Identifying candidate genes affecting developmental time in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>: pervasive pleiotropy and gene-by-environment interaction

Background  

Understanding the genetic architecture of ecologically relevant adaptive traits requires the contribution of developmental and evolutionary biology. The time to reach the age of reproduction is a complex life history trait commonly known as developmental time. In particular, in holometabolous insects that occupy ephemeral habitats, like fruit flies, the impact of developmental time on fitness is further exaggerated. The present work is one of the first systematic studies of the genetic basis of developmental time, in which we also evaluate the impact of environmental variation on the expression of the trait.     

Oriented covalent immobilization of antibodies on physically inert and hydrophilic support surfaces through their glycosidic chains

Immobilization of antibodies by their oxidized sugar chain on aminated supports is a very efficient methodology to have a properly oriented antibody. However, these supports may behave as anionic exchangers, producing the unspecific adsorption of other proteins and reducing the selectivity of the system. To overcome this problem, we have proposed two solutions based in tailor-made support surfaces to immobilize antihorseradish peroxidase (HRP). The first solution was the use of supports having a very low amount of amino groups. These amino groups need to be very reactive with the aldehyde groups generated in the protein sugar chains to be efficient. Using supports having 7 micromol EDA/g (e.g., ethylenediamine modified glyoxyl-agarose), the antibody may be immobilized, keeping over 90% of the anti-HRP functionality. Second, by mixing amino groups and carboxylic groups, a neutral surface of the support has been generated. Again, this support has been unable to adsorb proteins while oxidized anti-HRP could be immobilized, giving functional anti-HRP antibodies. Both preparations retained 100% functionality after 2 months of storage at 4 degrees C. This way, the tailoring of the support surfaces has permitted solving some limitations of the immobilization of sugar-chain oxidized antibodies on primary amino supports.     

Manipulating DNA probe presentation via enzymatic cleavage of diluent strands

We previously reported a system for the controlled redispersion of DNA-linked aggregates using secondary, competitive hybridization events and found that complete redispersion is contingent upon dilution of the active 20 base-long probe strands with 20 base-long nonhybridizing strands. Here, to reduce the steric interference of nonhybridizing or diluent strands on probe activity, we investigate the effect of shorter diluent strands on the hybridization activity of immobilized probes using the following two approaches: (1) simultaneously coupling shorter diluent strands and longer probe strands to microspheres and (2) simultaneously coupling diluent and probe strands of the same base length to microspheres and then clipping diluent strands with the restriction endonuclease AluI. Results indicate that one can reduce the duplex density down by 50-70% of its initial value, depending on the location of the recognition motif along the hybridization segment. In addition, tighter control over the number of probe-target duplexes is achieved with the enzyme-based approach.     

Simultaneous detection and quantification of the unculturable microbe Candidatus Glomeribacter gigasporarum inside its fungal host Gigaspora margarita

A combined approach based on quantitative and nested polymerase chain reaction (qPCR and nPCR, respectively) has been set up to detect and quantify the unculturable endobacterium Candidatus Glomeribacter gigasporarum inside the spores of its fungal host Gigaspora margarita. Four genes were targeted, two of bacterial origin (23S rRNA gene and rpoB) and two from the fungus (18S rRNA gene and EF1-alpha). The sensitivity of the qPCR protocol has proved to be comparable to that of nPCR, both for the fungal and the bacterial detection. It has been demonstrated that the last detected dilution in qPCR corresponded, in each case, to 10 copies of the target sequences, suggesting that the method is equally sensitive for the detection of both fungal and bacterial targets. As the two targeted bacterial genes are predicted to be in single copy, it can be concluded that the detection limit is of 10 bacterial genomes for each mixture. The protocol was then successfully applied to amplify fungal and bacterial DNA from auxiliary cells and extraradical and intraradical mycelium. For the first time qPCR has been applied to a complex biological system to detect and quantify fungal and bacterial components using single-copy genes, and to monitor the bacterial presence throughout the fungal life cycle.     

Potentiometric, spectrophotometric and calorimetric study on iron(III) and copper(II) complexes with 1,2-dimethyl-3-hydroxy-4-pyridinone

The iron(III)-1,2-dimethyl-3-hydroxy-4-pyridinone (Deferiprone) system is carefully characterized by a combined potentiometric-spectrophotometric procedure at 25 and 37 degrees C at different ionic strengths, and by thermochemical and quantum-chemical studies. The main purpose of this work was to determine how the temperature dependence of both complex-formation and protonation constants can affect the pFe values on going from 25 degrees C (pFe is normally calculated using 25 degrees C stability constants) to the physiological temperature of 37 degrees C at which chelating agents are active in vivo. The copper(II)-Deferiprone system is also studied and the iron(III)-Deferiprone distribution diagrams in presence of variable copper(II) amounts are shown so as to explain possible side effects due to a competing metal ion during the chelating therapy of iron overload.     

Potentiometric and spectrophotometric equilibrium study on Fe(III) and new catechol-bisphosphonate conjugates

The coordination properties of mixed catechol-bisphosphonates towards Fe(III) are presented. From the potentiometric and spectroscopic results it was possible to state that iron coordination takes place only on the bisphosphonate moiety at acidic pH, and involves both catechol and bisphosphonate groups on two different iron(III) ions at higher pH values. Steric constracts keep both groups from chelating the same metal ion. Quantum mechanical calculations confirm this statement and allow to determine the minimum length of the linker for a stable conformation of complexes in which the same iron(III) ion is coordinated by both catechol and bisphosphonate.     

Differential binding of human immunoagents and Herceptin to the ErbB2 receptor

Overexpression of the ErbB2 receptor is associated with the progression of breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized antibody directed to the ErbB2 receptor, has been proven to be effective in the immunotherapy of breast cancer. However, it can result in cardiotoxicity, and a large fraction of breast cancer patients are resistant to Herceptin treatment. We have engineered three novel, fully human, anti-ErbB2 immunoagents: Erbicin, a human single-chain antibody fragment; ERB-hRNase, a human immunoRNase composed of Erbicin fused to a human RNase; ERB-hcAb, a human 'compact' antibody in which two Erbicin molecules are fused to the Fc fragment of a human IgG1. Both ERB-hRNase and ERB-hcAb strongly inhibit the growth of ErbB2-positive cells in vivo. The interactions of the Erbicin-derived immunoagents and Herceptin with the extracellular domain of ErbB2 (ErbB2-ECD) were investigated for the first time by three different methods. Erbicin-derived immunoagents bind soluble extracellular domain with a lower affinity than that measured for the native antigen on tumour cells. Herceptin, by contrast, shows a higher affinity for soluble ErbB2-ECD. Accordingly, ErbB2-ECD abolished the in vitro antitumour activity of Herceptin, with no effect on that of Erbicin-derived immunoagents. These results suggest that the fraction of immunoagent neutralized by free extracellular domain shed into the bloodstream is much higher for Herceptin than for Erbicin-derived immunoagents, which therefore may be used at lower therapeutic doses than those employed for Herceptin.     

Evidence of biogeography in surface ocean bacterioplankton assemblages

Regardless of the importance of bacterial assemblages as essential components of ecosystems, little is known about how their populations are structured. We analyzed the composition and turnover rates, based on 16S rDNA sequences, of surface water oceanic bacterial assemblages of the fraction between 0.1 and 0.8 μm along a latitudinal gradient (45°6′42′′N in the North Atlantic to 15°8′37′′S in the South Pacific) including geographic distance, temperature, chlorophyll a and salinity. Here we show that oceanic bacterial assemblages between 0.1 and 0.8 μm, can be structured by a variety of environmental interactions that include separation by distance and chlorophyll a concentration in temperate North Atlantic coastal samples and temperature in tropical Atlantic and Pacific coastal and open ocean samples. Bacterial phyla composition diverged between temperate and tropical regions. This study suggests that some bacterial assemblages could be structured both by environmental and spatial factors, while others by environmental factors alone.