Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway.

GTPases of the Rab1 subclass are essential for membrane traffic between the endoplasmic reticulum (ER) and Golgi complex in animals, fungi and plants. Rab1-related proteins in higher plants are unusual because sequence comparisons divide them into two putative subclasses, Rab-D1 and Rab-D2, that are conserved in monocots and dicots. We tested the hypothesis that the Rab-D1 and Rab-D2 proteins of Arabidopsis represent functionally distinct groups. RAB-D1 and RAB-D2a each targeted fluorescent proteins to the same punctate structures associated with the Golgi stacks and trans-Golgi-network. Dominant-inhibitory N121I mutants of each protein inhibited traffic of diverse cargo proteins at the ER but they appeared to act via distinct biochemical pathways as biosynthetic traffic in cells expressing either of the N121I mutants could be restored by coexpressing the wild-type form of the same subclass but not the other subclass. The same interaction was observed in transgenic seedlings expressing RAB-D1 [N121I]. Insertional mutants confirmed that the three Arabidopsis Rab-D2 genes were extensively redundant and collectively performed an essential function that could not be provided by RAB-D1, which was non-essential. However, plants lacking RAB-D1, RAB-D2b and RAB-D2c were short and bushy with low fertility, indicating that the Rab-D1 and Rab-D2 subclasses have overlapping functions.

Mapping the proteome of Leishmania Viannia parasites using two-dimensional polyacrylamide gel electrophoresis and associated technologies.

In this study we have demonstrated the potential of two-dimensional electrophoresis (2DE)-based technologies as tools for characterization of the Leishmania proteome (the expressed protein complement of the genome). Standardized neutral range (pH 5-7) proteome maps of Leishmania (Viannia) guyanensis and Leishmania (Viannia) panamensis promastigotes were reproducibly generated by 2DE of soluble parasite extracts, which were prepared using lysis buffer containing urea and nonidet P-40 detergent. The Coomassie blue and silver nitrate staining systems both yielded good resolution and representation of protein spots, enabling the detection of approximately 800 and 1,500 distinct proteins, respectively. Several reference protein spots common to the proteomes of all parasite species/strains studied were isolated and identified by peptide mass spectrometry (LC-ES-MS/MS), and bioinformatics approaches as members of the heat shock protein family, ribosomal protein S12, kinetoplast membrane protein 11 and a hypothetical Leishmania-specific 13 kDa protein of unknown function. Immunoblotting of Leishmania protein maps using a monoclonal antibody resulted in the specific detection of the 81.4 kDa and 77.5 kDa subunits of paraflagellar rod proteins 1 and 2, respectively. Moreover, differences in protein expression profiles between distinct parasite clones were reproducibly detected through comparative proteome analyses of paired maps using image analysis software. These data illustrate the resolving power of 2DE-based proteome analysis. The production and basic characterization of good quality Leishmania proteome maps provides an essential first step towards comparative protein expression studies aimed at identifying the molecular determinants of parasite drug resistance and virulence, as well as discovering new drug and vaccine targets.

High host-plant nitrogen content: a prerequisite for the evolution of ant-caterpillar mutualism?

The amount of nitrogen required to complete an insect's life cycle may vary greatly among species that have evolved distinct life history traits. Myrmecophilous caterpillars in the Lycaenidae family produce nitrogen-rich exudates from their dorsal glands to attract ants for protection, and this phenomenon has been postulated to shape the caterpillar's host-plant choice. Accordingly, it was postulated that evolution towards myrmecophily in Lycaenidae is correlated with the utilization of nitrogen-rich host plants. Although our results were consistent with the evolutionary shifts towards high-nutrient host plants serving as exaptation for the evolution of myrmecophily in lycaenids, the selection of nitrogen-rich host plants was not confined to lycaenids. Butterfly species in the nonmyrmecophilous family Pieridae also preferred nitrogen-rich host plants. Thus, we conclude that nitrogen is an overall important component in the caterpillar diet, independent of the level of myrmecophily, as nitrogen can enhance the overall insect fitness and survival. However, when nitrogen can be obtained through alternative means, as in socially parasitic lycaenid species feeding on ant brood, the selective pressure for maintaining the use of nutrient-rich host plants is relaxed, enabling the colonization of nitrogen-poor host plants.

Chemoresistance of Plasmodium falciparum and Plasmodium vivax parasites in Brazil: consequences on disease morbidity and control

In Brazil, malaria still remains a clinically important febrile syndrome for local populations and travelers, occurring mostly in the Amazon Basin. This review aims to report the main efforts employed to control this disease since the 1940s and the emergence of Plasmodium falciparum and Plasmodium vivax chemoresistance to chloroquine and sulphadoxine-pyrimethamine among other drugs. Additionally, in vivo, in vitro and molecular studies as well as malaria chemoresistance consequences on disease morbidity and policy treatment guidelines were commented.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

Cardenolides in nectar may be more than a consequence of allocation to other plant parts: a phylogenetic study of Asclepias

The primary function of secondary plant metabolites is thought to be defence against herbivores. The frequent occurrence of these same noxious compounds in floral nectar, which functions primarily to attract pollinators, has been seen as paradoxical. Although these compounds may have an adaptive purpose in nectar, they may also occur as a nonadaptive consequence of chemical defence in other plant parts. If nectar chemistry reflects physiological constraints or passive leakage from other tissues, we expect that the identity and relative concentration of nectar cardenolides to be correlated with those of other plant parts; in contrast, discordant distributions of compounds in nectar and other tissues may suggest adaptive roles in nectar. We compared the concentrations and identities of cardenolides in the nectar, leaves and flowers of 12 species from a monophyletic clade of Asclepias. To measure putative toxicity of nectar cardenolides, we then examined the effects of a standard cardenolide (digoxin) on the behaviour of bumblebees, a common generalist pollinator of Asclepias. We found that the average cardenolide concentrations in nectar, leaves and flowers of the 12 Asclepias species were positively correlated as predicted by nonadaptive hypotheses. However, significant differences in the identities and concentrations of individual cardenolides between nectar and leaves suggest that the production or allocation of cardenolides may be independently regulated at each plant part. In addition, cardenolide concentrations in leaves and nectar exhibited no phylogenetic signal. Surprisingly, bumblebees did not demonstrate an aversion to digoxin-rich nectar, which may indicate that nectar cardenolides have little effect on pollination. Although the idea that discordant patterns of secondary metabolites across tissue types may signal adaptive functions is attractive, there is evidence to suggest constraint contributes to nectar secondary chemistry. Further work testing the ecological impacts of such patterns will be critical in determining the functional significance of nectar cardenolides

Genetic variability in a population of arbuscular mycorrhizal fungi causes variation in plant growth.

Different species of arbuscular mycorrhizal fungi (AMF) alter plant growth and affect plant coexistence and diversity. Effects of within-AMF species or within-population variation on plant growth have received less attention. High genetic variation exists within AMF populations. However, it is unknown whether genetic variation contributes to differences in plant growth. In our study, a population of AMF was cultivated under identical conditions for several generations prior to the experiments thus avoiding environmental maternal effects. We show that genetically different Glomus intraradices isolates from one AMF population significantly alter plant growth in an axenic system and in greenhouse experiments. Isolates increased or reduced plant growth meaning that plants potentially receive benefits or are subject to costs by forming associations with different individuals in the AMF population. This shows that genetic variability in AMF populations could affect host-plant fitness and should be considered in future research to understand these important soil organisms.

Apoptotic markers in protozoan parasites.

ABSTRACT: The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms.In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

In vitro and in vivo antimalarial activity and cytotoxicity of extracts, fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.

Characterization of a plant-derived peptide displaying water clarifying and antimicrobial activities

SUMMARY Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment- friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components was unknown. Here, we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Pseudomona, Streptococcus and Legionella species. Structural modeling of the peptide coupled to the functional analysis of synthetic peptide derivatives delineated distinct structural determinants for the flocculation and antibacterial activities. Our results suggest that a glutamine-rich portion of the polypeptide is involved in the sedimentation process; alternatively, the antibacterial activity depends on a amphiphilic loop. Assembly of multiple copies of this loop into a branched peptide derivative strongly enhances antibacterial activity without displaying hemolytic effect. In conclusion, this polypeptide displays the unprecedented feature of combining efficient water purification and disinfectant properties indicating different molecular mechanisms involved in each case. This work not only identified the features responsible for these activities but also provides useful information that has implications for the further development of this cationic polypeptide as a potent antibacterial agent. RESUME L'eau potable est actuellement une ressource limitée dans le monde. La production d'eau propre à la consommation exige des traitements complexes, incluant la clarification des particules en suspension ainsi que sa désinfection par des additifs chimiques. Les extraits de la graine d'un arbre tropical, Moringa oleifera, sont utilisés traditionnellement en Afrique afin de clarifier l'eau. Quoique la nature exacte des composants actifs était inconnue, on a pu mettre en évidence un polypeptide cationique contenu dans ces graines, capable de sédimenter de manière efficace des particules minérales en suspension ainsi que des bactéries. Ce travail a aussi mis en évidence que ce polypeptide a une activité bactéricide, permettant une désinfection d'eau fortement contaminée. De plus, nous avons démontré que ce polypeptide est efficace contre de nombreuses souches bactériennes pathogènes, également celles résistantes aux antibiotiques comme Pseudomonas, Streptococcus et Legionella. L'analyse de la structure moléculaire de ce polypeptide, couplée à son analyse fonctionnelle a mis en évidence deux domaines structuraux distinct, un pour l'activité de floculation et l'autre pour l'activité antibactérienne. Nos résultats suggèrent que le domaine riche en glutamine est impliqué dans le processus de sédimentation et que l'activité antimicrobienne dépend d'un domaine formant une boucle amphiphilique. En ramifiant plusieurs copies de cette boucle on a pu augmenter de manière significative l'activité antibactérienne. En conclusion, nous avons pu démontrer que ce polypeptide à la capacité unique de combiner des propriétés de purification et de désinfection de l'eau, ce qui implique des mécanismes moléculaires distincts pour ces deux activités. Ce travail a permis d'identifier les domaines du polypeptide qui sont responsables de ses activités et offre une perspective pour le développement d'un nouvel agent antimicrobien.

Novel Chlamydiales strains isolated from a water treatment plant.

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers (Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae; two closely related strains belonged to the Criblamydiaceae; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.

Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.

Ecology of cutaneous leishmaniasis in Sinai: linking parasites, vectors and hosts

Cutaneous leishmaniasis (CL) is a neglected clinical form of public health importance that is quite prevalent in the northern and eastern parts of Egypt. A comprehensive study over seven years (January 2005-December 2011) was conducted to track CL transmission with respect to both sandfly vectors and animal reservoirs. The study identified six sandfly species collected from different districts in North Sinai: Phlebotomus papatasi, Phlebotomus kazeruni, Phlebotomus sergenti, Phlebotomus alexandri, Sergentomyia antennata and Sergentomyia clydei. Leishmania (-)-like flagellates were identified in 15 P. papatasi individuals (0.5% of 3,008 dissected females). Rodent populations were sampled in the same districts where sandflies were collected and eight species were identified: Rattus norvegicus (n = 39), Rattus rattus frugivorous (n = 13), Rattus rattus alexandrinus (n = 4), Gerbillus pyramidum floweri (n = 38), Gerbillus andersoni (n = 28), Mus musculus (n = 5), Meriones sacramenti (n = 22) and Meriones crassus (n = 10). Thirty-two rodents were found to be positive for Leishmania infection (20.12% of 159 examined rodents). Only Leishmania major was isolated and identified in 100% of the parasite samples. The diversity of both the vector and rodent populations was examined using diversity indices and clustering approaches.