Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus

In the invertebrate chordate amphioxus, as in vertebrates, retinoic acid (RA) specifies position along the anterior/posterior axis with elevated RA signaling in the middle third of the endoderm setting the posterior limit of the pharynx. Here we show that AmphiHox1 is also expressed in the middle third of the developing amphioxus endoderm and is activated by RA signaling. Knockdown of AmphiHox1 function with an antisense morpholino oligonucleotide shows that AmphiHox1 mediates the role of RA signaling in setting the posterior limit of the pharynx by repressing expression of pharyngeal markers in the posterior foregut/midgut endoderm. The spatiotemporal expression of these endodermal genes in embryos treated with RA or the RA antagonist BMS009 indicates that Pax1/9, Pitx and Notch are probably more upstream than Otx and Nodal in the hierarchy of genes repressed by RA signaling. This work highlights the potential of amphioxus, a genomically simple, vertebrate-like invertebrate chordate, as a paradigm for understanding gene hierarchies similar to the more complex ones of vertebrates.     

Amplification of individual preferences in a social context: the case of wall-following in ants

Amplification processes are an essential component of the collective phenomena observed in social and gregarious species. In this paper, we tested the hypothesis that a weak individual wall-following tendency in ants can be amplified by communication through chemical trails, leading to a response to the spatial heterogeneities at the collective level. In our experiments, ants had to cross a diamond-shaped bridge along either of two branches of equal length to get from their nest to a food source. Two types of bridge were used: control bridges without a wall, and experimental bridges equipped with a wall along the inner edge of one of their branches. On the control bridges, ants collectively chose either branch of the bridge in most experiments, whereas on the experimental bridges, the branch with the wall was selected almost systematically. A mathematical model is proposed to assess, in various conditions, the importance of the amplification effect of the chemical trail on the wall-following tendency observed at the individual level. The model highlights the fact that the amplification process can lead to an overestimation of individual capabilities and, thus, that the results of experiments investigating individual preferences at group level in animals must be interpreted with caution.     

Towards writing the encyclopedia of life: an introduction to DNA barcoding

An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the 'Barcode of Life Initiative', to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1 = CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life.     

Identification of proteins binding the native tubulin dimer

Microtubules play an essential role in eukaryotic cells, where they perform a wide variety of functions. In this paper, we describe the characterization of proteins associated to tubulin dimer in its native form, using affinity chromatography and mass spectrometry. We used an immunoaffinity column with coupled-monoclonal antibody directed against the alpha-tubulin C-terminus. Tubulin was first loaded onto the column, then interphase and mitotic cell lysates were chromatographed. Tubulin-binding proteins were eluted using a peptide mimicking the alpha-tubulin C-terminus. Elution fractions were analyzed by SDS-PAGE, and a total of 14 proteins were identified with high confidence by mass spectrometry. These proteins could be grouped in four classes: known tubulin-binding proteins, one microtubule-associated protein, heat shock proteins, and proteins that were not shown previously to bind tubulin dimer or microtubules.     

Nanosecond dynamics of a mimicked membrane-water interface observed by time-resolved stokes shift of LAURDAN

We studied the dipolar relaxation of the surfactant-water interface in reverse micelles of AOT-water in isooctane in the nanosecond and subnanosecond time ranges by incorporating the amphipathic solvatochromic fluorescent probes LAURDAN and TOE. A negative component was observed in the fluorescence decays in the red edge of the emission spectrum-the signature of an excited state reaction-with LAURDAN but not for TOE. The deconvolution of the transient reconstructed spectra of LAURDAN based on a model constructed by adding together three log-normal Gaussian equations made it possible to separate the specific dynamic solvent response from the intramolecular excited state reactions of the probe. The deconvoluted spectrum of lowest energy displayed the largest Stokes shift. This spectral shift was described by unimodal kinetics on the nanosecond timescale, whereas the relaxation kinetics of water-soluble probes have been reported to be biphasic (on the subnanosecond and nanosecond timescales) due to the heterogeneous distribution of these probes in the water pool. Most of this spectral shift probably resulted from water relaxation as it was highly sensitive to the water to surfactant molar ratio (w(0)) (60-65 nm at w(0) = 20-30). A small part of this spectral shift (9 nm at w(0) = 0) probably resulted from dipolar interaction with the AOT polar headgroup. The measured relaxation time values were in the range of the rotational motion of the AOT polar headgroup region as assessed by LAURDAN and TOE fluorescence anisotropy decays.     

Unfolding and extraction of a transmembrane alpha-helical peptide: dynamic force spectroscopy and molecular dynamics simulations

An atomic force microscope (AFM) was used to visualize CWALP(19)23 peptides ((+)H(3)N-ACAGAWWLALALALALALALWWA-COO(-)) inserted in gel-phase DPPC and DSPC bilayers. The peptides assemble in stable linear structures and domains. A model for the organization of the peptides is given from AFM images and a 20 ns molecular dynamics (MD) simulation. Gold-coated AFM cantilevers were used to extract single peptides from the bilayer through covalent bonding to the cystein residue. Experimental and simulated force curves show two distinct force maxima. In the simulations these two maxima correspond to the extraction of the two pairs of tryptophan residues from the membrane. Unfolding of the peptide precedes extraction of the second distal set of tryptophans. To probe the energies involved, AFM force curves were obtained from 10 to 10(4) nm/s and MD force curves were simulated with 10(8)-10(11) nm/s pulling velocities (V). The velocity relationship with the force, F, was fitted to two fluctuation adhesive potential models. The first assumes the pulling produces a constant bias in the potential and predicts an F approximately ln (V) relationship. The second takes into account the ramped bias that the linker feels as it is being driven out of the adhesion complex and scales as F approximately (ln V)2/3.     

Editorial

Editorial

Editorial     

Isolation of a cDNA encoding the alpha-subunit of CAAX-prenyltransferases from Catharanthus roseus and the expression of the active recombinant protein farnesyltransferase

Crfta/ggt_Ia (AF525030), a cDNA encoding the ?-subunit of the two types of CaaX-prenyltransferase (CaaX-PTase), i.e. protein farnesyltransferase (PFT) and type I protein geranylgeranyltransferase, was cloned from Catharanthus roseus via a PCR strategy. Crfta/ggt_Ia is 1381-bp long and bears a 999-bp open reading frame encoding a protein of 332 residues (FTA) that shares 66% identity with its Lycopersicon esculentum orthologue. Southern blot analysis revealed that FTA is encoded by a single gene copy per haploid genome. Co-expression of Crfta/ggt_Ia and Crftb encoding the beta-subunit of PFT yielded purified active recombinant PFT. This enzyme is able to prenylate proteins from C. roseus, and could be used as a potent tool for prenylated protein identification.     

HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse

Dendritic cells (DCs) are essential components of the early events of HIV infection. Here, we characterized the trafficking pathways that HIV-1 follows during its capture by DCs and its subsequent presentation to CD4(+) T cells via an infectious synapse. Immunofluorescence microscopy indicates that the virus-containing compartment in mature DCs (mDCs) co-labels for the tetraspanins CD81, CD82, and CD9 but contains little CD63 or LAMP-1. Using ratio imaging of pH-reporting fluorescent virions in live DCs, we show that HIV-1 is internalized in an intracellular endocytic compartment with a pH of 6.2. Significantly, we demonstrate that the infectivity of cell-free virus is more stable at mildly acidic pH than at neutral pH. Using electron microscopy, we confirm that HIV-1 accumulates in intracellular vacuoles that contain CD81 positive internal membranes but overlaps only partially with CD63. When allowed to contact T cells, HIV-1-loaded DCs redistribute CD81, and CD9, as well as internalized HIV-1, but not the immunological synapse markers MHC-II and T-cell receptor to the infectious synapse. Together, our results indicate that HIV-1 is internalized into a non-conventional, non-lysosomal, endocytic compartment in mDCs and further suggest that HIV-1 is able to selectively subvert components of the intracellular trafficking machinery required for formation of the DC-T-cell immunological synapse to facilitate its own cell-to-cell transfer and propagation.     

A comparison of RNA folding measures

Background  

In the last few decades there has been a great deal of discussion concerning whether or not noncoding RNA sequences (ncRNAs) fold in a more well-defined manner than random sequences. In this paper, we investigate several existing measures for how well an RNA sequence folds, and compare the behaviour of these measures over a large range of Rfam ncRNA families. Such measures can be useful in, for example, identifying novel ncRNAs, and indicating the presence of alternate RNA foldings.