Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.     

Molecular characterisation of the caprine (<Emphasis Type="Italic">Capra hircus</Emphasis>) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA

Background

Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response.

Results

The Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues.

Conclusion

Therefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species.

     

Inoculation of plant-growth-promoting rhizobacteria in Myracrodruon urundeuva Allemão supports in tolerance to drought stress

This study evaluated the influence of Azospirillum lipoferum on the growth of Myracroduon urundeuva (Anacardiaceae) plants under drought stress, by means of biometric, physical–chemical and biochemical parameters. The association of A. lipoferum with the roots of the plants provided increases of 30% root length, 50% root dry weight, 34% shoot dry weight and 10% soluble protein content. The inoculated plants still maintained 5% higher leaf water potential than those not inoculated and lower membrane damage. Furthermore, the inoculated plants shown less leaf fall and dark green leaves, confirmed by maintenance of the highest levels of chlorophyl a, b and total. On the other hand, superoxide dismutase activity was significantly lower in the inoculated plants, possibly due to the induction of a non-enzymatic protective feature. In this way, the inoculation of PGPR in M. urundeuva can be an alternative for the production of plants that are more tolerant to drought stress.     

The use of conspecific reproductive success for breeding patch selection in terrestrial migratory species

Classical models of breeding habitat selection rarely deal with the question of information gathering for patch quality assessment. In this paper, we present two models comparing the fitness outcomes of behavioural strategies based on conspecific reproductive success as a cue to assess local environmental quality before selecting a new breeding habitat. The models deal with two phases of the life-cycle of a territorial migratory species: recruitment to a breeding population (model 1) and breeding site fidelity of subsequent breeding attempts (model 2). The first model shows that prospecting breeding patches before recruiting is the best strategy if the environment is predictable and contains a low proportion of good patches, even if it implies losing a breeding opportunity. The second model shows that dispersing after a breeding attempt according to the patch's breeding success rather than the individual's own success is the bests own success is the best strategy if the environment is patchy. These results underline the importance of studying the spatio-temporal variations of factors affecting reproductive success when considering the importance of habitat selection strategies based on conspecifics. Moreover, they allow the understanding of individual behaviour patterns observed in natural populations and their potential consequences at the metapopulation level.     

Dipole moments of scorpion toxins direct the interaction towards small- or large-conductance Ca2+-activated K+ channels

Ca²⁺-activated K⁺ channels consist of alarge family of membrane proteins, among which twogroups have been characterized by electrophysiologicalcriteria, the small conductance (SK) and the largeconductance (BK) Ca²⁺-activated K⁺channels. Scorpion toxins that block K⁺ channelsexhibit a common three-dimensional structureconstituted of a short -helix connected bydisulfide bonds to a -sheet. The leiurotoxin I(LTX1) related toxins interact specifically with theSK channel via basic residues of their -helix,while the charybdotoxin (ChTX) family recognizes theBK channel with basic residues of their -sheet.In an attempt to better understand thestructure–activity relationships of these toxins andthe characteristics of the electrostatic interactionswith the receptor site, we investigated theelectrostatic potential supported by natural toxinsand a synthetic analogue to find out if it may help inunderstanding the molecular mechanisms involved inthis peptide–protein interaction.     

Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-l-phenylalanine precursor of pristinamycin I

Four pap genes ( papA , papB , papC , papM  ) were found by sequencing near to snbA , a Streptomyces pristinaespiralis gene which was previously shown to encode one of the pristinamycin I (PI) synthetases. Analysis of the homologies observed from the deduced amino acid sequences suggested that these four genes could be involved in the biosynthesis of the PI precursor 4-dimethylamino- l -phenylalanine (DMPAPA). This was first verified when disruption of papA in S. pristinaespiralis led to a PI⁻ phenotype, which was reversed by the addition of DMPAPA into the culture medium. Further confirmation was obtained when papM was overexpressed in Escherichia coli and the corresponding protein purified to homogeneity. It catalysed the two successive N -methylation steps of 4-amino- l -phenylalanine leading to DMPAPA via 4-methylamino- l -phenylalanine. These results allowed us to assign a function to each of the four pap genes and to propose a biosynthetic pathway for DMPAPA.