Cytotoxic and antiproliferative effects induced by a non terpenoid polar extract of A. indica seeds on 3T6 murine fibroblasts in culture

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. Its composition is very complex and the oil exhibits a number of biological activities. The most studied component is the terpenoid azadirachtin which is used for its insecticidal and putative antimicrobial properties. In this report we investigate the biological activity of partially purified components of the oil obtained from A. indica. We show that the semi-purified fractions have moderate to strong cytotoxicity. However, this is not attributable to azadirachtin but to other active compounds present in the mixture. Each fraction was further purified by appropriate extraction procedures and we observed a differential cytotoxicity in the various sub-fractions. This led us to investigate the mode of cell death. After treatment with the oil fractions we observed positivity to TUNEL staining and extensive internucleosomal DNA degradation both indicating apoptotic death. The anti-proliferative properties of the neem oil-derived compounds were also assayed by evaluation of the nuclear PCNA levels (Proliferating Cell Nuclear Antigen). PCNA is significantly reduced in cells treated with a specific fraction of neem oil. Finally, our results strongly suggest a possible involvement of the mitochondrial pathway in the apoptotic death.     

Patterns of endemism in south-eastern Pacific benthic polychaetes of the Chilean coast

Aim In this study we evaluate patterns of endemism for benthic polychaete species along the southeastern Pacific coast of Chile. Our goals were (1) to describe latitudinal gradients of endemism and identify areas of high endemism, (2) to evaluate the effect of biogeographical limits on endemism patterns, and (3) to evaluate indirectly the role played by evolutionary dynamics on patterns of endemism. Location South‐eastern Pacific coast of Chile, ranging from Arica (18° S) to Cape Horn (56° S). Methods We used a list of 178 species of endemic, shallow benthic polychaetes to evaluate patterns of endemism. Parsimony analysis of endemicity (PAE) and the endemism index (EI) were used to evaluate hierarchical relationships of endemism between different latitudinal bands, and to identify areas with high degrees of endemism and differences in endemism. We evaluated the effect of biogeographical limits on endemic polychaete fauna by testing for the existence of geometric constraints (mid‐domain effect). The role of evolutionary dynamics on latitudinal patterns of endemism was evaluated with nestedness analysis (NA) using the temperature index. Results The PAE analysis indicated two large, separate areas of endemism: (1) the northern area between 18° S and 38° S, and (2) the southern area between 39° S and 56° S. The endemism index showed a maximum value (32 species) around 39°–41° S. Species‐richness curves of each 3° band of latitude showed a clear mid‐domain effect (69%), but the two maximum points of species richness at mid‐latitudes (36° S to 38° S and 39° S to 41° S) did not correspond to the mid‐domain peak in species richness, presenting a greater number of species than expected by the mid‐domain effect. The nestedness analysis showed that the number of genera reaches a maximum of 70 at mid‐latitudes (36°–41° S), decreasing towards both the northern and southern areas. The spatial distribution of the entire data set of endemic species showed a nested pattern (T° = 24.5°, P < 0.0001). Main conclusions Our results strongly support the existence of a latitudinal gradient of endemism for benthic polychaete species along the Chilean coast. The shape of this gradient is clearly non‐linear, with a marked peak of endemism occurring at mid‐latitudes (36°–41° S, endemism hotspot), which also corresponds to a peak in species richness. Furthermore, this hotspot is the midpoint separating two distinct areas of endemism to the north and south. We suggest that the observed pattern of endemism for benthic polychaete taxa of the Chilean coast can be explained by a combination of geometric constraints and historical mechanisms, such as the processes that affected the Chilean coast during the Neogene (e.g. ENSO, oxygen minimum zone, glaciations).     

Effect of nucleotides on the oligomeric state of human lactoferrin

Lactoferrin (LF) is a multifunctional acute-phase protein involved in nonspecific defense against bacteria, viruses, and cancer diseases and is present in human barrier fluids, blood, and milk. Small-angle X-ray scattering (SAXS) and light scattering (LS) demonstrated for the first time that LF occurs in the form of oligomers, with a high monomer unit number in the solution. The degree of LF oligomerization depends on the LF concentration and the storage period of non-frozen neutral LF solutions. The average inertial radius of scattering particles (R _g) reaches 100–450 Å at LF concentrations comparable with those in human milk, while R _g of LF monomers is 26.7 Å. LF forms complexes with various nucleotides and hydrolyzes them. The addition of ATP or AMP to LF solutions accelerates LF oligomerization and increases R _g to 600–700 Å, regardless of the initial degree of LF oligomerization. According to the different models (sphere, plate, and cylinder) of LF aggregates, its complexes with such R _g presumably contain several tens to thousands of LF monomers. The possible role of oligomeric complexes in multiple biological functions of LF is discussed.     

Pollination processes and the Allee effect in highly fragmented populations: consequences for the mating system in urban environments

The urban environment was used to study the plant reproductive system in small fragmented populations as well as the potential adaptations of plants to urban conditions. We examined the effect of density on the pollination process and on reproduction in urban populations of the allogamous species Crepis sancta. The habitat is composed of small uncultivated square patches (c. 2 m2) regularly spaced along the pavement in streets of the city of Montpellier, France. Pollinator behaviour (the presence of pollinators, the number of flowers visited and the duration of each visit) and seed set as a function of the number of plants in patches and selfing rates, determined using progeny array analysis, were studied. The propensity for the urban populations to produce seeds by self-fertilization in insect-proof glasshouse was also analysed. We found strong evidence of reduced pollinator activities at low densities, resulting in reduced pollination and a reduction in seed set from 80 to 20% of ovules fertilized (the Allee effect). Progeny array analysis revealed a slight increase (marginally significant) in selfing rates in urban populations compared with large populations. In spite of lower pollinator activity, urban populations did not show a greater ability to self-fertilize compared with rural populations from the nearby countryside.     

Aquatic hyphomycete diversity and identity affect leaf litter decomposition in microcosms

We conducted a microcosm experiment with monocultures and all possible combinations of four aquatic hyphomycete species, Articulospora tetracladia, Flagellospora curta, Geniculospora grandis and Heliscus submersus, to examine the potential effects of species richness on three functional aspects: leaf litter decomposition (leaf mass loss), fungal production (ergosterol buildup) and reproductive effort (released spores). Both species richness and identity significantly affected fungal biomass and conidial production (number and biomass of released spores), whereas only species identity had a significant effect on leaf mass loss. In mixed cultures, all measures of fungal functions were greater than expected from the weighted performances of participating species in monoculture. Mixed cultures outperformed the most active monoculture for biomass accumulation but not for leaf mass loss and conidial production. The three examined aspects of aquatic hyphomycete activity tended to increase with species richness, and a complementary effect was unequivocally demonstrated for fungal biomass. Our results also suggest that specific traits of certain species may have a greater influence on ecosystem functioning than species number.     

Antiplatelet prenylflavonoids from Artocarpus communis

Four flavonoids, dihydroartomunoxanthone (1), artomunoisoxanthone (2), cyclocomunomethonol (3) and artomunoflavanone (4), together with three known compounds, artochamins B (5), D and artocommunol CC (6) were isolated from the cortex of the roots of Artocarpus communis. The structures of 1-4 were determined by spectroscopic methods. The antiplatelet effects of the flavonoids, 1-3, 5 and 6 on human platelet-rich plasma (PRP) were evaluated. Of the compounds tested in human PRP, compounds 1, 5 and 6 showed significant inhibition of secondary aggregation induced by adrenaline. It is concluded that the antiplatelet effect of 1, 5 and 6 is mainly owing to an inhibitory effect on thromboxane formation.     

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus)

The transition state of adenosine nucleosidase (EC 3.2.2.7) isolated from yellow lupin (Lupinus luteus) was determined based upon a series of heavy atom kinetic isotope effects. Adenosine labeled with 13C, 2H, and 15N was analyzed by liquid chromatography/electrospray mass spectrometry to determine kinetic isotope effects. Values of 1.024+/-0.004, 1.121+/-0.005, 1.093+/-0.004, 0.993+/-0.006, and 1.028+/-0.005 were found for [1'-13C], [1'-2H], [2'-2H], [5'-2H], and [9-15N] adenosine, respectively. Using a bond order bond energy vibrational analysis, a transition state consisting of a significantly broken C-N bond, formation of an oxocarbenium ion in the ribose ring, a conformation of C3-exo for the ribose ring, and protonation of the heterocyclic base was proposed. This transition state was found to be very similar to the transition state for nucleoside hydrolase, another purine metabolizing enzyme, isolated from Crithidia fasciculata.     

A spatially explicit model for an Allee effect: why wolves recolonize so slowly in Greater Yellowstone

A reduced probability of finding mates at low densities is a frequently hypothesized mechanism for a component Allee effect. At low densities dispersers are less likely to find mates and establish new breeding units. However, many mathematical models for an Allee effect do not make a distinction between breeding group establishment and subsequent population growth. Our objective is to derive a spatially explicit mathematical model, where dispersers have a reduced probability of finding mates at low densities, and parameterize the model for wolf recolonization in the Greater Yellowstone Ecosystem (GYE). In this model, only the probability of establishing new breeding units is influenced by the reduced probability of finding mates at low densities. We analytically and numerically solve the model to determine the effect of a decreased probability in finding mates at low densities on population spread rate and density. Our results suggest that a reduced probability of finding mates at low densities may slow recolonization rate.     

A mixed strategy model for the emergence and intensification of social learning in a periodically changing natural environment

Based on a population genetic model of mixed strategies determined by alleles of small effect, we derive conditions for the evolution of social learning in an infinite-state environment that changes periodically over time. Each mixed strategy is defined by the probabilities that an organism will commit itself to individual learning, social learning, or innate behavior. We identify the convergent stable strategies (CSS) by a numerical adaptive dynamics method and then check the evolutionary stability (ESS) of these strategies. A strategy that is simultaneously a CSS and an ESS is called an attractive ESS (AESS). For certain parameter sets, a bifurcation diagram shows that the pure individual learning strategy is the unique AESS for short periods of environmental change, a mixed learning strategy is the unique AESS for intermediate periods, and a mixed learning strategy (with a relatively large social learning component) and the pure innate strategy are both AESS's for long periods. This result entails that, once social learning emerges during a transient era of intermediate environmental periodicity, a subsequent elongation of the period may result in the intensification of social learning, rather than a return to innate behavior.     

Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate–DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection.