Biomedical subjects
Publications and source records attributed to M Bergers.
We report the quantification of a membrane-bound phospholipase C in human epidermis which is active against the physiologically relevant substrate, phosphatidylinositol 4,5-bisphosphate. The level of this enzyme is significantly increased in the psoriatic lesion, both on a weight and protein basis. Etiological implications of this observation are discussed.
We describe the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a particulate rat brain preparation in the presence of the cationic detergent, cetrimide. The optimum cetrimide concentration was in the range 0.2-0.4 mg/ml; at higher or lower concentrations, the reaction rate diminished abruptly, suggesting that the electrical charge density of the micelle is critical for enzymatic attack. In other respects, such as its partial requirement for Ca++ and its pH optimum of about 7.0, the particulate enzyme seems similar to soluble preparations which have been reported previously. Interestingly, the particulate preparation could be stimulated about fourfold by a soluble brain extract in the presence of 1 mM guanosine triphosphate, confirming that the enzyme is the catalytic subunit in a membrane-bound signal-transduction system.
It is now well established that epidermis, like many other tissues, contains a phospholipase A2 that is responsible for the initiation of the arachidonic acid cascade. Here we report that human epidermis also contains a second, quite distinct enzyme of the phospholipase A2 group, which is unique in its extreme activity against phospholipids in true solution. It also differs from the classic cutaneous enzyme in that (a) its activity is not reduced by pretreatment of the skin with corticosteroids in vivo nor by treatment of the epidermal homogenate with alkaline phosphatase in vitro, and (b) its activity is reduced, rather than increased, in the lesions of inflammatory diseases such as psoriasis. The enzyme seems to occur mainly in fully differentiated keratinocytes, its level being low in the basal cell layer of epidermis and in keratinocytes cultured in vitro. On the basis of these observations, we suggest that this new phospholipase A2 is responsible for the degradation of phospholipids that accompanies the terminal keratinization process.
We describe a new approach to the quantification of tissue phospholipase A2 based on the observation that the Vmax is increased by several orders of magnitude in the presence of high concentrations of dimethyl sulphoxide. The enzyme from human skin showed optimal activity at 30% dimethyl sulphoxide and pH 6-7.5, and had an absolute dependence on Ca2+ (apparent Km = 1.4 mmol/l). The relationship between reaction velocity and substrate concentration did not follow Michaelis-Menten kinetics. The enzyme was inhibited by p-bromophenacyl bromide, half-maximal inhibition occurring at 2.4 mumol/l. Surprisingly, assay of specimens of various human tissues indicated that, with the exception of pancreas, skin contained higher levels of phospholipase A2 activity than any other tissue investigated. Equally surprising was that primate skin (in particular human) seems considerably more active than that of other mammals. Speculatively, these findings may be related to the high levels of free fatty acids occurring on the skin surface.
We report an investigation of peripheral blood monocytes from untreated patients with mild, quiescent psoriasis. Possible metabolic changes were monitored by the determination of 3 enzymes representing different pathways of glucose metabolism and 2 lysosomal enzymes. Signal processing was evaluated by the measurement of cyclic AMP levels before and after hormonal stimulation. Luminol-amplified chemiluminescence provided an objective approach to assessing phagocytic capacity. Finally, the pattern of maturation of normal and psoriatic monocytes has been compared during culture in vitro. Our results were uniformly and wholly negative; we conclude that the concept of an "intrinsic" abnormality of the psoriatic monocyte may be excluded. Possible reasons for discrepancies in the literature are discussed.
Keratinocytes have been isolated from rabbit cornea, esophagus, and skin by trypsinization. The freshly isolated cells differed in their morphology, growth characteristics in culture, transglutaminase activity (a marker for differentiation), and the composition of glycoprotein-derived fucopeptides. A clear relationship has been shown between the proportion of low-molecular-weight fucopeptides and the pattern of keratinization, being minimal in cornea and maximal in skin. Comparison with earlier literature suggests that this relationship may be a general feature of epithelial tissue. After several passages in culture, all differences between the three cell types disappeared. The unusual elution pattern of the fucopeptides on gel filtration suggests the possibility of a block at an early stage of glycoprotein synthesis under culture conditions.
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Adherent monolayer cultures of monocytes at two stages of maturation were exposed to long-wave ultra-violet radiation in the presence and absence of 8-methoxypsoralen. A remarkably predictable, dose-related lethal effect was seen, suggesting that mesenchymal cells in general may be sensitive to photochemotherapy (PUVA). Both monocytes and macrophages were lethally damaged with doses that may reach the dermis in the course of normal clinical therapy.
Monocytes purified with cell scatter monitored counterflow centrifugation were cultured in plastic (adherent) and in teflon culture bags (suspension). Sequential changes were monitored during 15 days by measuring intracellular activity of three enzymes of intermediary metabolism: glucose-6-phosphate dehydrogenase (G-6-PDH), phosphohexose isomerase (PHI) and isocitrate dehydrogenase (ICDH), and the two acid hydrolases: acid phosphatase (ACP) and N-acetyl-beta-glucosaminidase (NAG). In teflon grown macrophages a significantly lower G-6-PDH activity was seen after 15 days in comparison to plastic adherent macrophages (P less than 0.0002). For the other enzymes similar values for both culture modalities were found. The significantly, cycloheximide insensitive, higher values for G-6-PDH, PHI and ICDH in 2 h plastic adherent monocytes in comparison with plastic non-adherent monocytes, suggest a relationship between adherent capacity and the level of intermediary metabolism. The overall yield of plastic adherent macrophages after 15 days was 35% in contrast with 89% for the in suspension cultured macrophages. This corroborates the existence of adherent and non-adherent monocytes, both capable of differentiation in vitro. In 14 patients with advanced Hodgkin's disease (HD) and 14 normal controls, monocyte differentiation was studied applying both culture modalities. The enzyme levels, reflecting growth and intermediary metabolism, were similar for both groups. The adherent capacity and yield, both in teflon and in plastic, after 15 days was comparable for both groups. It was concluded that in vitro monocyte differentiation in the presence of autologous serum was qualitatively and quantitatively normal in advanced HD; this is in favour of an intrinsically normal function of monocytes in HD.
The glycocalyx of epidermal keratinocytes from psoriatic patients has been investigated by means of lectins. Striking changes were found in the levels of glucose and/or mannose (concanavalin A) and of N-acetylglucosamine and/or sialic acid (wheat germ agglutinin) on the surface of cells from the psoriatic lesion. Smaller but significant changes were seen in the clinically uninvolved epidermis of the patient. A marked increase in the affinity of the cell surface for Ulex europus agglutinin (fucose-specific) confirms our previous reports of structural alterations in fucose-containing oligosaccharides in psoriasis.
We report the uptake of four labelled sugars by keratinocytes isolated from normal epidermis, psoriatic 'uninvolved' skin and psoriatic lesions. Our findings include the following: (1) The rate of uptake of all sugars by the psoriatic lesion is increased. (2) This abnormally high uptake diminishes dramatically during 22 h incubation in vitro. (3) There is a striking abnormality in the metabolism of fucose by psoriatic keratinocytes; our data suggest an increased rate of incorporation of fucose into glycoconjugates.
A substantial proportion (20-50%) of radioactive sugar incorporated into glycoconjugates by normal human keratinocytes was soluble in chloroform-methanol; using (14C)-galactose as precursor about half of this fraction was neutral lipid. The incorporation of labelled sugars into the lipid fraction was consistently increased in keratinocytes derived from psoriatic lesions. The abnormality in fucose metabolism which we reported previously has been confirmed. In particular we have shown that the molecular weight of fucosylated glycopeptides appears to be abnormally high in the untreated psoriatic lesion, possibly reflecting an increased degree of branching. In psoriatic uninvolved epidermis and in treated psoriatic lesions the situation is reversed, the molecular weight being significantly lower than normal.
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A method is described for the preparation of isolated keratinocytes suitable for subsequent biochemical studies. Scanning electron microscopy showed that the maturation process is accompanied by an increase in cell size and a shortening and eventual loss of microvilli. Psoriatic keratinocytes are distinguishable by exhibiting longer microvilli at all levels of maturation.
Cyclic AMP levels have been determined for the first time in isolated keratinocytes. Values were more reproducible than those reported using epidermal slices. Evidence is presented to show that damage to hormone receptors is minimal. Other observations include the following: (1) Keratinocytes from psoriatic lesions showed reduced 'resting' levels of cyclic AMP as well as a diminished response to adrenaline. (2) Cyclic AMP levels were maximal in the basal cells, falling dramatically in fully differentiated keratinocytes. (3) The topical application of a corticosteroid (fluocinolone acetonide) did not modulate the response of adenyl cyclase to hormonal stimulation.