Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum.

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.

A micromechanical technique for monitoring cell-substrate adhesiveness: measurements of the strength of red blood cell adhesion to glass and polymer test surfaces.

We report a novel method for rapid comparison of the relative strength of adhesion of cells to different solid surfaces. A vertically oscillating micropipette is brought above an individual cell in such a manner that it makes contact with the cell at the lower limit of its travel. The pressure within the micropipette is gradually reduced until the cell attaches to the micropipette by suction and is lifted from the solid surface. The reduction in pressure required to detach a cell depends on the specific cell/substrate combination and serves as a relative measure of the strength of cell adhesion. A particular advantage of this approach over conventional methods is the ability to select particular cells from a population. As a test of the reproducibility of the method and its ability to distinguish the strength of adhesion of cells to different solid surfaces, we have used it to measure the adhesiveness of human red blood cells to hydrophilic glass, tissue culture grade polystyrene, polyethylene terephthalate, and polymethyl methacrylate. We find that results for the same surface are highly reproducible and that the method is capable of distinguishing small differences in the adhesiveness of red blood cells to the above surfaces.

Benign hemangiomyoma of the uterus.

A benign cavernous hemangiomyoma of the uterus in a 52-year-old woman is described. The tumor consisted of large venous channels within a stroma composed chiefly of smooth muscle and was confined to the corpus, without involvement of the endometrium or cervix. Considerable thrombosis had occurred within the vascular spaces. Hemangiomas of the uterus are very uncommon and the great majority are benign. A classification of uterine hemangiomas is presented, together with a discussion of their histogenesis and their relation to malformations.