Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

Extraction buffer effects on detection of transient gene expression in white spruce

β-Glucuronidase (GUS) and luciferase (LUC) reporter genes were introduced into white spruce (Picea glauca [Moench] Voss) cultured cells via particle bombardment. Transient expression of these genes was evaluated by extracting the enzymes using 3 buffers. Different buffers resulted in significantly different sensitivities of GUS and LUC detection. In the case of cobombardment, the buffer that gave high levels of expression of one reporter gene did not necessarily result in a better detection of the second reporter gene. This study indicates that appropriate buffers should be used for maximum detection of reporter genes.     

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.     

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.     

Vertebrate development: Et in Arkadia

The similarities in organiser formation in Xenopus and mouse embryos have remained elusive. Recent evidence suggests a common mechanism, in which an intracellular protein, Arkadia, is required for formation of the mouse organiser and potentiates the effects of the signalling protein Nodal.     

Temporal progression of hypothalamic patterning by a dual action of BMP

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. Here, we demonstrate how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. Our studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; here, we propose that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs.     

Tensile strength of bone fixation of hydroxyapatite coated Schanz screws of the Heidelberg External Fixation System (HEFS)--comparative torque measurements in clinical use and in cadaver tibia]

It is claimed in the literature that hydroxyapatite(HA)-coated screws of external fixators have superior fixation strength in bone, which is postulated to lead to a substantial decrease in loosening and infection rates. We report on a study of the maximum torque values developed while inserting and removing 30 HA-coated Schanz screws of 8 Heidelberg external fixation systems applied to the tibia to correct leg length differences and axial deformities. The infection rate was determined in accordance with defined criteria, and was found to be about 20% for the HA-coated screws. Screws without infection showed an extraction torque above insertion torque, screws with infection an extraction torque below. A significant correlation (p = 0.05) was seen between infection and decrease in fixation strength (quotient: loosening torque/tightening torque). To exclude the impact of such biological processes as osteointegration and bone remodelling, the clinical results were compared with the torques measured for coated and uncoated Schanz screws in a human cadaveric tibia. A significantly higher fixation strength in bone was found for HA-coated screws in comparison with uncoated screws (p = 0.002). These data warrant a clinical study directly comparing HA-coated and uncoated Schanz screws.     

Airway patterning: A paradigm for restricted signalling.

Intercellular signalling is limited by the range of cell responsiveness, often mediated by repressors. A recently identified repressor, Sprouty, inhibits MAP kinase signalling in flies, mice and humans and has a conserved function in patterning the airways of these divergent species.     

Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise

Red blood cell (RBC) susceptibility to oxidative and osmotic stress in vitro was investigated in cells from trained and untrained men before and after submaximal exercise. Whilst no significant change in peroxidative haemolysis occurred immediately after 1 h of cycling at 60% of maximal aerobic capacity ( _max), a 20% increase was found 6 h later in both groups (P<0.05). The RBC osmotic fragility decreased by 15% immediately after exercise (P<0.001) and this was maintained for 6 h (Ps<0.001). There was an associated decrease in mean cell volume (P<0.05). Training decreased RBC susceptibility to peroxidative haemolysis (P<0.025) but it did not influence any other parameter. These exercise-induced changes were smaller in magnitude but qualitatively similar to those found in haemopathological states involving haem-iron incorporation into membrane lipids and the short-circuiting of antioxidant protection. To explore this similarity, a more strenuous and mechanically stressful exercise test was used. Running at 75% _max for 45 min reduced the induction time of O₂ uptake (peroxidation), consistent with reduced antioxidation capacity, and increased the maximal rate of O2 uptake in RBC challenged with cumene hydroperoxide (P<0.001). The proportion of high-density RBC increased by 10% immediately after running (P<0.001) but no change in membrane-incorporated haem-iron occurred. In contrast, treatment of RBC with oxidants (20–50 mol·l^–1 in vitro increased cell density and membrane incorporation of haem-iron substantially. These results showed that single episodes of submaximal exercise caused significant changes in RBC susceptibility to oxidative and osmotic stress. Such responses may account for the increase in RBC turnover found in athletes undertaking strenuous endurance training.