Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations

Molecular Biology Reports - Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic....     

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.     

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.     

Effect of landscape features on genetic structure of the goitered gazelle (<Emphasis Type="Italic">Gazella subgutturosa</Emphasis>) in Central Iran

The populations of goitered gazelle suffered significant decline due to natural and anthropogenic factors over the last century. Investigating the effects of barriers on gene flow among the remaining populations is vital for conservation planning. Here we adopted a landscape genetics approach to evaluate the genetic structure of the goitered gazelle in Central Iran and the effects of landscape features on gene flow using 15 polymorphic microsatellite loci. Spatial autocorrelation, isolation by distance (IBD) and isolation by resistance (IBR) models were used to elucidate the effects of landscape features on the genetic structure. Ecological modeling was used to construct landscape permeability and resistance map using 12 ecogeographical variables. Bayesian algorithms revealed three genetically homogeneous groups and restricted dispersal pattern in the six populations. The IBD and spatial autocorrelation revealed a pattern of decreasing relatedness with increasing distance. The distribution of potential habitats was strongly correlated with bioclimatic factors, vegetation type, and elevation. Resistance distances and graph theory were significantly related with variation in genetic structure, suggesting that gazelles are affected by landscape composition. The IBD showed greater impact on genetic structure than IBR. The Mantel and partial Mantel tests indicated low but non-significant effects of anthropogenic barriers on observed genetic structure. We concluded that a combination of geographic distance, landscape resistance, and anthropogenic factors are affecting the genetic structure and gene flow of populations. Future road construction might impede connectivity and gene exchange of populations. Conservation measures on this vulnerable species should consider some isolated population as separate management units.     

Nest-site selection and its impact on breeding success of the Lesser Kestrel,Falco naumanni,in the North of Iran (Aves: Falconiformes)

The Lesser Kestrel (Falco naumanni) is a small migratory falcon which nests coloni- ally on inland cliffs and ruined buildings. Bandar Turkman city holds one of the most important breeding colonies in Iran. It nests in holes in roofs, walls or on ledges of buildings throughout of the city. We tried to find out whether there is a relationship between nest-site selection and breeding success. Although height from the ground is the most important factor in nest-site selection by F. naumanni, height from the floor and distance to the nearest neighbour are other main factors which affect breeding success. This could be the result of accessibility by human and natural predators such as domestic cats which threaten nests. The number of possible nest-sites in the study area is limited as many old, ruined buildings are being replaced by new construc- tions; this colonially breeding bird species therefore suffers from a lack of highly suitable nest-sites and this directly affect breeding success.     

Leishmania major: Reactive oxygen species and interferon gamma induction by soluble lipophosphoglycan of stationary phase promastigotes

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is membrane glycoconjugate lipophosphoglycan (mLPG). In addition, it has been shown that mLPG could be used as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we attempted to compare the immunological properties of culture supernatant and membrane LPG prepared from stationary phase promastigotes of Leishmania major. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by thin layer chromatography. The effect of sLPG and mLPG on the production of reactive oxygen species (ROS) was studied using PBMCs isolated from healthy individuals. In addition, induction of IL-12, IFN-gamma and IL-10 secretion in the presence of sLPG and mLPG was investigated. Reactive oxygen species in addition to IL-10 and IL-12 were induced by both sLPG and mLPG. However, IFN-gamma production was promoted only in response to sLPG suggesting its ability to promote Th1 response and implication in vaccine design.     

Selective leishmanicidal effect of 1,3,4-thiadiazole derivatives and possible mechanism of action against Leishmania species

With the aim of determining selectivity and the possible target(s) of nitroheteroaryl-1,3,4-thiadiazoles considered as possible leads for the development of anti-leishmanial agents, we studied 5-nitroimidazole, 5-nitrofuran and 5-nitrothiophene analogs of N-substituted-piperazinyl-1,3,4-thiadiazoles. We investigated 21 representative compounds 1-3(a-g) for the following properties: selectivity and efficiency against different Leishmania wild type species and intracellular parasite, toxicity against host cells and inhibition of topoisomerases I and II. Our results indicate that the nitroimidazole analogs 1a and 1f, and nitrofuran derivatives 2a, 2b, 2c, 2f, and 2g exhibited low toxicity against the host cells (IC₅₀ ? 80 μM), but high selectivity against intracellular amastigotes (selectivity index > 12). Leishmania topoisomerases revealed impressive sensitivity to the agents (%inhibition >50 at IC₅₀ doses of compounds against Leishmania). Our findings showed that at least part of leishmaniacidal effect of the compounds could be attributed to disruption DNA-relaxed activities of topoisomerases I and II, and cleavable-complex formation.     

Investigation on the surface hydrophobicity and aggregation kinetics of human calprotectin in the presence of calcium

Calcium and zinc binding protein, calprotectin is a multifunctional protein with broad spectrum antimicrobial and antitumoural activity. It was purified from human neutrophil, using a two-step ion exchange chromatography. Since surface hydrophobicity of calprotectin may be important in membrane anchoring, membrane penetration, subunits oligomerization and some biological roles of protein, in this study attempted to explore the effect of calcium in physiological range on the calprotectin lipophilicity. Incubation of human calprotectin (50 microg/ml) with different calcium concentrations showed that 1-anilino-8-naphthalene sulfonic acid (ANS) fluorescence intensity of the protein significantly elevates with calcium in a dose dependent manner, suggesting an increase in calprotectin surface hydrophobicity upon calcium binding. Our study also indicates that calcium at higher concentrations (6, 8 and 10 mM) induces aggregation of human calprotectin. Our finding demonstrates that the starting time and the rate constant of calprotectin aggregation depend on the calcium concentration.