Novel dissociation mechanism of a polychaetous annelid extracellular haemoglobin

The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of approximately 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or 'linker' subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS/PAGE, UV-visible spectrophotometry, electrospray-ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.     

Change of conformation of the DNA-binding domain of p53 is the only key element for binding of and interference with p73

Xenopus p53 has biological and biochemical properties similar to those of human p53, except for optimal temperature. The frog protein is fully active at 30 degrees C and inactive at 37 degrees C, leading to a temperature-sensitive behavior similar to that of the human mutant p53Ala(143) and the murine mutant p53Val(135). Using hybrid proteins between human and Xenopus expressed from artificial p53 minigenes, we have been able to demonstrate that change of conformation of the DNA-binding domain is the major determinant of this heat sensitivity. It has been reported that some human tumor-derived p53 mutants can engage in a physical association with p73, thus inhibiting its transactivating properties. The mechanism of this association remains to be elucidated. The nature of the mutant p53 that can engage in this association also remains controversial. Using the unique opportunity of the temperature sensitivity of Xenopus p53, we demonstrate that binding of and interference with p73 require a change of conformation in the p53 protein. This interaction occurs through the DNA-binding domain of p53 only when it is in a denatured state. These results reinforce the notion that mutant p53 with a conformational change can act as a down-regulator of the p73 pathway in human cancer and could confer a selective advantage to the tumor.     

Entry of viruses through the epithelial barrier: pathogenic trickery

Mucosal surfaces--such as the lining of the gut or the reproductive tract--are the main point of entry for viruses into the body. As such, almost all viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. In addition to protein receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the host cell.     

Interfacial kinetic and binding properties of the complete set of human and mouse groups I,II, V,X, and XII secreted phospholipases A2

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.     

CFTR transgene expression in primary DeltaF508 epithelial cell cultures from human nasal polyps following gene transfer with cationic phosphonolipids

Cystic fibrosis (CF) is the most common autosomal lethal recessive disorder in the Caucasian population. The major cause of mortality is lung disease, owing to the failure of a functional protein from the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Today, even though the knowledge about the CFTR genomic is extensive, no efficient treatment has been developed yet. In this context, gene therapy represents a potential important advance on condition that it could develop efficient and safe transfection agents. Even though viral vectors have been used in most clinical trials owing to their high transfection efficiency, random integration and immunogenicity are still critical side effects. Consequently, all of these drawbacks brought forth the development of nonviral transfection systems. Although they engender few toxicity and immunogenicity problems, their low transfection efficiency is a hurdle that must be overcome. Over the past decade, we have developed an original family of monocationic lipids, cationic phosphonolipids, whose efficiency has been previously demonstrated both in vitro and in vivo. In this report, we observe that a new cationic phosphonolipid (KLN 30) can lead to the restoration of the CFTR protein following the ex vivo transfection of epithelial cells issuing from a F508 homozygous patient. The transgene expression and the cytotoxicity correlate with the charge ratio of the lipoplex. A kinetic study was performed, and a luminescent signal was detected until 35 d after transfection.     

Centrophobism/thigmotaxis, a new role for the mushroom bodies in Drosophila

Insects, like vertebrates, exhibit spatially complex locomotor activity patterns when foraging or navigating. Open field studies recently showed that Drosophila avoids central zones and stays at the periphery, an effect that can be interpreted as centrophobism and/or thigmotaxis. In this study, we further characterized this phenomenon and studied the responsible underlying neural mechanisms. The implication of the Drosophila mushroom bodies (MBs) in olfactory learning and memory processes is well documented. In an open field situation in which fly locomotor activity is recorded by video tracking, we show that center avoidance is greatly diminished in flies with hydroxyurea-ablated MBs, suggesting a new role for these structures. Furthermore, the temperature-sensitive allele of the dynamin gene shibire was expressed in various enhancer-trap P[GAL4] lines, disrupting synaptic transmission in different MB lobes. Specifically blocking the gamma lobes alters centrophobism/thigmotaxis while blocking the alpha/beta lobes does not, suggesting a functional specialization of MB lobes. Drosophila may serve as a new model system for elucidating the genetic and neural bases of such complex phenomena as centrophobism/thigmotaxis.     

Optimal calibration marker mesh for 2D X-ray sensors in 3D reconstruction

Image intensifiers suffer from distortions due to magnetic fields. In order to use this X-ray projections images for computer-assisted medical interventions, image intensifiers need to be calibrated. Opaque markers are often used for the correction of the image distortion and the estimation of the acquisition geometry parameters. Information under the markers is then lost. In this work, we consider the calibration of image intensifiers in the framework of 3D reconstruction from several 2D X-ray projections. In this context, new schemes of marker distributions are proposed for 2D X-ray sensor calibration. They are based on efficient sampling conditions of the parallel-beam X-ray transform when the detector and source trajectory is restricted to a circle around the measured object. Efficient sampling are essentially subset of standard sampling in this situation. The idea is simply to exploit the data redundancy of standard sampling and to replace some holes of efficient schemes by markers. Optimal location of markers in the sparse efficient sampling geometry can thus be found. In this case, the markers can stay on the sensor during the measurement with--theoretically--no loss of information (when the signal-to-noise ratio is large). Even if the theory is based on the parallel-beam X-ray transform, numerical experiments on both simulated and real data are shown in the case of weakly divergent beam geometry. We show that the 3D reconstruction from simulated data with interlaced markers is essentially the same as those obtained from data with no marker. We show that efficient Fourier interpolation formulas based on optimal sparse sampling schemes can be used to recover the information hidden by the markers.