Energy landscape of chelated uranyl: antibody interactions by dynamic force spectroscopy

We used dynamic force spectroscopy (DFS) to explore the energy landscape of interactions between a chelated uranyl compound and a monoclonal antibody raised against the uranyl-dicarboxy-phenanthroline complex. We estimated the potential energy barrier widths and the relevant thermodynamic rate constants along the dissociation coordinate. Using atomic force microscopy, four different experimental setups with or without the uranyl ion in the chelate ligand, we have distinguished specific and nonspecific binding in the binding affinity of the uranyl compound to the antibody. The force loading rates for our system were measured from 15 to 26,400 pN/s. The results showed two regimes in the plot of the most probable unbinding force versus the logarithm of the loading rate, revealing the presence of two (at least) activation barriers. Analyses of DFS suggest parallel multivalent binding present in either regime. We have also built a molecular model for the variable fragment of the antibody and used computational graphics to dock the chelated uranyl ion into the binding pocket. The structural analysis led us to hypothesize that the two regimes originate from two interaction modes: the first one corresponds to an energy barrier with a very narrow width of 0.5 +/- 0.2 A, inferring dissociation of the uranyl ion from its first coordination shell (Asp residue); the second one with a broader energy barrier width (3.9 +/- 0.3 A) infers the entire chelate compound dissociated from the antibody. Our study highlights the sensitivity of DFS experiments to dissect protein-metal compound interactions.     

Efficacy of moclobemide in a rat model of neurotoxicant-induced edema

The potent antidepressant effect of moclobemide, a selective and reversible type A monoamine oxidase (MAO) inhibitor, is clinically established. In view of the ongoing debate on the neuroprotective properties of MAO inhibitors, the present study was undertaken to further define the protective effect of moclobemide in a rat model of neurotoxicant-induced edema. In this model, daily oral triethyltin (TET) administration for 5 consecutive days strongly perturbed the rat behaviour and induced a cerebral edema at the 5th day. Oral coadministration of moclobemide (2 x 100 mg.kg-1.day-1) with TET blocked the development of brain edema and the increase in the cerebral chloride content induced by TET. Moreover, moclobemide reduced the increase in the cerebral sodium content and attenuated the neurological deficit. In conclusion, moclobemide possesses potent protective properties in this rat model of cerebral edema, suggesting potential clinical utility as a neuroprotectant.     

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.     

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.     

Two measurement methods of leaf dry matter content produce similar results in a broad range of species

BACKGROUND AND AIMS: Leaf dry matter content (LDMC) is widely used as an indicator of plant resource use in plant functional trait databases. Two main methods have been proposed to measure LDMC, which basically differ in the rehydration procedure to which leaves are subjected after harvesting. These are the 'complete rehydration' protocol of Garnier et al. (2001, Functional Ecology 15: 688-695) and the 'partial rehydration' protocol of Vendramini et al. (2002, New Phytologist 154: 147-157). METHODS: To test differences in LDMC due to the use of different methods, LDMC was measured on 51 native and cultivated species representing a wide range of plant families and growth forms from central-western Argentina, following the complete rehydration and partial rehydration protocols. KEY RESULTS AND CONCLUSIONS: The LDMC values obtained by both methods were strongly and positively correlated, clearly showing that LDMC is highly conserved between the two procedures. These trends were not altered by the exclusion of plants with non-laminar leaves. Although the complete rehydration method is the safest to measure LDMC, the partial rehydration procedure produces similar results and is faster. It therefore appears as an acceptable option for those situations in which the complete rehydration method cannot be applied. Two notes of caution are given for cases in which different datasets are compared or combined: (1) the discrepancy between the two rehydration protocols is greatest in the case of high-LDMC (succulent or tender) leaves; (2) the results suggest that, when comparing many studies across unrelated datasets, differences in the measurement protocol may be less important than differences among seasons, years and the quality of local habitats.     

Resource availability regulates reproductive meristem activity, development of reproductive structures and seed set in buckwheat (Fagopyrum esculentum)

Grain yields in buckwheat ( Fagopyrum esculentum ) are consistently low despite a profuse and long-lasting flowering. The mechanisms underlying this reproductive failure are still largely unknown. In this study, performed in strictly controlled conditions, the possible implication of assimilate availability in the regulation of the reproductive development was investigated in the 'La Harpe' cultivar, by manipulating sink/source relationships through excisions of selected leaves and/or selected inflorescences and hand pollinations of selected and precisely identified flowers. Increasing or decreasing source/sink organ ratio in buckwheat, respectively, enhanced or reduced the production of racemes by the shoot apical meristem and of cymes by the racemes, ultimately modulating the number of flowers per plant. The effect on seed production was essentially indirect and related to flower production. Seeds developing on a plant also affected reproductive morphogenesis, limiting flower production. Three stages at which flower development to mature seed may fail have been identified. First, an abnormal morphogenesis resulted in flowers with an undersized and sterile gynoecium. Secondly, after anthesis, numerous flowers with a normal sized gynoecium, were unable to develop a seed after hand pollination. Finally, a small proportion of flowers exhibited a later abortion of the seed. Flower fate is dependent on position in the raceme and time of opening: chance to develop normally and produce a ripe seed is higher for flowers at the base than at the top of the inflorescence. A mechanism, internal to the raceme, regulates flower fate, independently of assimilate availability. This mechanism may be disturbed by drastic reductions in nutrient supply.     

Let the concept of trait be functional!

In its simplest definition, a trait is a surrogate of organismal performance, and this meaning of the term has been used by evolutionists for a long time. Over the last three decades, developments in community and ecosystem ecology have forced the concept of trait beyond these original boundaries, and trait-based approaches are now widely used in studies ranging from the level of organisms to that of ecosystems. Despite some attempts to fix the terminology, especially in plant ecology, there is currently a high degree of confusion in the use, not only of the term "trait" itself, but also in the underlying concepts it refers to. We therefore give an unambiguous definition of plant trait, with a particular emphasis on functional trait. A hierarchical perspective is proposed, extending the "performance paradigm" to plant ecology. "Functional traits" are defined as morpho-physio-phenological traits which impact fitness indirectly via their effects on growth, reproduction and survival, the three components of individual performance. We finally present an integrative framework explaining how changes in trait values due to environmental variations are translated into organismal performance, and how these changes may influence processes at higher organizational levels. We argue that this can be achieved by developing "integration functions" which can be grouped into functional response (community level) and effect (ecosystem level) algorithms.     

Listeria Phage and Phage Tail Induction Triggered by Components of Bacterial Growth Media (Phosphate,LiCl, Nalidixic Acid,and Acriflavine)

The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na₂HPO₄ at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na₂HPO₄ and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.     

Combined effects of nutrients and temperature on the production of fermentative aromas by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> during wine fermentation

Volatile compounds produced by yeast during fermentation greatly influence the organoleptic qualities of wine. We developed a model to predict the combined effects of initial nitrogen and phytosterol content and fermentation temperature on the production of volatile compounds. We used a Box–Behnken design and response surface modeling to study the response of Lalvin EC1118® to these environmental conditions. Initial nitrogen content had the greatest influence on most compounds; however, there were differences in the value of fermentation parameters required for the maximal production of the various compounds. Fermentation parameters affected differently the production of isobutanol and isoamyl alcohol, although their synthesis involve the same enzymes and intermediate. We found differences in regulation of the synthesis of acetates of higher alcohols and ethyl esters, suggesting that fatty acid availability is the main factor influencing the synthesis of ethyl esters whereas the production of acetates depends on the activity of alcohol acetyltransferases. We also evaluated the effect of temperature on the total production of three esters by determining gas–liquid balances. Evaporation largely accounted for the effect of temperature on the accumulation of esters in liquid. Nonetheless, the metabolism of isoamyl acetate and ethyl octanoate was significantly affected by this parameter. We extended this study to other strains. Environmental parameters had a similar effect on aroma production in most strains. Nevertheless, the regulation of the synthesis of fermentative aromas was atypical in two strains: Lalvin K1M® and Affinity? ECA5, which produces a high amount of aromatic compounds and was obtained by experimental evolution.