Effects of hyperbaric oxygen on Leishmania amazonensis promastigotes and amastigotes

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.     

Ferns and Melastomataceae as indicators of vascular plant composition in rain forests of Colombian Amazonia

In a case-study from Colombian Amazonia, species information from ferns and Melastomataceae was used to explain the compositional patterns of other vascular plant species in 40 widely distributed 0.1-ha plots. Canonical correspondence analysis was applied to regress vascular plant species composition in the forests against information from these two indicator groups (summarized as axes of principal coordinate analyses), together with that from soils, landscape, and the spatial sampling design. In total, 53,941 individuals of 2480 vascular plant species were recorded. Of these, 17,473 individuals and 132 species were from ferns and Melastomataceae. In 19 well-drained upland (tierra firme) plots 19,622 vascular plant individuals and 1716 species were found, with 3793 plants and 91 species from ferns and Melastomataceae. In both the set of all landscapes and the subset of tierra firme forests the principal PCoA axes of the two indicator groups were highly related to the main patterns of forest species composition. In principle, therefore, ferns and Melastomataceae can be used to detect and forecast changes in the forest composition of the study area. However, evidence was not obtained that ferns and Melastomataceae show more potential to predict the main patterns in species composition of forests than soil, landscape, and spatial variables. The partioning of the total variation in forest composition showed that the correlation of ferns and Melastomataceae with other forest plants was quite independent from that of soil, landscape, and space. Direct effects of ferns and Melastomataceae on other plants might be obtained from experimental studies of between-plant interactions, concentrating on the seedling or juvenile stages of trees and lianas, both above-ground as well as in the rooting environment.     

Mammalian embryonic cerebrospinal fluid proteome has greater apolipoprotein and enzyme pattern complexity than the avian proteome

During early stages of embryo development, the brain cavity is filled with Embryonic Cerebro-Spinal Fluid, which has an essential role in the survival, proliferation and neurogenesis of the neuroectodermal stem cells. We identified and analyzed the proteome of Embryonic Cerebro-Spinal Fluid from rat embryos (Rattus norvegicus), which includes proteins involved in the regulation of Central Nervous System development. The comparison between mammalian and avian Embryonic Cerebro-Spinal Fluid proteomes reveals great similarity, but also greater complexity in some protein groups. The pattern of apolipoproteins and enzymes in CSF is more complex in the mammals than in birds. This difference may underlie the greater neural complexity and synaptic plasticity found in mammals. Fourteen Embryonic Cerebro-Spinal Fluid gene products were previously identified in adult human Cerebro-Spinal Fluid proteome, and interestingly they are altered in patients with neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis may contribute to our understanding of Central Nervous System development and evolution, and these human diseases.     

A Tn7-based broad-range bacterial cloning and expression system

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.     

The membrane proteome of Halobacterium salinarum

The identification of 114 integral membrane proteins from Halobacterium salinarum was achieved using liquid chromatography/tandem mass spectrometric (LC/MS/MS) techniques, representing 20% of the predicted alpha-helical transmembrane proteins of the genome. For this experiment, a membrane preparation with only minor contamination by soluble proteins was prepared. From this membrane preparation a number of peripheral membrane proteins were identified by the classical two dimensional gel electrophoresis (2-DE) approach, but identification of integral membrane proteins largely failed with only a very few being identified. By use of a fluorescently labeled membrane preparation, we document that this is caused by an irreversible precipitation of the membrane proteins upon isoelectric focusing (IEF). Attempts to overcome this problem by using alternative IEF methods and IEF strip solubilisation techniques were not successful, and we conclude that the classical 2-DE approach is not suited for the identification of integral membrane proteins. Computational analysis showed that the identification of integral membrane proteins is further complicated by the generation of tryptic peptides, which are unfavorable for matrix assisted laser desorption/ionization time of flight mass spectrometric peptide mass fingerprint analysis. Together with the result from the analysis of the cytosolic proteome (see preceding paper), we could identify 34% (943) of all gene products in H. salinarum which can be theoretically expressed. This is a cautious estimate as very stringent criteria were applied for identification. These results are available under www.halolex.mpg.de.     

Potentials for win-win alliances among animal agriculture and forest products industries: Application of the principles of industrial ecology and sustainable development

Commercial forests in many parts of the world are deficient in nitrogen and phosphorus. These nutrient-deficient forests often exist in close proximity to large animal feeding operations, meat processing and other food, textile, or other biomass-processing plants, and municipal waste treatment facilities. Many of these facilities produce large surpluses of nitrogen, phosphorus, and organic matter as gaseous ammonia, urea, uric acid, phosphorus compounds, bacterial sludges, and partially treated municipal wastewaters. These co-existing and substantial nutrient deficiencies and surpluses offer ready-made opportunities for discovery, demonstration, and commercial development of science-based, technology-facilitated, environmentally sound, economically viable, and socially acceptable "win-win alliances" among these major industries based on the principles of industrial ecology and sustainable development. The major challenge is to discover practical means to capture the surplus nutrients and put them to work in forest stands from which value-added products can be produced and sold at a profit.     

Golgi and endoplasmic reticulum functions take place in different subcellular compartments of Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite that causes dysentery in developing countries of Africa, Asia, and Latin America. The lack of a defined Golgi apparatus in E. histolytica as well as in other protists led to the hypothesis that they had evolved prior to the acquisition of such organelle even though glycoproteins, glycolipids, and antigens have been detected, the latter of which react with antibodies against Golgi apparatus proteins of higher eukaryotes. We here provide direct evidence for Golgi apparatus-like functions in E. histolytica as well as for components of glycoprotein folding quality control. Using a combination of bioinformatic, cell biological, and biochemical approaches we have (a) cloned and expressed the E. histolytica UDP-galactose transporter in Saccharomyces cerevisiae; its K(m) for UDP-galactose is 2.9 microm; (b) characterized vesicles in an extract of the above protist, which transport UDP-galactose into their lumen with a K(m) of 2.7 microm;(c) detected galactosyltransferase activity(ies) in the lumen of the above vesicles with the K(m) for UDP-galactose, using endogenous acceptors, being 93 microm;(d) measured latent apyrase activities in the above vesicles, suggesting they are in the lumen; (e) characterized UDP-glucose transport activities in Golgi apparatus and endoplasmic reticulum-like vesicles with K(m)s for UDP-glucose of approximately 2-4 microm. Although the endoplasmic reticulum-like fraction showed UDP-glucose: glycoprotein glucosyltransferase activity, the Golgi apparatus-like fraction did not. This fraction contained other glucosyltransferases. Together, these studies demonstrate that E. histolytica has different vesicles that play a role in protein glycosylation and folding quality control, analogous to the above organellar functions of higher eukaryotes.     

The detection of shared and ancestral polymorphisms

There is increasing evidence that closely related species contain many polymorphisms that were present in their common ancestral species. Use of a more distant relative as an outgroup increases the ability to detect such ancestral polymorphisms. We describe a method for further improving estimates of the fraction of polymorphisms that are ancestral, and illustrate this with reference to data on Drosophila pseudoobscura and D. miranda. We also derive formulae for the proportion of fixations arising from ancestral polymorphisms and new mutations, respectively. The results should be useful for tests of selection based on the levels of expected and observed ancestral polymorphisms.     

A novel conotoxin framework with a helix-loop-helix (Cs alpha/alpha) fold

Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6A, which is a conserved structural feature in K(+) channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.