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Cells of Escherichia coli strain B develop large intracellular vacuoles and exhibit an abnormally long lag phase when inoculated into a defined medium to glucose and salts containing 3 x 10 6 M Cd2+. Early in this lag, about 95% of the cells fail to form colonies when plated on nutrient broth-NaCl-agar. Prior to the initiation of proliferation, the morphology of these cells becomes normal. They regain viability in the absence of deoxyribonucleic acid replication. The rate and extent of growth are normal once proliferation begins. This reversible phenomenon of accommodation to a growth-inhibiting concentration of Cd2" does not appear to result from a selection of mutant cells. Cells which are proliferating in the presence of Cd2+ accumulate the ion to a very high concentration. In accommodated cells, 56%c of the Cd2+ is associated with the cell wall, 13% in the membranes and 31% in the cytoplasm. In unaccommodated cells, the figures are 2%, 75%/, and 23%., respectivelv. The activity of alkaline phosphatase, a zinc-metalloenzyme which is inhibited by cadmium and is located between cell wall and membrane, is not abnormally low in accommodated cells, suggesting that the cadmium is compartmentalized in these cells. Molecular sieve chromatography of cell extracts shows that the Cd2+ is associated with two classes of macromolecules. It appears that accommodation of E. coli to the presence of Cd2+ involves exclusion of the ion from the cell and reversal of damage caused by prior exposure to the ion.

Three fluorescein-labeled lectins have been shown to exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. An isolectin from seeds of Bandeiraea simplicifolia with specificity for a-D-galactosyl end groups labeled the basal and lower spinous cells; a lectin from Ulex europaeus exhibiting specificity for a-L-fucosyl units outlines the surface of spinous cells, and a second lectin from B. simplicifolia, with specificity for N-acetyl-Dglucosamine, labels the cornified cells. Appropriate blocking experiments have confirmed the specific nature of the binding.The investigation of the differentiative process in mammalian epidermis would be assisted by the availability of a technique that would specifically identify different strata. Lectins, which have an affinity for specific sugars (cf. refs. 1 and 2), have been used to probe the nature of the complex carbohydrates associated with cell surfaces (cf. ref.3) and to investigate the role of the cell surface in cellular functions such as growth (4, 5), adhesion (6), pinocytosis (7), antigenicity (8), fertilization (9), and differentiation (10).It seemed possible that specific lectins could be found that would associate with the cell surface during various stages of epidermal differentiation if changes in surface carbohydrates, occurred during this process. Fluorescein-labeled concanavalin A attaches to the surfaces of cells in all layers of the human epidermis (11) and oral mucosa (12) except the stratum corneum. The pattern of binding for concanavalin A and pemphigus antibody are similar (13); however, there are differences in the binding of lectins to the cell surface of carcinomas and wounds in the human oral epithelium (14). In the normal oral mucosa (15), human epidermis (16), and gastrointestinal mucosa of the rat (17, 18), keratinocytes in different differentiative stages exhibit specificity for the binding of particular lectins to their surfaces. The loss of the nucleus from differentiating erythrocytes of the rat results in a change in the labeling of the surface by concanavalin A (10).Three lectins have now been identified that do exhibit specificity for the surface of cells in different layers of the epidermis in the newborn rat. One lectin, with specificity for a-D-galactosyl groups, binds to basal and lower spinous cells; another, specific for a-L-fucosyl units, outlines spinous cells, and the third, specific for N-acetyl-D-glucosamine, labels cornified cells. FITC-Ulex I was diluted to 125-150 g/ml. As controls, Lfucose (Sigma) or N-acetyl-D-glucosamine (Aldrich) at a concentration of 0.5 M in phosphate-buffered saline was preincubated with the lectin. Dorsal skin from newborn rats (CFN strain) was stretched over the surface of a watch glass and frozen to -30'C. Small strips (3 mm X 5 mm) were subsequently cut with a scalpel and set into Tissue-Tek II O.C.T. embedding medium (Miles Laboratories, Naperville, IL), exposing a cross section of skin. Sections were cut 4-6 Mm thick in a cryostat and placed o...

When cutaneous sections from the newborn rat were treated with a-fucosidase, Ulex europeus agglutinin I (UEA) binding to the cell surface of the differentiated cells in the epidermis was diminished and there was an appearance in these cell layers of binding by Bandeiraea simplicifolia I-B4 lectin (BS I-B4), which normally is specific for the basal cells. A similar treatment with a-galactosidase resulted in a loss of BS I-B4 binding, but had no effect on UEA binding. Glycoproteins isolated from the membranes of epidermal cells showed a threefold increase in the ratio of binding to UEA versus BS I-B4 affinity columns as the proteins were derived from the more differentiated cell populations. These data suggest that a-fucosyl residues are added to the glycoproteins on the cell surfaces of differentiated cells, thus blocking a-galactosyl residues and changing the lectin binding specificity as epidermal cells move out of the basal cell layer.

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