Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research II. Secondary Lichen Compounds

Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.     

Identification of bifidobacteria isolated from Asian elephant (Elephas maximus)

Bifidobacteria are considered as one of the key genera in intestinal tracts of animals, and their species composition vary depending on the host. The aim of this study was to identify faecal bifidobacteria from Asian elephants (Elephas maximus), housed in Zoological gardens (Ostrava, Czech Republic). Using culturing, bifidobacteria were found in counts 7.60?±?0.56 log CFU/g. Twenty-six pure strains were isolated from faeces of Asian elephant. The isolates were clustered into two groups according to fingerprinting profiles and fermentation characteristic. Bacteria were identified by a combination of MALDI-TOF MS, PCR methods and sequencing as B. boum (12 isolates) and B. adolescentis (14 isolates). Elephant strains showed different fingerprinting profiles than type and collection strains. Since these two species are frequently isolated from gastrointestinal tract of herbivores, they seem to be typical of animals fed plant diets.     

Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

STEPWISE EVOLUTION OF RESISTANCE TO TOXIC CARDENOLIDES VIA GENETIC SUBSTITUTIONS IN THE NA+/K+‐ATPASE OF MILKWEED BUTTERFLIES (LEPIDOPTERA: DANAINI)

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na⁺/K⁺‐ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so‐called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide‐containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na⁺/K⁺‐ATPase gene and by enzymatic assays with extracted Na⁺/K⁺‐ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom‐up selection by plant compounds and top‐down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.     

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Human dihydrolipoamide dehydrogenase (LADH, E3) is a component in the pyruvate-, alpha-ketoglutarate- and branched-chain ketoacid dehydrogenase complexes and in the glycine cleavage system. The pathogenic mutations of LADH cause severe metabolic disturbances, called E3 deficiency that often involve cardiological and neurological symptoms and premature death. Our laboratory has recently shown that some of the known pathogenic mutations augment the reactive oxygen species (ROS) generation capacity of LADH, which may contribute to the clinical presentations. A recent report concluded that elevated oxidative stress generated by the above mutants turns the lipoic acid cofactor on the E2 subunits dysfunctional. In the present contribution we generated by molecular dynamics (MD) simulation the conformation of LADH that is proposed to be compatible with ROS generation. We propose here for the first time the structural changes, which are likely to turn the physiological LADH conformation to its ROS-generating conformation. We also created nine of the pathogenic mutants of the ROS-generating conformation and again used MD simulation to detect structural changes that the mutations induced in this LADH conformation. We propose the structural changes that may lead to the modulation in ROS generation of LADH by the pathogenic mutations.     

Genetic differentiation of the Euglossini (Hymenoptera, Apidae) populations on a mainland coastal plain and an island in southeastern Brazil

Euglossini bees are among the main pollinators of plant species in tropical and subtropical forests in Central and South America. These bees are known as long-distance pollinators due to their exceptional flight performance. Here we assessed through microsatellite loci the gene variation and genetic differentiation between populations of four abundant Euglossini species populations sampled in two areas, Picinguaba (mainland) and Anchieta Island, Ubatuba, São Paulo State, southeastern Brazil. There was no significant genetic differentiation between the island and mainland samples of Euglossa cordata (Fst = 0.008, P = 0.60), Eulaema cingulata (Fst = 0.029, P = 0.29) and Eulaema nigrita (Fst = 0.062, P = 0.38), but a significant gene differentiation between mainland and island samples of Euglossa stellfeldi (Fst = 0.028, P = 0.016) was detected. As expected, our results showed that the water body that separates the island from the mainland does not constitute a geographic barrier for these Euglossini bees. The absence of populational structuring of three out the four species studied corroborates previous reports on those bees, characterized by large populations, with high gene diversity and gene flow and very low levels of diploid males. But the Eg. stellfeldi results clearly point that dispersal ability is not similar to all euglossine bees, what requires the development of different conservationist strategies to the Euglossini species.     

Pollen Harvest by Stingless Bee Foragers (Hymenoptera,Apidae, Meliponinae)

Data on pollen load capacity and flower constancy are discussed for nine stingless bee species. The foragers present high levels of flower constancy and often visit only one flower type (on average 97% of bee foragers), rarely a few flower types (on average 3% of bee foragers), during the same foraging trip. The latter foragers exhibit no tendencies for choosing similar sources, related either to flower type or to pollen type. Pollen load capacity (the ratio between pollen load weight/worker weight) decreases as forager body weight increases, so it is larger in smaller stingless bees species and smaller in larger ones. Nevertheless, it seems that specific load capacity also depends on pollen types.     

Cyanobacterial dominance in Brazil: distribution and environmental preferences

Based on a literature survey, we evaluated the periods of cyanobacterial dominance in Brazil. We hypothesized that variability of environmental forces along the country will promote or facilitate temporal and spatial mosaic in cyanobacterial dominance. The most striking outcomes are related to the dominance of Cylindrospermopsis, Dolichospermum, and Microcystis. Although they share important adaptive strategies (e.g., aerotopes, large size and toxins production), our findings suggest that they have different environmental preferences. Dolichospermum and Microcystis dominated mainly in warm-rainy periods whereas Cylindrospermopsis was more common during dry periods and in mixed systems, or formed perennial dominance. Maximum phosphorus concentrations were observed in reservoirs dominated by Cylindrospermopsis. Although the main genera reached high biomass levels individually, different abilities to form dominance and co-dominance were observed. The number of co-dominance of Chroococales and Nostocales was almost the same as the individual occurrence of the main genera from these groups. This dataset reveals patterns of dominance of these cyanobacteria and also indicates that physiological features will cause differences in the mechanisms of interactions between species. The understanding of these processes and their relationship to environmental conditions will promote better understanding of cyanobacterial dominance and increase our ability to predict and manage these events.