. This study emphasizes both the importance of agoutis in dispersing seeds of A. aculeatissimum and the collapse in seed dispersal of this palm in areas where agoutis are already extinct.

Are tortoises important seed dispersers in Amazonian forests?

According to most studies on seed dispersal in tropical forests, mammals and birds are considered the main dispersal agents and the role played by other animal groups remains poorly explored. We investigate qualitative and quantitative components of the role played by the tortoise Chelonoidis denticulata in seed dispersal in southeastern Amazon, and the influence of seasonal variation in tortoise movement patterns on resulting seed shadows. Seed shadows produced by this tortoise were estimated by combining information on seed passage times through their digestive tract, which varied from 3 to 17 days, with a robust dataset on movements obtained from 18 adult C. denticulata monitored with radio transmitters and spoon-and-line tracking devices. A total of 4,206 seeds were found in 94 collected feces, belonging to 50 seed morphotypes of, at least, 25 plant genera. Very low rates of damage to the external structure of the ingested seeds were observed. Additionally, results of germination trials suggested that passage of seeds through C. denticulata`s digestive tract does not seem to negatively affect seed germination. The estimated seed shadows are likely to contribute significantly to the dispersal of seeds away from parent plants. During the dry season seeds were dispersed, on average, 174.1 m away from the location of fruit ingestion; during the rainy season, this mean dispersal distance increased to 276.7 m. Our results suggest that C. denticulata plays an important role in seed dispersal in Amazonian forests and highlight the influence of seasonal changes in movements on the resulting seed shadows.

Analysis of a hyper-diverse seed dispersal network: modularity and underlying mechanisms

Mutualistic interactions involving pollination and ant-plant mutualistic networks typically feature tightly linked species grouped in modules. However, such modularity is infrequent in seed dispersal networks, presumably because research on those networks predominantly includes a single taxonomic animal group (e.g. birds). Herein, for the first time, we examine the pattern of interaction in a network that includes multiple taxonomic groups of seed dispersers, and the mechanisms underlying modularity. We found that the network was nested and modular, with five distinguishable modules. Our examination of the mechanisms underlying such modularity showed that plant and animal trait values were associated with specific modules but phylogenetic effect was limited. Thus, the pattern of interaction in this network is only partially explained by shared evolutionary history. We conclude that the observed modularity emerged by a combination of phylogenetic history and trait convergence of phylogenetically unrelated species, shaped by interactions with particular types of dispersal agents.

Melatonin and the time window for the expression of the alpha 8 nicotinic acetylcholine receptor in the membrane of chick retinal cells in culture

We have previously shown that melatonin influences the development of alpha 8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha 8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48 h suppresses the development of functional alpha 8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha 8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23 pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60 min, or 12 h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha 8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10 nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha 8 nAChR subtype in full functional form. (C) 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

Ca(2+)-activated transbilayer movement of plasma membrane phospholipids in Leishmania donovani during ionomycin or thapsigargin stimulation

The protozoan parasite Leishmania causes serious infections in humans all over the world. After being inoculated into the skin through the bite of an infected sandfly, Leishmania promastigotes must gain entry into macrophages to initiate a successful infection. Specific, surface exposed phospholipids have been implicated in Leishmania-macrophage interaction but the mechanisms controlling and regulating the plasma membrane lipid distribution remains to be elucidated. Here, we provide evidence for Ca(2+)-induced phospholipid scrambling in the plasma membrane of Leishmania donovani. Stimulation of parasites with ionomycin increases intracellular Ca(2+) levels and triggers exposure of phosphatidylethanolamine at the cell surface. We found that increasing intracellular Ca(2+) levels with ionomycin or thapsigargin induces rapid transbilayer movement of NBD-labelled phospholipids in the parasite plasma membrane that is bidirectional, independent of cellular ATP and not specific to the polar lipid head group. The findings suggest the presence of a Ca(2+)-dependent lipid scramblase activity in Leishmania parasites. Our studies further show that lipid scrambling is not activated by rapid exposure of promastigotes to higher physiological temperature that increases intracellular Ca(2+) levels. (C) 2011 Elsevier B.V. All rights reserved.

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (similar to 50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (similar to 30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca(2+) and Mg(2+) ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation. (C) 2010 Elsevier Ltd. All rights reserved.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of < 49 and a parts per thousand yen20 KDa (M. hominis) and < 102 and a parts per thousand yen58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Interaction of membrane mimetic vesicles with the antimicrobial peptide gomesin and its analogues

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sequence-specific reconstruction from fragmentary databases using seed sequences: implementation and validation on SAGE, proteome and generic sequencing data

Motivation: DNA assembly programs classically perform an all-against-all comparison of reads to identify overlaps, followed by a multiple sequence alignment and generation of a consensus sequence. If the aim is to assemble a particular segment, instead of a whole genome or transcriptome, a target-specific assembly is a more sensible approach. GenSeed is a Perl program that implements a seed-driven recursive assembly consisting of cycles comprising a similarity search, read selection and assembly. The iterative process results in a progressive extension of the original seed sequence. GenSeed was tested and validated on many applications, including the reconstruction of nuclear genes or segments, full-length transcripts, and extrachromosomal genomes. The robustness of the method was confirmed through the use of a variety of DNA and protein seeds, including short sequences derived from SAGE and proteome projects.

Protection against high-dose homologous infection in calves immunized with intestine or membrane extracts from Haemonchus placei

Control of Haemonchus placei, one of the most important cattle nematodes in Brazil, relies on the use of anthelmintics. However, there is a need for integrated control, which includes active immunization. The aim of this work was to assess the protection afforded to calves by immunization with adult H. placei extracts against a high-dose challenge infection, a condition frequently found in the tropics. Holstein calves aged 8-10 months were immunized four times with intestinal extracts (Group D) or with a Triton X-100-soluble fraction of adult H. placei (Group A), challenge-infected with 120,000 infective larvae and sacrificed 40 days later. Immunized animals had higher IgG titers than the controls against tested fractions after the 2nd immunization, peaking after the 4th. Sera from both immunized groups recognized bands of similar apparent mass in both antigenic preparations, some of which were similar in molecular weight to Haemonchus contortus antigens with known protective effect to sheep. Egg counts were 49% and 57% lower in Groups A and D than in controls, respectively. High levels of protection were observed in two of the four calves in Group D, as evidenced by very low worm numbers recovered at necropsy, absence of eggs in the uteri of the recovered females and reduced worm length. Group D animals also showed milder signs of anemia than the other infected animals. Results demonstrate that protection against homologous high-dose challenge can be achieved by immunizing calves with H. placei gut antigens. (C) 2007 Elsevier B.V. All rights reserved.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.