Concanavalin A-induced chemiluminescence in rat thymus lymphocytes. Its origin and role in mitogenesis.

Incubation of a particulate preparation from potato tissue culture cells with UDP-beta-L-[1-3H] arabinose yielded a glycoprotein fraction containing labelled material with the characteristics of hydroxyproline arabinosides. The sugar-protein linkage was resistant to hot alkaline hydrolysis, and the hydrolytic products showed similar electrophoretic and chromatographic behavior to authentic hydroxyproline-arabinosides prepared from potato tissue culture cell walls. Incorporation of arabinose into glycoprotein was stimulated by the addition of de-arabinosylated potato lectin. The product of the incubation co-migrated with native potato lectin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The subcellular distribution of the arabinosyl-transferase was investigated by fractionating potato tissue culture membranes on a discontinuous sucrose gradient in the presence or absence of Mg2+. Under both fractionation conditions the highest specific activity of the enzyme was found in the Golgi-enriched fraction. The results are discussed in relation to the synthesis of the hydroxy-proline-rich glycoprotein component of plant cell walls.     

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.     

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.     

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.     

The floor plate: multiple cells, multiple signals

One of the key organizers in the CNS is the floor plate - a group of cells that is responsible for instructing neural cells to acquire distinctive fates, and that has an important role in establishing the elaborate neuronal networks that underlie the function of the brain and spinal cord. In recent years, considerable controversy has arisen over the mechanism by which floor plate cells form. Here, we describe recent evidence that indicates that discrete populations of floor plate cells, with characteristic molecular properties, form in different regions of the neuraxis, and we discuss data that imply that the mode of floor plate induction varies along the anteroposterior axis.     

FGF-dependent midline-derived progenitor cells in hypothalamic infundibular development

The infundibulum links the nervous and endocrine systems, serving as a crucial integrating centre for body homeostasis. Here we describe that the chick infundibulum derives from two subsets of anterior ventral midline cells. One set remains at the ventral midline and forms the posterior-ventral infundibulum. A second set migrates laterally, forming a collar around the midline. We show that collar cells are composed of Fgf3(+) SOX3(+) proliferating progenitors, the induction of which is SHH dependent, but the maintenance of which requires FGF signalling. Collar cells proliferate late into embryogenesis, can generate neurospheres that passage extensively, and differentiate to distinct fates, including hypothalamic neuronal fates and Fgf10(+) anterior-dorsal infundibular cells. Together, our study shows that a subset of anterior floor plate-like cells gives rise to Fgf3(+) SOX3(+) progenitor cells, demonstrates a dual origin of infundibular cells and reveals a crucial role for FGF signalling in governing extended infundibular growth.     

Opponent activities of Shh and BMP signaling during floor plate induction in vivo.

We performed in vivo experiments in chick embryos that examined whether application of an exogenous source of Shh protein mimics the ability of the notochord to induce ectopic floor plate cells in the neural tube. Shh cannot act alone to induce a floor plate. However, coapplication of Shh and chordin, a BMP antagonist normally coexpressed with Shh in the notochord, results in a marked switch from dorsal to ventral cell fate, including a dramatic and widespread induction of floor plate cells. These data provide in vivo evidence that notochord-derived BMP antagonists may normally generate a permissive environment for the Shh-mediated induction of floor plate. Further experiments performed to address the source of BMPs that are inhibited by the action of chordin suggest that they derive specifically from the surface ectoderm and dorsal-most neuroepithelium. These data indicate that, at neural groove stages, dorsally derived BMPs affect ventral-most regions of the neural plate, suggesting a novel long-range action of BMPs. Together, these studies suggest that the balance of dorsally derived signals and notochord-derived signals determines the extent of floor plate cell induction.     

Patterns of accumulation of duvatrienediols in shoot cultures of tobacco

Summary The accumulation of duvatrienediols associated with the glandular exudate of tobacco trichomes was investigated in tobacco shoot cultures. The pattern of accumulation was related to culture growth. and duvatrienediols showed the same pattern of accumulation appearing in a ratio of 21 increasing to 31 during senescence. Both epimers were absent from newly subcultured tissue but started to accumulate two weeks after subculture and increased in parallel with fresh weight until the stationary phase was reached. A further increase (two-fold) in the yield of both epimers was observed during the stationary phase. However, with the onset of senescence both epimers began to disappear. The use of sterile cultures precluded the possibility of losses of duvatrienediols due to microbial degradation although duvatriendiols could be subject either to chemical or metabolic degradation/turnover continuously or during senescence and should not be regarded as stable end products of metabolism.     

Non-cell-autonomous role for Cripto in axial midline formation during vertebrate embryogenesis

Several membrane-associated proteins are known to modulate the activity and range of potent morphogenetic signals during development. In particular, members of the EGF-CFC family encode glycosyl-phosphatidylinositol (GPI)-linked proteins that are essential for activity of the transforming growth factor beta (TGFbeta) ligand Nodal, a factor that plays a central role in establishing the vertebrate body plan. Genetic and biochemical studies have indicated that EGF-CFC proteins function as cell-autonomous co-receptors for Nodal; by contrast, cell culture data have suggested that the mammalian EGF-CFC protein Cripto can act as a secreted signaling factor. Here we show that Cripto acts non-cell-autonomously during axial mesendoderm formation in the mouse embryo and may possess intercellular signaling activity in vivo. Phenotypic analysis of hypomorphic mutants demonstrates that Cripto is essential for formation of the notochordal plate, prechordal mesoderm and foregut endoderm during gastrulation. Remarkably, Cripto null mutant cells readily contribute to these tissues in chimeras, indicating non-cell-autonomy. Consistent with these loss-of-function analyses, gain-of-function experiments in chick embryos show that exposure of node/head process mesoderm to soluble Cripto protein results in alterations in cell fates toward anterior mesendoderm, in a manner that is dependent on Nodal signaling. Taken together, our findings support a model in which Cripto can function in trans as an intercellular mediator of Nodal signaling activity.     

Orchestrating ontogenesis: variations on a theme by sonic hedgehog

Embryonic development is an emergent process in which increasing complexity is generated by sequential cellular interactions. Recently, it has become clear that such interactions are mediated by just a few families of signalling molecules; but how does this limited repertoire elicit the diversity of form that is characteristic of multicellular organisms? Here we review the various ways in which a member of one such family, the sonic hedgehog (SHH) protein, is deployed during embryonic development. These examples of SHH function provide paradigms for inductive interactions that should help to inform attempts to recapitulate cellular programming and organogenesis in vitro.