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Several problems have frustrated the isolation of lamellar bodies (LB) from mammalian epidermis. We obtained pellets enriched in intact LB by utilizing the staphylococcal epidermolytic toxin to provide intact, outer epidermal sheets, by controlled homogenization in a cell disrupter, and by passage of homogenates through a graded series of nuclepore filters (Science 221:962, 1983). Such preparations contained more intact LB than did fractions prepared by a variety of differential or sucrose/metrizamide discontinuous centrifugation methods. Initial characterization of the enzymatic content of this fraction revealed it to be enriched in certain hydrolytic enzymes (acid phosphatase, carboxypeptidase, cathepsin B, acid lipase, sphingomyelinase, and phospholipase A), but strikingly depleted in all sulfatases, beta-glucuronidase, and the non-lysosomal protease, plasminogen activator. Thus, LB show some properties of lysosomes, although certain characteristic lysosomal enzymes are strikingly absent. Lamellar body fractions contained 2-3 times more lipid per unit weight than did homogenates, and were enriched in phospholipids, free sterols, and glycosphingolipids, but not in other neutral lipids or ceramides. In summary, whereas some of the enzymes in LB could participate in the metabolism of LB lipid precursors to hydrophobic barrier constituents, others may attack intercellular constituents, ultimately resulting in desquamation. The lipid profile of these organelles suggests that they deliver precursors of permeability barrier lipids to intercellular domains.

We have examined the possibility that proteases such as plasminogen activator (PA) contribute to the extraordinary motile capability of neural crest cells. We show that trunk neural crest cells that migrate from isolated neural tubes in vitro produce PA and that the level of cell-associated PA increases dramatically after 8 days in culture. This increase is not the result of differentiation or time in culture, because neural crest cell clusters that form on top of the neural tube and differentiate into pigment cells but are immotile produce very low levels of PA. If these clusters are removed from the neural tube and replated on a plastic substratum where they migrate, the level of PA associated with the cells increases dramatically, suggesting that PA production is associated with motility. Inhibitors of PA/plasmin activity significantly reduce neural crest cell motility in vitro, further supporting the idea that proteases are important in neural crest cell migration.

The effects of ionizing radiation on smell acuity were assessed in 12 patients in whom the olfactory mucosa was exposed to radiation in the course of treatment for nasopharyngeal carcinoma or pituitary adenoma. Olfactory detection thresholds for two odorants (amyl acetate and eugenol) were determined before the start of radiation therapy, within a week of termination of therapy, and 1, 3, and 6 months later. The results show clearly that smell acuity is profoundly affected by therapeutic irradiation. Thresholds had increased in all 12 patients by the end of treatment and were still high one month later. Varying degrees of recovery were noted in most patients three to six months after cessation of treatment. The fate of the sense of smell deserves more attention when considering the disability caused by irradiation to certain head and neck tumors.

Results of olfactory function tests (threshold determination and odor identification) in three cases of bilateral and one case of unilateral choanal atresia are reported. All four patients underwent successful repair of choanal atresia at relatively advanced ages (8 to 31 years). Test results showed that patients who had suffered from bilateral atresia had permanent olfactory deficits, while the patient who had suffered from unilateral atresia appeared to have normal olfactory acuity. Although these results should be interpreted with caution due to the small number of cases examined, they suggest the possibility that early sensory exposure might be needed for the normal development of central olfactory functions in analogy to the visual system.

The in vitro incubation of murine keratinocytes in the presence of 1 alpha,25-dihydroxyvitamin D3 enhanced the rapid hydrolysis of the prelabeled keratinocyte polyphosphoinositides (polyPtdIns) when compared to untreated cells. The rapid hydrolysis of the polyPtdIns and the release of the inositol phosphates (particularly InsP3 and InsP2) precede the onset of differentiation of these cells. These data therefore suggest that 1 alpha,25-dihydroxyvitamin D3 functions in vitro to initiate the rapid generation of InsP3 from cellular polyPtdIns; this in turn may mobilize intracellular Ca2+, thus providing the signal which program the murine keratinocytes from a proliferating mode into a differentiating mode.

The relationship between the turnover of inositol phospholipids (PtdIns) and the growth and differentiation of normal murine keratinocytes in culture was studied. Addition of myo-[U-14C]inositol to freshly plated cells resulted in a linear incorporation of radiolabel into the inositol phospholipids of the proliferating basal cells in culture during the initial 36 h, after which time the rate of radiolabel incorporation into the cells declined. The decrease in the incorporation of the radiolabel into the PtdIns, particularly the more highly phosphorylated PtdIns-4P and PtdIns4,5P2, correlated with the marked hydrolysis of these polyphosphoinositides and the rapid hydrolytic release of the inositol phosphates (InsP2 and InsP3). The transient accumulation of the InsPs correlated with the onset of differentiation of these cells. To ascertain whether the above observations of keratinocytes that were undergoing normal proliferative and differentiating phases in culture are consistent with the more synchronized populations of proliferative and differentiating cells, we investigated the turnover of PtdIns in a Ca2+-regulated system of homogenous populations of proliferating mouse keratinocytes in 0.09 mM Ca2+, and a differentiating population in 1.8 mM Ca2+. Our data from system revealed rapid hydrolysis of the PtdIns in the prelabeled low-Ca2+ proliferating cells immediately after a switch from the low to normal extracellular Ca2+ medium. Associated with this hydrolysis was the rapid and transient accumulation of the InsPs (maximum of 60 sec). The hydrolysis of the PtdIns and the accumulation of the InsP3 were not observed when the prelabeled proliferating cells were switched from a low to a low extracellular Ca2+ medium. These results suggest that the rapid hydrolysis of the PtdIns, particularly PtdIns4,5P2, which was accompanied by the hydrolytic release of InsP3, could be the initiating signal to program proliferating keratinocytes into differentiation.

The origin of arachidonic acid (AA) found in the epidermis is not known. Two possibilities exist: either de novo synthesis within the epidermal keratinocyte, or transport of AA formed at distant tissue sites. The current study examined the ability of cultured murine and human keratinocytes to metabolize exogenously added linoleic acid (LA). Conversion of radiolabeled substrate (14C-LA) into 18:3(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) (AA) was noted. The conversion of non-radiolabeled 18:3(n-6) or 20:2(n-6) was also examined and the pattern of metabolites synthesized suggests that the preferred metabolic pathway for conversion of linoleic acid into arachidonic acid is via the classically described pathway in which a delta 6 desaturase constitutes the initial reaction. Although cultured skin fibroblasts are known to convert linoleic acid into arachidonic acid, the current study demonstrates that cultured epidermal keratinocytes can also avidly metabolize exogenous linoleic acid. The ability of cultured keratinocytes, and not of whole epidermis in vivo, to convert linoleic acid into arachidonic acid suggests that specific enzymatic activities may be induced by the tissue culture system itself. Hence, findings of metabolic capabilities in cultured cells may not necessarily be extrapolated to the in vivo situation.