Use of degenerate primers and touchdown PCR for construction of cDNA libraries

Optimized construction of low-redundancy cDNA mini-libraries using low-stringency RT-PCR is described cDNAs are generated using arbitrary consensus-degenerate hybrid oligonucleotide primers and nanogram amounts of Schistosoma mansoni mRNA. A number of conditions such as temperature and salt concentration are combined to create balanced, low-stringency conditions that permit a normalized amplification of a diversified, random set of sequences. On average, 350 different sequences are obtained per mini-library, which represents a significantly higher diversity of messages per library when compared to previously published conditions (ie., 20-40 sequences/ mini-library). The optimized high-throughput approach described here is likely to help in the discovery of expressed genes in any complex organism.     

An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Characterization of pregnancy-associated glycoproteins extracted from zebu (Bos indicus) placentas removed at different gestational periods

In the present work, two biochemical approaches were used to characterize PAGs isolated from Bos indicus fetal cotyledons removed at different gestational ages. The first procedure included acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies and the second included pepstatin-agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western blotting, before transfer to a polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. Use SDS-PAGE and Western blotting, different isoforms of PAG with apparent molecular masses of 51 to 69 kDa and isoelectric points varying from 4.4 to 6.7 were identified in the placentas from different gestational ages. N-terminal microsequencing (10 to 25 aa long) indicates the expression of one single terminal amino acid sequence in the Bos indicus placenta, which is 100% identical to the bovine PAG-1.     

Animal movement in the absence of predation: environmental drivers of movement strategies in a partial migration system

Animal movement strategies including migration, dispersal, nomadism, and residency are shaped by broad‐scale spatial‐temporal structuring of the environment, including factors such as the degrees of spatial variation, seasonality and inter‐annual predictability. Animal movement strategies, in turn, interact with the characteristics of individuals and the local distribution of resources to determine local patterns of resource selection with complex and poorly understood implications for animal fitness. Here we present a multi‐scale investigation of animal movement strategies and resource selection. We consider the degree to which spatial variation, seasonality, and inter‐annual predictability in resources drive migration patterns among different taxa and how movement strategies in turn shape local resource selection patterns. We focus on adult Galapagos giant tortoises Chelonoidis spp. as a model system since they display many movement strategies and evolved in the absence of predators of adults. Specifically, our analysis is based on 63 individuals among four taxa tracked on three islands over six years and almost 10⁶ tortoise re‐locations. Tortoises displayed a continuum of movement strategies from migration to sedentarism that were linked to the spatio‐temporal scale and predictability of resource distributions. Movement strategies shaped patterns of resource selection. Specifically, migratory individuals displayed stronger selection toward areas where resources were more predictable among years than did non‐migratory individuals, which indicates a selective advantage for migrants in seasonally structured, more predictable environments. Our analytical framework combines large‐scale predictions for movement strategies, based on environmental structuring, with finer‐scale analysis of space‐use. Integrating different organizational levels of analysis provides a deeper understanding of the eco‐evolutionary dynamics at play in the emergence and maintenance of migration and the critical role of resource predictability. Our results highlight that assessing the potential benefits of differential behavioral responses first requires an understanding of the interactions among movement strategies, resource selection and individual characteristics.     

Dispersal of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in an urban endemic dengue area in the State of Rio de Janeiro,Brazil

Experimental releases of female Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus were performed in August and September 1999, in an urban area of Nova Igua u, State of Rio de Janeiro, Brazil, to estimate their flight range in a circular area of 1,600 m where 1,472 ovitraps were set. Releases of 3,055 Ae. aegypti and 2,225 Ae. albopictus females, fed with rubidium (Rb)-marked blood and surgically prevented from subsequent blood-feeding, were separated by 11 days. Rb was detected in ovitrap-collected eggs by atomic emission spectrophotometry. Rb-marked eggs of both species were detected up to 800 m from the release point. Eggs of Ae. albopictus were more numerous and more heterogeneously distributed in the area than those of Ae. aegypti. Eggs positively marked for Rb were found at all borders of the study area, suggesting that egg laying also occurred beyond these limits. Results from this study suggest that females can fly at least 800 m in 6 days and, if infected, potentially spread virus rapidly.     

Resource-related variables drive individual variation in flowering phenology and mediate population-level flowering responses to climate in an asynchronously reproducing palm

Many tropical plant species show wide intra-population variation in reproductive timing, resulting in the protracted presence of flowering and fruiting individuals. Various eco-evolutionary drivers have been proposed as ultimate causes for asynchronous phenology, yet little is known about the proximate factors that control reproductive onset among individuals or that influence the proportion of trees producing new inflorescences within a population. We employed a nine-year phenological record from 178 individuals of the hyperdominant, asynchronously flowering canopy palm, Oenocarpus bataua (Arecaceae)¸ to assess whether resource-related variables influence individual- and population-level flowering phenology. Among individuals, access to sunlight increased rates of inflorescence production, while the presence of resource sinks related to current investment in reproduction—developing infructescences—reduced the probability of producing new inflorescences. At the population level, climate anomalies induced by El Niño Southern Oscillation (ENSO) affected the proportion of the population producing inflorescences through time. Moreover, the effects of ENSO anomalies on flowering patterns depended on the prevalence of developing infructescences in the population, with stronger effects in periods of low developing-infructescence frequency. Taken together, these results suggest that resource-related variables can drive phenological differences among individuals and mediate population-level responses to larger-scale variables, such as climate anomalies. Consequently, a greater focus on the role of resource levels as endogenous cues for reproduction might help explain the frequent aseasonal phenological patterns observed among tropical plants, particularly those showing high intra-population asynchrony.     

Stem Mortality Following Fire in Kalahari Sand Vegetation: Effects of Frost,Prior Damage,and Tree Neighbourhoods

Fire is a key factor affecting the survivorship and dynamics of woody plants in savannas, but few empirical studies in savanna vegetation have investigated correlates of mortality following fire at the level of individual stems. A study of stem mortality as a function of size, neighbourhood effects, and prior damage (mainly caused by elephants) was undertaken in an area of Kalahari sand vegetation in western Zimbabwe. Stem and whole-plant mortality were quantified for the dominant stems of 557 trees in 4 plots following dry season unplanned fires in 2001. Two plots were located in areas that had been affected by frost earlier in the season, and 2 in areas that had not. Mortality was also recorded for 1762 trees in 20 unburned reference plots, also classified according to the presence of frost damage. Mortality estimators were constructed with a maximum likelihood regression method. Whole-plant mortality was low (on the order of 1–2%) compared with stem mortality, which in burned plots approximated 100% for the smallest size classes and declined as a function of stem diameter. Fire-driven mortality was lower in stems protected by the crowns of larger trees than in stems that were in the open. There was also evidence to suggest that the effects of fire are exacerbated by the prior action of frost and elephant herbivory.     

Benthic community metabolism and trophic conditions of four South American lakes

Oxygen uptake rates of undisturbed sediment columns have been used as an integrative measure of the metabolic activities of benthic communities. Since the intensity of metabolic processes of profundal lake is dependent on the production of organic matter in the pelagic zone, oxygen uptake rates reflect the trophic condition of the whole lake. Four small lakes of central Chile, differing strongly in trophic conditions, provided a possibility to compare benthic oxygen uptake rates, under different oxygen conditions (Quiñenco, Grande, Chica and Lleulleu). Our objective was to establish the relationship between the oxygen uptake rates and bottom characteristics of lakes with different trophic conditions. At 8 mg O₂ l^-1 in the overlying water of the cores studied, the oxygen uptake rates of the sediment were: Quiñenco 51.2–56.0 mg O₂ m² h^-1 (eutrophic), Grande 41.2–46.4 mg O₂ m² h^-1 (mesotrophic), Chica 23.2–18.1 mg O₂ m² h^-1 (mesotrophic) and Lleulleu 11.7–16.0 mg O₂ m² h^-1 (oligotrophic). By exposing the sediments to different oxygen levels in the laboratory, it was found that benthic community metabolism decreased with oxygen concentrations. The slope of regression lines, relating oxygen uptake rates to oxygen concentrations, differed for the different sites investigated, closely related with the trophic conditions of the lakes. It was positively correlated with the organic matter content of the sediment of the cores (r ²= 0.78, p<0,05) and the nutrients of the bottom waters (total-P: r ²= 0.73, p<0,05; total-N: r ²= 0.73, p<0,05), and negatively with the redox potential of the sediments (r ²= 0.88, p<0,05).     

Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A-TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A-TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation.