Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

Site-directed mutagenesis of the basic N-terminal cluster of pancreatic bile salt-dependent lipase. Functional significance

Previous studies have postulated the presence of a heparin-binding site on the bile salt-dependent lipase (BSDL), whereas two bile salt-binding sites regulate the enzyme activity. One of these sites may overlap with the tentative heparin-binding site at the level of an N-terminal basic cluster consisting of positive residues Lys(32), Lys(56), Lys(61), Lys(62), and Arg(63). The present study uses specific site-directed mutagenesis to determine the functional significance of this basic cluster. Mutations in this sequence resulted in recombinant enzymes that were able to bind to immobilized and to cell-associated heparin before moving throughout intestinal cells. Recombinant BSDL was fully active on soluble substrate, but mutants were less active on micellar cholesteryl oleate in comparison with the wild-type enzyme. Activation studies by primary (sodium taurocholate) and by secondary (sodium taurodeoxycholate) bile salts revealed that the activation of BSDL by sodium taurocholate at concentrations below the critical micellar concentration, and not that evoked by micellar bile salts, was affected by substitutions, suggesting that this N-terminal basic cluster likely represents the specific bile salt-binding site of BSDL. Substitutions also affected the activation of the enzyme promoted by anionic phospholipids, extending the function of this site to that of a cationic regulatory site susceptible to accommodate anionic ligands.     

Evaluation of the global optimisation method within the upper limb kinematics analysis

The aim of this study is to assess the performances of the global optimisation (GO) method (Bone position estimation from skin marker co-ordinates using GO with joint constraints. Journal of Biomechanics 32, 129-134) within the upper limb kinematics analysis. First the model of the upper limb is presented. Then we apply GO method in order to reduce skin movement artefacts that imply relative movement between markers and bones. The performances of the method are then evaluated with the help of simulated movements of the upper limb. Results show a significant reduction of the errors and of the variability due to skin movement.     

Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo

Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo . Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.     

Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.     

Development of a phenomenological modeling approach for prediction of growth and xanthan gum production using Xanthomonas campestris

An unstructured kinetic model for xanthan production is described and fitted to experimental data obtained in a stirred batch reactor. The culture medium was composed of several nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) consumed sequentially. The model proposed is able to describe this sequential consumption of nitrogen sources, the consumption of inorganic phosphate and carbon, the evolution of biomass, and production of xanthan. The parameter estimation has been performed by fitting the kinetic model in differential form to experimental data. Runs of the model for simulating xanthan gum production as a function of the initial concentration of inorganic phosphate have shown the positive effect of phosphate limitation on xanthan yield, though diminishing rates of production. The model was used to predict the kinetic parameters for a medium containing a 2-fold lower initial phosphate concentration. When tested experimentally, the measured fermentation parameters were in close agreement with the predicted model values, demonstrating the validity of the model.     

Notch activity is required to maintain floorplate identity and to control neurogenesis in the chick hindbrain and spinal cord

Notch signalling plays a major role in many invertebrate and vertebrate patterning systems. In this paper, we use high-titre, non-replicative pseudotype viruses to show that the two Notch ligands, Delta1 and Serrate1 (Jagged1), have differing activities in the developing chick spinal cord and hindbrain. In the walls of the neural tube, Serrate1 appears not to affect neurogenesis, in contrast to Delta1 which mediates lateral inhibition as elsewhere in the nervous system. In the floorplate we find that there is also a requirement for Notch, but with a different type of dependence on the two Notch ligands: cells with a floorplate character are lost when Notch activity is blocked with dominant-negative, truncated forms of either Delta1 or Serrate1. Our results are consistent with ligand-receptor specificity within the Notch signalling pathway, Serrate1 recognising selectively Notch2 (which is expressed in the floorplate), and Delta1 acting on both Notch2 and Notch1 (which is expressed in the walls of the neural tube).     

Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity

Stimulation of the Ras/extracellular signal-regulated kinase (ERK) pathway can modulate cell growth, proliferation, survival, and motility. The p90 ribosomal S6 kinases (RSKs) comprise a family of serine/threonine kinases that lie at the terminus of the ERK pathway. Efficient RSK activation by ERK requires its interaction through a docking site located near the C terminus of RSK, but the regulation of this interaction remains unknown. In this report we show that RSK1 and ERK1/2 form a complex in quiescent HEK293 cells that transiently dissociates upon mitogen stimulation. Complex dissociation requires phosphorylation of RSK1 serine 749, which is a mitogen-regulated phosphorylation site located near the ERK docking site. Using recombinant RSK1 proteins, we find that serine 749 is phosphorylated by the N-terminal kinase domain of RSK1 in vitro, suggesting that ERK1/2 dissociation is mediated through RSK1 autophosphorylation of this residue. Consistent with this hypothesis, we find that inactivating mutations in the RSK1 kinase domains disrupted the mitogen-regulated dissociation of ERK1/2 in vivo. Analysis of different RSK isoforms revealed that RSK1 and RSK2 readily dissociate from ERK1/2 following mitogen stimulation but that RSK3 remains associated with active ERK1/2. RSK activity assays revealed that RSK3 also remains active longer than RSK1 and RSK2, suggesting that prolonged ERK association increased the duration of RSK3 activation. These results provide new evidence for the regulated nature of ERK docking interactions and reveal important differences among the closely related RSK family members.     

Genomic sequencing reveals the structure of the Kcnk6 and map3k11 genes and their close vicinity to the sipa1 gene on mouse chromosome 19

In this report we present the analysis of two overlapping mouse cosmid clones that contain the entire Kcnk6, Map3k11 and Pcnxl3 genes, as well as part of the Sipa1 gene. The sequence and genomic organisation of the Kcnk6 and Map3k11 genes are described in detail. Sipa1 and Map3k11, which have independently been mapped with low resolution to the centromeric region of mouse chromosome 19, are shown here to lie close to each other and to the Kcnk6 gene, which has not previously been mapped. This gene cluster maps to the vicinity of the Dancer (Dc) mutation, which involves inner ear abnormalities and circling phenotypes. Since potassium channels have been implicated in deafness disorders, we have analysed the Kcnk6 gene, which encodes a two-P domain potassium channel, in the Dc mutant. No Dc-causing mutation in the Kcnk6 coding region could be identified. However, we detected a polymorphism in the Kcnk6 gene that leads to a C-terminal extension of the encoded protein by eight amino acids.     

The attendance cycles of the Cory's Shearwater Calonectris diomedea borealis on Selvagem Grande

In the Cory's Shearwater Calonectris diomedea borealis of Selvagem Grande (30 degrees 09'N, 15 degrees 52'W), the visits the birds pay to their colonies are cyclic throughout the breeding period. The number of visitors varies regularly between a minimum and a maximum following a sinusoid, the half-period of which is 4.8 +/- 1.5 d (2-8 d). No variation exists according to the status of the birds (breeders or non-breeders) or to the stage of the breeding cycle (pre-egg, incubation or chick rearing) and moon phases have no influence. Among Procellariids, such attendance cycles are related to the length of the pre-egg stage. They are absent when it is short (less than 50 d), present when it is long (more than 60 d), and indifferently present or absent when it is intermediate (50-60 d). Considering the comparative scarcity of the visits of the birds to the colonies during the pre-egg stage, these cycles are likely to play a part essentially in the synchronisation of individuals in colonies and of mates in pairs.