Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg^-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass^®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg^-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes’ DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes’ DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes’ embryo development impacting fertility of lactating dairy cows.     

Vegetation responses to past volcanic disturbances at the Araucaria araucana forest‐steppe ecotone in northern Patagonia

Volcanic eruptions play an important role in vegetation dynamics and its historical range of variability. However, large events are infrequent and eruptions with a significant imprint in today''s vegetation occurred far in the past, limiting our understanding of ecological processes. Volcanoes in southern Andes have been active during the last 10 ka and support unique ecosystems such as the Araucaria–Nothofagus forest. Araucaria is an endangered species, with a fragmented distribution and well‐adapted to fire and volcanic disturbances. Yet, it was suggested that volcanism might have increased the fragmentation. Through the use of pollen and tephra analysis from a sedimentary record, this paleoecological study aims to provide an insight into the vegetation responses to past volcanic disturbances, to assess the role of volcanic disturbance on the vegetation dynamics and to determine if the current fragmentation has been caused by volcanism. Results show that during the last 9 kyr, 39 tephra falls buried the vegetation around Lake Relem, more frequently between 4 and 2 ka. The pollen percentage indicates that the vegetation changed after small tephra fall but seldom caused significant changes. However, the large eruption of Sollipulli volcano (~3 ka) changed the environmental conditions affecting severely the vegetation. Ephedra dominated the early successional stage, perhaps facilitating Nothofagus recovering after ~500 years. Slight increase of Araucaria and Nothofagus obliqua‐type pollen percentages suggests that forest resisted without permanent changes and recovered relatively fast after the large eruption, perhaps because of sparse biological legacies distributed in the landscape. In the study area, the relative stability of Araucaria pollen after several tephra fall suggests no change in its past distribution at the current forest‐steppe ecotone, thus not affecting its current conservation status. Perhaps, random factors, the colonization patterns of the high elevations in the Andes after deglaciation and topography might play a more important role than previously thought.     

Induction of Cell Cycle Arrest and Apoptosis by Ruthenium Complex cis-(Dichloro)tetramineruthenium(III) Chloride in Human Lung Carcinoma Cells A549

Lung cancer is one of the leading causes of death in the world, and non-small cell lung carcinoma (NSCLC) accounts for approximately 75-85% of all lung cancers. In the present work, we studied the cytotoxic activity, cell cycle arrest and induction apoptosis of the compound cis-(dichloro)tetramineruthenium(III) chloride {cis-[RuCl(2)(NH(3))(4)]Cl} in human lung carcinoma tumor cell line A549. The results of MTT and trypan blue assays showed that cis-[RuCl(2)(NH(3))(4)]Cl causes reduction in the viability of A549 cells when treating with 95 and 383 μM of the compound for 48 and 72 h. Lower concentrations of the compound (19, 3.8 and 0.38 μM), however, only slightly affected cell viability. The IC(50) value for the compound was about 383 μM. Survival analysis of the A549 cells after treatment with ruthenium(III) compound using long term clonogenic assay showed that it reduced colony formation ability at concentrations of 0.38 and 3.8 μM, and at concentrations of 95 and 383 μM no colonies were observed. Cell cycle analysis showed that compound ruthenium led to an accumulation of A549 cells in S phase and increased in the sub-G1 peak. In addition, cis-(dichloro)tetramineruthenium(III) chloride treatment induced apoptosis, as observed by the increased numbers of annexin V-positive cells and increased messenger RNA expression of caspase-3.     

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8+ T-Cell Response: Reversal by Adenoviral Vaccine

MHC class Ia-restricted CD8⁺ T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8⁺ T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8⁺ T cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8⁺ T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8⁺ T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8⁺ cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8 T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8⁺ T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.     

Natural Regeneration in Plantations of Native Trees in Lowland Brazilian Atlantic Forest: Community Structure,Diversity, and Dispersal Syndromes

Plantations of native‐tree species are often recommended for ecological restoration, but the understanding of how these techniques catalyze natural ecological processes is limited. We investigated natural regeneration in five plantations of native trees in the Poço das Antas Biological Reserve (PABR) in the state of Rio de Janeiro, Brazil. The plantations were 9–11 years old, and contained 8–14 native‐tree species with different compositions and relative density of species. We analyzed floristic composition, structure (density and basal area) of overstory and understory strata, as well as other ecological attributes (dispersal syndromes, fruit or seed size, and the availability of fruit for frugivores). Zoochorous species comprised 77% of the community, with a prevalence of the two smallest size classes of propagules (< 0.6 and 0.6–1.6 cm) in natural regeneration. The density of zoochorous plants in the understory was positively correlated with their density in the overstory, indicating their influence on natural regeneration (r² = 0.36; p < 0.0002). Fruit availability for frugivores (density and richness of plants fruiting during the year) was also positively correlated with the density of stems in the understory. Therefore, attributes such as dispersal syndrome and fruiting season should be considered in selecting species to be planted. The differences in natural regeneration observed in each of the native‐tree plantations indicated that the performance of plantations as a restoration strategy may differ, depending on initial species composition, planting density, and site conditions.     

Emerging Threats and Opportunities for Large‐Scale Ecological Restoration in the Atlantic Forest of Brazil

Over the past 150 years, Brazil has played a pioneering role in developing environmental policies and pursuing forest conservation and ecological restoration of degraded ecosystems. In particular, the Brazilian Forest Act, first drafted in 1934, has been fundamental in reducing deforestation and engaging private land owners in forest restoration initiatives. At the time of writing (December 2010), however, a proposal for major revision of the Brazilian Forest Act is under intense debate in the National Assembly, and we are deeply concerned about the outcome. On the basis of the analysis of detailed vegetation and hydrographic maps, we estimate that the proposed changes may reduce the total amount of potential areas for restoration in the Atlantic Forest by approximately 6 million hectares. As a radically different policy model, we present the Atlantic Forest Restoration Pact (AFRP), which is a group of more than 160 members that represents one of the most important and ambitious ecological restoration programs in the world. The AFRP aims to restore 15 million hectares of degraded lands in the Brazilian Atlantic Forest biome by 2050 and increase the current forest cover of the biome from 17% to at least 30%. We argue that not only should Brazilian lawmakers refrain from revising the existing Forest Law, but also greatly step up investments in the science, business, and practice of ecological restoration throughout the country, including the Atlantic Forest. The AFRP provides a template that could be adapted to other forest biomes in Brazil and to other megadiversity countries around the world.     

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.     

Morphology and molecular taxonomy of Evlachovaea-like fungi,and the status of this unusual conidial genus

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.     

Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution

Root system architecture depends on nutrient availability, which shapes primary and lateral root development in a nutrient-specific manner. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis thaliana roots to iron (Fe). Relative to a homogeneous supply, localized Fe supply in horizontally separated agar plates doubled lateral root length without having a differential effect on lateral root number. In the Fe uptake-defective mutant iron-regulated transporter1 (irt1), lateral root development was severely repressed, but a requirement for IRT1 could be circumvented by Fe application to shoots, indicating that symplastic Fe triggered the local elongation of lateral roots. The Fe-stimulated emergence of lateral root primordia and root cell elongation depended on the rootward auxin stream and was accompanied by a higher activity of the auxin reporter DR5-β-glucuronidase in lateral root apices. A crucial role of the auxin transporter AUXIN RESISTANT1 (AUX1) in Fe-triggered lateral root elongation was indicated by Fe-responsive AUX1 promoter activities in lateral root apices and by the failure of the aux1-T mutant to elongate lateral roots into Fe-enriched agar patches. We conclude that a local symplastic Fe gradient in lateral roots upregulates AUX1 to accumulate auxin in lateral root apices as a prerequisite for lateral root elongation.     

Monte Carlo Study of Structural and Thermodynamic Properties of Liquid Chloroform Using a Five Site Model

A five site potential model combining Lennard–Jones plus Coulomb potential functions has been developed for chloroform molecule. The partial charges needed for Coulombic interactions were derived using the chelpg procedure implemented in the gaussian 92 program. These calculations were performed at the MP2 level with MC-311G* basis set for Cl and 6-311G** for C and H atoms. The parameters for the Lennard–Jones potentials were optimized to reproduce experimental values for the density and enthalpy of vaporization of the pure liquid at 298 K and 1 atm. The statistical mechanics calculations were performed with the Monte Carlo method in the isothermic and isobaric (NpT) ensemble. Besides the values obtained for density, ρ, and molar enthalpy of vaporization at constant pressure, Δ H_V, for liquid chloroform, results for molar volume, V_m, molar heat capacity, C_p, isobaric thermal expansivity, α_p, and isothermal compressibility, κ_T, for this pure liquid are also in very good agreement with experimental observations. Size effects on the values of thermodynamic properties were investigated. The potential model was also tested by computing the free energy for solvating one chloroform molecule into its own liquid at 298 K using a statistical perturbation approach. The result obtained compares well with the experimental value. Site–site pair correlation functions were calculated and are in good accordance with theoretical results available in the literature. Dipole–dipole correlation functions for the present five site model were also calculated at different carbon–carbon distances. These correlations were compared to those obtained using the four site model reported in the literature. An investigation of the solvent dependence of the relative free energy for cis/trans conversion of a hypothetical solute in TIP4P water and chloroform was accomplished. The results show strong interaction of water and chloroform molecules with the gauche conformer. The value obtained for the free energy barrier for cis/trans rotation in TIP4P water is higher than that for chloroform. This result is in agreement with the continuous theory for solvation as the conformer with higher dipole moment is more favoured by the solvent with higher dieletric constant. The results also show an increase in entropy as the solute goes from the cis to the trans geometry and this result is more appreciable in the aqueous solution. This revised version was published online in June 2006 with corrections to the Cover Date.