Biomedical subjects
Publications and source records attributed to W R Otto.
A growth assay using the fluorescent dye Hoechst 33258 has been developed which allows sensitive and rapid analysis of the DNA content of a variety of cell types including keratinocytes and mucus-secreting cells, and which requires a minimum of liquid handling. The assay can detect as few as 500 diploid human cells, and is compatible with the simultaneous detection of [3H]thymidine incorporation in the same cultures.
After assessment in mice, a biological skin equivalent consisting of cultured fibroblasts in a collagen gel overlain with cultured keratinocytes was applied to three patients who had had tattoos excised. In all patients the grafts took well with good cosmetic results and little or no contracture. A biopsy at 4 weeks showed that the central graft area had a fully differentiated epidermis and a mature dermis without adnexae.
We have developed an assay to study the effect of drugs on the proliferation of neonatal human skin-derived keratinocytes in vitro. Expanding populations of neonatal keratinocytes were cultured in low concentrations (0.5%) of fetal calf serum for up to 12 d. Growth of the cultures was determined by measurement of DNA using a sensitive fluorimetric assay. Addition of 10(-9)-10(-6) M 12(RS)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(RS)-HETE) neither stimulated keratinocyte proliferation nor enhanced the incorporation of [3H]thymidine. The ability of neonatal keratinocytes in low serum medium to respond to exogenous factors was demonstrated by increased growth in response to a mixture of cholera toxin, hydrocortisone, and epidermal growth factor. Confluent keratinocyte cultures in 10% human AB serum exposed to 12(S)-HETE for 72 h also showed no changes in DNA, [3H]thymidine incorporation, or labeling index. Metabolism of 12(S)-[3H]HETE was greater in cultures containing low concentrations of serum but there was no evidence for the formation of 12,20-dihydroxyeicosatetraenoic acid.
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1. Groups of lean and genetically obese (ob/ob) mice were adapted to varying energy intakes and the rates of total protein turnover in liver, gut and kidney were measured. 2. Lean mice gained less weight when fed above maintenance and lost less weight when fed below maintance than obese mice. 3. Hepatic protein turnover (mg/d) was sigmoidally related to digestible energy intake in lean mice but showed no significant changes with dietary intake in obese mice. 4. The changes in protein turnover resulted from changes in both the half-lives of protein synthesis and catabolism and in tissue protein content. 5. In the lean mice, protein turnover in kidney and gut was not significantly changed with increasing energy intake until the highest level was reached. 6. The findings suggest that protein turnover may be an important cycle for the regulation of energy balance in mice and that this cycle is impaired in the genetically obese (ob/ob) mice.
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Using histochemical techniques an abnormal programme of epidermal differentiation has been well documented in psoriasis. In order to characterise further the biochemistry of this process we have cultured dermal fibroblasts and epidermal keratinocytes from involved psoriatic skin. This has facilitated metabolic radiolabelling of skin cells and analysis of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The expression of keratin and differentiation markers was identical to that of normal keratinocytes, suggesting that psoriatic epidermal differentiation is not truncated in vitro as has been postulated to be the case in vivo. Low molecular mass components (5-8.5 kDa), previously shown to be upregulated in suprabasal keratinocytes, were detected in epidermal fractions from psoriatic skin enriched for basal cells. Of special interest was a component of 26 kDa, pI 5.9, which was highly upregulated in psoriatic as compared to normal cultured keratinocytes and was not detected in fibroblasts. These findings are in accord with a qualitatively abnormal pattern of differentiation for keratinocytes in the involved psoriatic epidermis.