Mitochondria and nuclei of pig and human epidermis: isolation and lipid composition.

Pig and human epidermis were mechanically disrupted with a Polytron homogenizer and mitochondria and nuclei were isolated from homogenates in buffered sucrose and 2.5% citric acid solution, respectively, by differential and density-gradient centrifugation. Examination of the mitochondrial preparations by electron microscopy and the assay of several marker enzymes indicated that they consisted mostly of mitochondria with some microsomal contamination. The nuclear preparations were substantially free from contaminants as judged by particle counts with the aid of phase and electron microscopy. The lipid compositions of the mitochondria from both species were characterized by a high concentration of cardiolipin and low concentration of cholesterol and sphingomyelin and an absence of glycosphingolipids as compared with the lipid composition of the whole epidermal cell. The lipid compositions of the nuclei were characterized by a high proportion of phosphatidyl choline and an absence of glycosphingolipids. The neutral lipids accounted for an unusually high proportion of the total lipids especially in human epidermal nuclei. Although the technical difficulties in isolating subcellular organelles from mammalian epidermis limit the yield and quality of the mitochondria, these preparations as well as those of nuclei are superior to preparations reported previously and are pure enough for a valid comparison with other membrane systems of the epidermal cell.

Raised epidermal phospholipase A2 activity in rheumatoid arthritis.

Epidermal phospholipase A2 (PLA2) in RA patients was significantly higher than in normals (p less than 0.0001) but lower than in psoriatic patients (p less than 0.05). No relationship was observed between PLA2 activity and commonly used measures of rheumatoid disease activity in a cross-sectional study. However, in a longitudinal study change in PLA2 activity correlated strongly with changes in disease activity.

Epidermal lipids.

Synopsis From the time an epidermal cell leaves the basal layer to the time it is desquamated, the cell lipids change dramatically, both qualitatively and quantitatively. The most abundant lipid class in basal cells is phospholipid with the remaining lipid being accounted for by roughly equal proportions of cholesterol, non-esterified fatty acid, triacylglycerol and glycolipid: minor components include cholesteryl esters and ceramide. In contrast, approximately half of the lipid in a desquamated cell consists of ceramide, with the remainder consisting largely of cholesterol and non-esterified fatty acid. Immediately before desquamation, small concentrations of cholesteryl sulphate and glycolipid have been found and there is evidence that these polar lipids are important components of the water barrier and also contribute towards the physical integrity of the lower part of the stratum corneum. The change in lipid content as cornification proceeds is no less dramatic than the change in lipid composition. A basal cell contains about 10 pg lipid, whereas a desquamated stratum corneum cell contains approximately six times this amount. The change in lipid composition of a cell undergoing cornification results, therefore, largely from de novo synthesis of lipid, especially cholesterol, non-esterified fatty acid and ceramide. Les lipides de l'épiderme.

Changes in dry weight and projected area of human epidermal cells undergoing keratinization as determined by scanning interference microscopy.

Cells, obtained from human leg by successive treatments with trypsin, were air dried on microscope slides before mounting in glycerol. Dry weights and projected areas of individual cells were measured using a Vickers M 86 scanning microinterferometer. The dry weights of cells varied from 100 pg for basal cells to 700 pg for large squames. Corresponding projected areas varied from 100 to 1500 mum2.

Lipid compositions of cells isolated from pig, human, and rat epidermis.

Epidermal slices from pig, human, and rat skin were treated with dilute buffered trypsin solution (0.005%, w/v), and suspensions of mixed basal and spinous cells were obtained in good yield. Total lipids accounted for approximately 8% of the pig, 10% of the human, and 20% of the rat epidermal cell (dry weight). Phospholipids in pig, human, and rat cells accounted for, respectively, 62%, 53%, and 35% of the total lipids. Phosphatidylcholine (34-38%), phosphatidylethanolamine (18-23%), and sphingomyelin (17-21%) were major compounds in all species. The major neutral lipids were sterols (mostly cholesterol) and triglycerides. Free fatty acids were a major lipid class in pig and human cells, whereas wax esters were a major component in rat epidermal cells. Nearly half (45%) of the sterols in rat cells but less than 10% of those in pig and human cells were esterified. Cholest-7-ene-3beta-ol accounted for 20% of the total sterols in rat cells. Cholesteryl sulfate and ceramide were minor lipids in the three species. The predominant glycosphingolipid (greater than 99%) was glucosylceramide, which accounted for 7% and 9%, respectively, of the total lipids in pig and human cells. A significant proportion (pig, 17%; human, 11%) of the fatty acids in the glucosylceramides were C26:0 and C28:0.

Different populations of pig epidermal cells: isolation and lipid composition.

Preparations representing populations of (a) basal and spinous cells, (b) granular cells, and (c) stratum corneum cells were obtained by successive treatments of epidermal slices from pig skin with dilute buffered trypsin solutions. Total lipids accounted for about 8% of the cell dry weight in each of the three populations. Phospholipids, which predominated in the basal and spinous cells, accounted for only 21% of the total lipids in the granular cells and less than 0.1% in the stratum corneum. The latter cells contained more cholesterol (23% of total lipid) than either the granular cells (18%) or the basal and spinous cells (8%). The proportion of ceramide was also much higher in the stratum corneum (17%) and granular cells (9%) than in the basal and spinous cells (1%). The relative amounts of glycosphingolipid (glucosylceramide) and cholesteryl sulfate in the total lipids of stratum corneum cells were less than half those in the granular cells and basal and spinous cells. A novel phospholipid was a major component (26% of total) of the phospholipids from granular cells. The compound, which was partially characterized, contained phosphorus, fatty acids, and glycerol (molar ratio 1:3:2) and appeared to be a neutral derivative of phosphatidic acid.

Brittle fracture of epidermal cells by liquid shear.

A suspension of epidermal cells obtained from pig tail skin by trypsinization was subjected to high liquid-shear forces in a French press. The material issuing from the press was examined by phase-contrast microscopy, transmission electron microscopy and scanning electron microscopy. The cytoskeleton of tonofibrils retained the shape of cell fragments, and subcellular organelles remained enmeshed in the network of tonofibrils. Examination of some cell fragments by scanning electron microscopy revealed the internal organization of the tonofibrils. The relevance of these findings to the problem of isolating subcellular fractions from epidermis is discussed.

Lipids in the laminated layer of liver, lung and daughter hydatid cysts of equine Echinococcus granulosus (Cestoda).

Lipids extracted from the laminated layers of horse liver and lung hydatids, including a daughter liver cyst, were analysed using TLC. No differences in lipid composition was detected in 11 liver cysts, whether from the same or different livers, and di- and triacylglycerols, cholesterol, wax and steryl esters, oleic acid, sphingomyelin, phosphatidyl choline, phosphatidyl inositol and ceramide hexosides were detected. The daughter cyst differed from its "parent" cyst in lacking diacylglycerols and wax and steryl esters. The lung cyst differed from the liver cysts in that cholesterol, wax and steryl esters and diacylglycerols were not detected.

The plasma membrane of Malpighian cells from pig epidermis: isolation and lipid and protein composition.

A plasma membrane fraction from Malpighian cells has been isolated by differential and density gradient centrifugation of a pig epidermal homogenate. It was enriched in the marker enzymes 2-naphthylamidase, 5'-nucleotidase, phosphodiesterase I and acid phosphatase and depleted of NADH-ferricyanide reductase and cytochrome c oxidase. It had a protein to lipid ratio of 3:2 by weight. The protein composition was complex with compounds ranging from a molecular weight of 150,000 down to 13,000. Major components with molecular weights 120,000 to 90,000 were glycoproteins. Two other components had molecular weights of 39,000 (actin ?) and 24,000. There were minor components with molecular weights from 63,000 to 46,000. About 76% of the total lipid was present as phospholipid, which was enriched in sphingomyelin. Most of the neutral lipids were accounted for by cholesterol, triacylglycerols and fatty acids: very little glycosphingolipid was present. The preparation was probably derived from non-desmosomal areas of the plasma membrane of Malpighian cells, as desmosomes were not seen in the preparation.

Epidermal phospholipase A2 activity is raised in the uninvolved skin of psoriasis.

In uninvolved epidermis from nine psoriatic subjects, the mean phospholipase A2 activity was significantly greater (P less than 0.01) than in nine controls. The increased activity of this enzyme could lead to an increase in the concentration of free arachidonic acid which, in turn, could lead to increased concentrations of inflammatory mediators.

Inhibition of normal and psoriatic epidermal phospholipase A2 by picomolar concentrations of recombinant human lipocortin I.

Normal human and psoriatic epidermal phospholipase A2 (PLA2) was inhibited by human recombinant lipocortin I when the substrate was present in a several million-fold molar excess. Inhibition was not total, even at relatively high concentrations of lipocortin I. It is therefore suggested that human epidermis contains two species of PLA2: one that is controlled by lipocortin I and one that is not.

Utilization of epidermal phospholipase A2 inhibition to monitor topical steroid action.

The effect of several steroid creams on epidermal phospholipase A2 (PLA2) activity in symptomless psoriatic and normal epidermis was studied. The magnitude of PLA2 inhibition produced by the steroids was directly proportional to the initial level of the enzyme activity. This differential inhibition resulted in PLA2 activity approaching or attaining the normal range regardless of its initial level. Clobetasol propionate 0.05% (Dermovate) produced more enzyme inhibition than betamethasone valerate 0.1% (Betnovate) but there was no difference in inhibition between this latter steroid and clobetasone butyrate 0.05% (Eumovate). All were more inhibitory than hydrocortisone I% (Efcortelan).

Modulation of phospholipase A2 activity in extracts of lesion-free psoriatic epidermis by alkaline phosphatase and a protein phosphatase inhibitor.

Phospholipase A2 activity is raised in non-lesional psoriatic epidermis compared with normal epidermis. It has been shown that the activity of this enzyme is controlled by an inhibitory protein the inhibitory effect of which is increased by dephosphorylation. Treatment of epidermal extracts with alkaline phosphatase reduced the phospholipase A2 activity, both in normal and in lesion-free psoriatic epidermis. Inclusion of pyrophosphate, a protein phosphatase inhibitor, in the homogenizing medium caused the activity of phospholipase A2 in epidermal extracts from normal and lesion-free epidermis to be raised to the same high level. These results are consistent with the hypothesis that the raised phospholipase A2 activity in psoriatic epidermis is due to hyperphosphorylation of an endogenous inhibitor as a result of defective control of a phosphorylation/dephosphorylation mechanism. The relevance of these findings to other work is discussed.