Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin

Purified rabbit reticulocyte transferrin receptors were incorporated into phosphatidylcholine vesicles containing varying amounts of cholesterol. The binding of transferrin to the receptor in the reconstituted vesicles had three distinct characteristics: (1) The binding of transferrin exhibited the two components characteristic of transferrin binding to erythroid cells, a saturable, specific component and a nonsaturable, nonspecific component. (2) Transferrin binding exhibited positive cooperativity at low cholesterol/phospholipid (C/P) molar ratios. However, the cooperativity diminished and then disappeared as the C/P molar ratios were increased to the levels found in circulating red blood cells. (3) The amount of specific transferrin binding to the reconstituted vesicles also decreased as the C/P molar ratio was increased. These results indicate that in the reconstituted system the lipid environment plays a significant role in the expression of transferrin receptors.     

Inhibition of phosphate uptake in barley roots by hydroxy-benzoic acids

The influence of 15 hydroxy-benzoic acids upon active inorganic phosphate absorption by barley roots was examined. For each compound an inhibition constant (k_i) was determined, i.e. the concentration of compound required to bring about a 50% inhibition of absorption. The k_i values of the benzoic acids were strongly correlated with their octanol—water partition coefficients and their pK_a values. This suggests that the inhibition of normal membrane functions, brought about by benzoic acids, results from a generalized increase in cell membrane permeability. Salicylate derivatives were generally more inhibitory than would be predicted from their partition coefficients; their pronounced toxicity probably arises from structural impediments to their detoxication.     

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0     

Regulation of phosphate influx in barley roots: Effects of phosphate deprivation and reduction of influx with provision of orthophosphate

Barley plants were grown in nutrient solutions, which were maintained at either 0 (-P) or 15 μ M orthophosphate (+P). After 11 days phosphate influx into the intact roots of the -P plants began to increase by comparison with +P plants. During this period differences became apparent between the treatments in absolute growth rates, as well as in the root:shoot ratios. Phosphate influx in the -P plants continued to increase as a function of time, to a maximum value of 2.4 μmol (g fresh wt)^-1h^-1 at 16 days after germination. This rate was 6 times higher than influx values for +P plants of the same age. During the period of enhanced uptake phosphate was strongly correlated (r²= 0.77) with root organic phosphate concentration. – The enhancement of inorganic phosphate influx into intact roots of -P plants was rapidly reduced by the provision of 15 μ M orthophosphate. Typically, within 4 h of exposure to this concentration of phosphate, influx values fell from 1.80 ± 0.20 to 0.75 ± 0.03 μmol (g fresh wt)^-1 h^-1, while inorganic phosphate concentrations of the roots increased from 0.12 to 1.15 μmol (g fresh wt)^-1 during the same period. Hill plots of the influx data obtained during this period, treating root inorganic phosphate as an inhibitor of influx, gave Hill coefficients close to 2. The rapidity of the reduction of influx associated with increased root inorganic phosphate together with the Hill plot data provide evidence for an allosteric inhibition of influx by internal inorganic phosphate.