Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.     

External factors involved in the regulation of an extracellular proteinase synthesis in Bacillus megaterium

Summary A mathematical model was formulated to describe the kinetics and stoichiometry of growth and proteinase production in Bacillus megaterium. Synthesis of the extracellular proteinase in a batch culture is repressed by amino acids. The specific rate of formation of the enzyme (r _E) can be described by the formula {ie373-1}, where k ₂ and k ₃ stand for the non-repressible and repressible part of enzyme synthesis respectively, k _{S 2} is a repression coefficient and S ₂ indicates the concentration of amono acids; the values of k ₂ and k _{S 2} depend on the composition of the mixture of amino acids. Even in a high concentration, a single amino acid is less effective than a mixture of amino acids. The dependence of the proteinase repression on the concentration of an external amino acid (leucine) follows the same course as its rate of incorporation into proteins, approaching saturation at concentrations higher than 50 M (half saturation approximately 10 M). However, the total uptake of leucine did not exhibit any saturation even at 500 M external concentration.Symbols X biomass concentration, g/l - E proteinase concentration, unit/l - t time, h - S ₁ concentration of glucose, g/l - S ₂ concentration of amino acids, g/l - specific growth rate, l/h - rE specific rate of enzyme production, unit/g/h - k ₁ growth kinetic constant, l/h - k ₂ product formation kinetic constant (for non-repressible part of enzyme synthesis), unit/g - k ₃ product formation kinetic constant (for repressible portion of enzyme synthesis), unit/g - k _{S 1} saturation constant, g/l - k _{S 2} repression coefficient for a certain amino acid or amino acids mixture, g/l     

Arsenic in the water,sediment and fish in the Neretva River Delta,Croatia

The Neretva River Delta in Croatia is under constant threat of pollution from various sources along the river watercourse, such as the aluminium industry and bauxite mining, intensive agriculture and untreated sewage from towns. The area is also an important fishing ground and food source for the local residents, whereby the suitability of fish for human consumption is always in question. In this paper the presence of arsenic from six sources was analysed: in water, sediment and fish organs (kidneys, liver, muscles and gonads) of 11 fish species: Lepomis gibbosus, Carassius auratus gibelio, Cyprinus carpio, Anguilla anguilla, Ameiurus nebulosus, Mugil cephalus, Leuciscus svallize, Oncorhynchus mykiss, Salmo trutta, Tinca tinca and Scardinius plotiza. The research showed that arsenic concentrations varied significantly from one source to another in the water, sediment and organs of different fish species. Average concentrations in water and sediment were 18.1 μg L^?1 and 32.7 μg kg^?1, respectively. Average arsenic levels in the fish organs were 115.9, 105.8, 76.1 and 61.9 μg kg^?1 in muscles, kidneys, gonads and livers, respectively. These values are below legally permitted concentrations, although individuals with higher than average concentrations were recorded.     

4-[6-(2-Aminoethyl)naphthalen-2-yl]benzonitriles are potent histamine H3 receptor antagonists with high CNS penetration

4-[6-(2-Tertiaryaminoethyl)naphthalen-2-yl]benzonitriles are conformationally constrained histamine H3 receptor antagonists with high potency and selectivity. The analogs were designed around a naphthalene core, with the goal of enhancing lipophilicity and CNS penetration, as compared to a previously reported benzofuran series. The SAR of the tertiary amine moiety is similar to that reported for the benzofuran series, with analogs bearing a 2-methylpyrrolidine substituent possessing the greatest rat and human H3 receptor binding affinities.     

An Image-Based Drug Susceptibility Assay Targeting the Placental Sequestration of Plasmodium falciparum-Infected Erythrocytes

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.     

Control of Photosynthesis and Stomatal Conductance in Ricinus communis L. (Castor Bean) by Leaf to Air Vapor Pressure Deficit

Castor bean (Ricinus communis L.) has a high photosynthetic capacity under high humidity and a pronounced sensitivity of photosynthesis to high water vapor pressure deficit (VPD). The sensitivity of photosynthesis to varying VPD was analyzed by measuring CO₂ assimilation, stomatal conductance (g_s), quantum yield of photosystem II (_II), and nonphotochemical quenching of chlorophyll fluorescence (q_N) under different VPD. Under both medium (1000) and high (1800 micromoles quanta per square meter per second) light intensities, CO₂ assimilation decreased as the VPD between the leaf and the air around the leaf increased. The g_s initially dropped rapidly with increasing VPD and then showed a slower decrease above a VPD of 10 to 20 millibars. Over a temperature range from 20 to 40°C, CO₂ assimilation and g_s were inhibited by high VPD (20 millibars). However, the rate of transpiration increased with increasing temperature at either low or high VPD due to an increase in g_s. The relative inhibition of photosynthesis under photorespiring (atmospheric levels of CO₂ and O₂) versus nonphotorespiring (700 microbars CO₂ and 2% O₂) conditions was greater under high VPD (30 millibars) than under low VPD (3 millibars). Also, with increasing light intensity the relative inhibition of photosynthesis by O₂ increased under high VPD, but decreased under low VPD. The effect of high VPD on photosynthesis under various conditions could not be totally accounted for by the decrease in the intercellular CO₂ in the leaf (C_i) where C_i was estimated from gas exchange measurements. However, estimates of C_i from measurements of _II and q_N suggest that the decrease in photosynthesis and increase in photorespiration under high VPD can be totally accounted for by stomatal closure and a decrease in C_i. The results also suggest that nonuniform closure of stomata may occur in well-watered plants under high VPD, causing overestimates in the calculation of C_i from gas exchange measurements. Under low VPD, 30°C, high light, and saturating CO₂, castor bean (C₃ tropical shrub) has a rate of photosynthesis (61 micromoles CO₂ per square meter per second) that is about 50% higher than that of tobacco (C₃) or maize (C₄) under the same conditions. The chlorophyll content, total soluble protein, and ribulose-1,5-bisphosphate carboxylase/oxygenase level on a leaf area basis were much higher in castor bean than in maize or tobacco, which accounts for its high rates of photosynthesis under low VPD.     

Ecological divergence of <Emphasis Type="Italic">Chaetopteryx rugulosa</Emphasis> species complex (Insecta,Trichoptera) linked to climatic niche diversification

Climate change may affect species diversity and, consequently, ecological processes such as leaf decomposition. We evaluated the effects of increased temperature and carbon dioxide (CO₂) on fungal biomass, leaf breakdown, and on survival and growth of the shredder Phylloicus elektoros. We hypothesized that climatic changes would result in lower survival and growth of shredders and lower leaf consumption by these organisms. On the other hand, we predicted an increase in fungal biomass in response to climatic changes. We conducted an experiment in Manaus, Brazil, using four microcosms that simulate real-time air temperature and CO₂ (control chamber), as well as three other chambers subjected to fixed increases in temperature and CO₂ as compared to the control chamber. The “extreme” condition represented an increase of ~4.5°C in temperature and ~870 ppm in CO₂ in relation to the control chamber. Total and shredder leaf-breakdown rates, fungal biomass, and shredder survival rates were significantly lower in warmer and CO₂ concentrated atmospheres. Shredder growth rate and leaf breakdown by microorganisms were similar among all climatic conditions. With climatic changes, we found an increase in the relative importance of microorganisms on leaf-breakdown rates as compared to shredders. Thus, lower leaf breakdown and a change in the main decomposer due to future climatic conditions may result in major changes in the pathways of organic matter processing and, consequently, in aquatic food webs.     

VipD of Legionella pneumophila Targets Activated Rab5 and Rab22 to Interfere with Endosomal Trafficking in Macrophages

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.