Novel RasGRF1-derived Tat-fused peptides inhibiting Ras-dependent proliferation and migration in mouse and human cancer cells

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.     

Impaired Temperature Stress Response of a Streptococcus thermophilus deoD Mutant

An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20°C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.     

Agrometeorological parameters for prediction of the maturation period of Arabica coffee cultivars

The objective of this study was to determine the harvest period of coffee fruits based on the relationship between agrometeorological parameters and sucrose accumulation in the seeds. Over the crop years 2004/2005 and 2006/2007, from 150 days after flowering (DAF) onwards, samples of 50 fruits of cultivars Mundo Novo IAC 376-4, Obat? IAC 1669-20 and Catuaí Vermelho IAC 144 were collected from coffee trees located in Campinas, Brazil. The endosperm of the fruits was freeze-dried, ground and analyzed for sucrose content by high-performance liquid chromatography. A weather station provided data to calculate the accumulated growing degree-day (GDD) units, and the reference (ET(o)) and actual (ET(r)) evapotranspiration rates. The results showed that the highest rates of sucrose accumulation occurred at the transition from the cane-green to the cherry phenological stage. Models for the estimation of sucrose content during maturation based on meteorological variables exhibited similar or better performance than the DAF variable, with better results for the variables GDD and ET(o). The Mundo Novo cultivar reached the highest sucrose level in the endosperm after 2,790 GDD, while cultivar Catuaí attained its maximum sucrose concentration after the accumulated evapotranspiration rate has reached a value of 870 mm. As for cultivar Obat?, the maximum sucrose concentration was predicted with the same degree of accuracy using any of the parameters investigated. For the Obat? cultivar, the values of the variables calculated for the maximum sucrose concentration to be reached were 249 DAF, 3,090 GDD, 1,020 ET(o) and 900 ET(r).     

Impaired temperature stress response of a Streptococcus thermophilus deoD mutant

An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20 degrees C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.     

Spatial variability of leaf wetness duration in different crop canopies

The spatial variability of leaf wetness duration (LWD) was evaluated in four different height-structure crop canopies: apple, coffee, maize, and grape. LWD measurements were made using painted flat plate, printed-circuit wetness sensors deployed in different positions above and inside the crops, with inclination angles ranging from 30 to 45°. For apple trees, the sensors were installed in 12 east-west positions: 4 at each of the top (3.3 m), middle (2.1 m), and bottom (1.1 m) levels. For young coffee plants (80 cm tall), four sensors were installed close to the leaves at heights of 20, 40, 60, and 80 cm. For the maize and grape crops, LWD sensors were installed in two positions, one just below the canopy top and another inside the canopy. Adjacent to each experiment, LWD was measured above nearby mowed turfgrass with the same kind of flat plate sensor, deployed at 30 cm and between 30 and 45°. We found average LWD varied by canopy position for apple and maize (P<0.05). In these cases, LWD was longer at the top, particularly when dew was the source of wetness. For grapes, cultivated in a hedgerow system and for young coffee plants, average LWD did not differ between the top and inside the canopy. The comparison by geometric mean regression analysis between crop and turfgrass LWD measurements showed that sensors at 30 cm over turfgrass provided quite accurate estimates of LWD at the top of the crops, despite large differences in crop height and structure, but poorer estimates for wetness within leaf canopies.     

The Influence of Catalysis on Mad2 Activation Dynamics

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process.     

The isolated catalytic hairpin of the Ras-specific guanine nucleotide exchange factor Cdc25Mm retains nucleotide dissociation activity but has impaired nucleotide exchange activity

Cdc25Mm is a mammalian Ras-specific guanine nucleotide exchange factor (GEF). By homology modeling we show that it shares with Sos-GEF the structure of the putative catalytic HI hairpin where the dominant negative T1184E mutation is located. Similarly to Cdc25MmT1184E, the isolated wild-type and mutant hairpins retain the ability to displace Ras-bound nucleotide, originate a stable Ras/GEF complex and downregulate the Ras pathway in vivo. These results indicate that nucleotide re-entry and Ras/GEF dissociation--final steps in the GEF catalytic cycle--require GEF regions different from the HI hairpin. GEF down-sizing could lead to development of novel Ras inhibitors.     

Climate change and the ecophysiology of Bertholletia excelsa seedlings

Plant Growth Regulation - The Bertholletia excelsa a species native to the Amazon region of great national and international interest, mainly for the nutritional benefits of its seeds. However,...     

Polar Constituents,Essential Oil and Antioxidant Activity of Marsh Woundwort (Stachys palustris L.)

Stachys palustris, well‐known as marsh woundwort, is a perennial herb growing in wet environments of Europe. Its tubers, leaves and seeds are eaten raw or cooked. Alike other Stachys species, the plant is also used as a traditional remedy. Despite S. palustris has been consumed for centuries, little is known about its chemical constituents. In this work the main secondary metabolites of S. palustris from Hungary and France have been analysed. From the plant ethanolic extracts, ethanoid glucosides, isoscutellarein derivatives, caffeoyl‐quinic acids and iridoids have been isolated and structurally characterized by NMR. The essential oils were analysed by GC/MS and showed (E)‐phytol, fatty acids and carbonylic compounds as the most abundant compounds. The radical scavenging capacity of plant ethanolic extracts, as evaluated by the DPPH assay, was noteworthy, with IC₅₀ values of 92.08 – 105.42 μg/ml.     

Simple agrometeorological models for estimating Guineagrass yield in Southeast Brazil

The objective of this work was to develop and evaluate agrometeorological models to simulate the production of Guineagrass. For this purpose, we used forage yield from 54 growing periods between December 2004–January 2007 and April 2010–March 2012 in irrigated and non-irrigated pastures in São Carlos, São Paulo state, Brazil (latitude 21°57′42″ S, longitude 47°50′28″ W and altitude 860 m). Initially we performed linear regressions between the agrometeorological variables and the average dry matter accumulation rate for irrigated conditions. Then we determined the effect of soil water availability on the relative forage yield considering irrigated and non-irrigated pastures, by means of segmented linear regression among water balance and relative production variables (dry matter accumulation rates with and without irrigation). The models generated were evaluated with independent data related to 21 growing periods without irrigation in the same location, from eight growing periods in 2000 and 13 growing periods between December 2004–January 2007 and April 2010–March 2012. The results obtained show the satisfactory predictive capacity of the agrometeorological models under irrigated conditions based on univariate regression (mean temperature, minimum temperature and potential evapotranspiration or degreedays) or multivariate regression. The response of irrigation on production was well correlated with the climatological water balance variables (ratio between actual and potential evapotranspiration or between actual and maximum soil water storage). The models that performed best for estimating Guineagrass yield without irrigation were based on minimum temperature corrected by relative soil water storage, determined by the ratio between the actual soil water storage and the soil water holding capacity.irrigation in the same location, in 2000, 2010 and 2011. The results obtained show the satisfactory predictive capacity of the agrometeorological models under irrigated conditions based on univariate regression (mean temperature, potential evapotranspiration or degree-days) or multivariate regression. The response of irrigation on production was well correlated with the climatological water balance variables (ratio between actual and potential evapotranspiration or between actual and maximum soil water storage). The models that performed best for estimating Guineagrass yield without irrigation were based on degree-days corrected by the water deficit factor.