Extraction and characterization of soluble protein fractions from Phaseolus lunatus L seeds

Legume proteins as a potential source of valuable nutrients, are the object of several studies in order to obtain the best use. A basic knowledge becomes more important for those proteins from species not wholly utilized, before using them as food ingredients. The objective of this work was to determine several structural and nutritional characteristics of the protein fractions from Phaseolus lunatus, separated in different solvents. The relative amount of extraction for the albumins (ALB), globulins (GLB), prolamines (PRL), and glutelins (GLT) was 62.3, 34.8, 1.4 and 1.5%, respectively. The SDS-PAGE electrophoretic profile of both ALB and GLB, showed seven common bands in intervals from 10 to 95 kDa, and 14 to 99 kDa, respectively; the amino acids profile showed that PRL was the rich fraction in sulfurated amino acids (11.5 g/100 g protein); the content of lysine in the fraction of ALB was smaller than expected but the requirement of the FAO in the fractions of GLB and GLT was covered. In general, the fraction of GLB had the best balance of amino acids and digestibility (80%); however, it had a relationship of calculated protein efficiency ratio (C-PER) of 0.11, smaller than the ratio in ALB (0.97). The calorimetric analysis showed denatured temperatures around 90 degrees C for the ALB, GLB, and GLU fractions. The PRL fraction probably did not present a thermal transition because the proteins were denaturalized by the extraction conditions.     

Detection and Discrimination of Conspecific Scents by the Anguid Slow-Worm Anguis fragilis

We tested the ability of male slow-worms, Anguis fragilis, a limbless anguid lizard with secretive, semifossorial habits, to detect chemical associated with conspecifics by using a T-maze in the laboratory. Male slow-worms discriminated conspecific male and female scent deposits. Males selected the arm with female scent, suggesting that scent deposits may be used to locate potential mates. Also, male slow-worms did not avoid the chemicals of other males, suggesting that they are not territorial. However, males discriminated their own scent from those of other males, and spent more time exploring the arm with other male scent, which suggests that scent marks may bear information that could be used in future intrasexual social contexts. We conclude that discrimination of conspecifics based on scents may be more widespread than previously expected among lizards inhabiting visually restricted environments.     

Structural Diversity and Defensive Properties of Norditerpenoid Alkaloids

We have tested the insect antifeedant and toxic activity of 43 norditerpenoid alkaloids on Spodoptera littoralis and Leptinotarsa decemlineata including eserine (physostigmine), anabasine, and atropine. Antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedants to L. decemlineata were 1,14-diacetylcardiopetaline (9) and 18-hydroxy- 14-O-methylgadesine (33), followed by 8-O-methylconsolarine (12), 14-O-acetyldelectinine (27), karakoline (7), cardiopetaline (8), 18-O-demethylpubescenine (13), 14-O-acetyldeltatsine (18), takaosamine (21), ajadine (24), and 8-O-methylcolumbianine (6) (EC50 < 1 microg/cm2). This insect showed a moderate response to atropine. S. littoralis had the strongest antifeedant response to 24, 18, 14-O-acetyldelcosine (19), and delphatine (29) (EC50 < 3 microg/cm2). None of the model substances affected the feeding behavior of this insect. The most toxic compound to L. decemlineata was aconitine (1), followed by cardiopetalidine (10) (% mortality > 60), 14-deacetylpubescenine (14), 18-O-benzoyl-18-O-demethyl-14-O-deacetylpubescenine (17), 14-O-acetyldelcosine (19), 14-deacetylajadine (25) and methyllycaconitine (30) (% mortality > 45). Orally injected S. littoralis larvae were negatively affected by 1, cardiopetaline (8), 10, 1,14-O-acetylcardiopetalidina (11), 12, 14, 1,18-O-diacetyl-19-oxo-gigactonine (41), olivimine (43), and eserine in varying degrees. Their antifeedant or insecticidal potencies did not parallel their reported nAChR binding activity, but did correlate with the agonist/antagonist insecticidal/antifeedant model proposed for nicotininc insecticides. A few compounds [14, tuguaconitine (38), 14-demethyldelboxine (40), 19, dehydrodelsoline (36), 18-O-demethylpubescenine (13), 41, 9, and delcosine (23)] had selective cytotoxic effects to ward insect-derived Sf9 cells. None were cytotoxic to mammalian CHO cells and none increased Trypanosoma cruzi mortality. The selective cytotoxic effects of some structures indicate that they can act on biological targets other than neuroreceptors.     

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.     

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.     

Simulation of electron energy loss near-edge structure at the Al and N K edges and Al L(23) edge in cubic aluminium nitride

Calculations of electron energy near edge structures (ELNES) are compared with experimental data obtained in a high-resolution transmission electron microscope. This study concerns small precipitates of aluminium nitride in low carbon steel. The ELNES technique allows to clearly establish that these precipitates crystallize in a cubic rather than in a hexagonal crystallographic cell. The influence on simulated spectra of different parameters are investigated: the size of the atomic shell and its relation with the electron inelastic mean free path. We also examine the influence of the core hole and the sensitivity to cell parameters. We particularly examine the Al L(23) near edge structure and features relating to the different transition channels (A(1g), E(g) and T(2g)). Results of a multiple scattering and band structure calculations using ICXANES and WIEN97 codes, respectively, are compared in the region from 0 to 30 eV above the edge onsets. Both calculations are in a rather good agreement.     

Thermal AFM: a thermopile case study

In this work, an atomic force microscope (AFM) with an integrated thermal sensor has been used to obtain the local spatial distribution of temperatures in a micromachined thermopile with submicron resolution. In this communication, we will show how the dimensional, structural and functional characteristics of a thermopile suits well with the requirements for AFM thermal imaging, and how a deeper insight of the thermopile operation can be gained with the aid of these advanced scanning probe-based tools.     

An analytic method to predict the thermal map of cryosurgery iceballs in MR images

This paper presents a newly developed method to estimate, in magnetic resonance (MR) images, the temperatures reached within the volume of an iceball produced by a cryogenic probe. Building on the direct measurements of the MR signal intensity and its correlation with independent temperature variations at the phase transition from liquid to solid, the thermal information embedded in the images was accessed. The volume and diameter of the growing iceball were estimated from a time series of MR images. Using regressions over the volume in the time and thermal domains, this method predicted the cryogenic temperatures beyond the range of sensitivity of the MR signal itself. We present a validation of this method in samples of gelatin and ex vivo pig liver. Temperature predictions are shown to agree with independent thermosensor readings over a range extending from 20 degrees C down to -65 degrees C, with an average error of less than 6 degrees C.