Kinetics and mechanics of stem gravitropism in Coprinus cinereus

Using video recordings we have completed the first kinetic analysis of mushroom stem gravitropism. The stem became gravireceptive after completion of meiosis, beginning to bend within 30 minutes of being placed horizontal. Stem bending first occurred in the apical 15% of its length, then the position of the bend moved rapidly towards the base, traversing 40% of stem length in 2.5 h. Meanwhile, the stem elongated by 25%, mostly in its upper half but also in basal regions. If the apex was pinned horizontally the stem base was elevated but overshot the vertical, often curling through more than 300 degrees. When the base was pinned to the horizontal (considered analogous to the normal situation), 90% of the initial bend was compensated as the stem brought its apex accurately upright, rarely overshooting the vertical. The apex had to be free to move for this curvature compensation to occur. Stems transferred to a clinostat after some minutes gravistimulation showed curvature which increased with the length of initial gravistimulation, indicating that continued exposure to the unilateral gravity vector was necessary for continued bending. Such gravistimulated stems which bent on the clinostat subsequently relaxed back towards their original orientation. Reaction kinetics were unaffected by submergence in water, suggesting that mechanical events do not contribute, but submerged stems bent first at the base rather than apex. In air, the gravitropic bend appeared first near the apex and then moved towards the base, suggesting basipetal movement of a signal. In water, the pattern of initial bending was changed (from apex to base) without effect on kinetics. Taken together these results suggest that bending is induced by a diffusing chemical growth factor (whose extracellular propagation is enhanced under water) which emanates from the apical zone of the stem. The apex is also responsible for regulating compensation of the bend so as to bring the tip to the vertical. The nature of this latter stimulus is unknown but it is polarized (the apex must be free to move for the compensation to occur) and it may not require reference to the unilateral gravity vector.     

Lactation-associated redistribution of the glial fibrillary acidic protein within the supraoptic nucleus

Summary Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca²⁺-dependent and Ca²⁺-independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.Dedicated to Prof. Dr. W. Lamprecht on the occasion of his 60th birthday     

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.     

A simple phenomenological thermodynamic model for protein partitioning in reversed micellar systems

A simple thermodynamic model is developed for the partitioning of proteins between a bulk aqueous solution and a reversed micellar organic phase by assuming that a pseudo-chemical equilibrium is established when proteins in solution interact with a non-integral number of empty micelles to form the protein-micelle complex. From the equilibrium constant for this reaction, which is related to both the chemical and electrical free energy changes associated with the transfer of the proteins between the two phases, a simple expression is derived for the partition coefficient as a function of pH and surfactant concentration. Assumptions include a linear variation in protein net charge with pH, and a linear decrease in protein-micelle complex size with increasing protein charge. Results on the solubilization of ribonuclease-a and concanavalin-a in Aerosol-OT/isooctane organic solvents were well-correlated by the model equation, and the estimated parameters were of the expected order of magnitude as estimated based on the known physical properties of the system components.List of Symbols F C/mol Faraday's constant - G J/mol standard free energy change on solubilization - G J/mol standard free energy change in the absence of charge effects - K partition coefficient - K _eq (mol/m³)^–n equilibrium constant for pseudo-reaction (1) - M micelle - N _ag empty micelle aggregation number - n number of empty micelles required to form protein/micelle complex - n ₀ number of empty micelles required at zero net protein charge - P protein - PM protein/micelle complex - pI protein isoelectric point - R J/mol K gas law constant - S surfactant - z protein charge - slope of protein titration curve - change in micelle size, n, per unit change in charge - V electrostatic potential difference     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.