Sphingomyelinase in pig and human epidermis.

The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine.     

FIBRINOLYSIS IN RELATION TO DERMATOLOGY

SUMMARY.– An attempt has been made to show the possible importance of the fibrinolytic system in dermatology. The complex mechanisms of the system have been briefly explained and emphasis has been laid on the interrelationship between the fibrinolysis, coagulation, vaso-active polypeptide and complement systems. The various methods for the study of fibrinolytic activity in the blood and in tissues are explained and their interpretation critically evaluated. The pitfalls in the study of blood fibrinolysis are emphasized. The fibrinolytic activity in normal human skin is located around the blood vessels and such activity in the epidermis has not been convincingly demonstrated. Damage to the skin in vivo by stripping with Scotch Tape appears to inhibit fibrinolysis. Homogenates of epidermis can be shown to contain an inhibitor of fibrinolysis.     

Microdisc-electrophoretic study of deoxyribonucleases in cow snout epidermis

Acid and neutral deoxyribonucleases (DNases) of the cow snout epidermis were investigated by the microdisc-electrophoresis of polyacrylamide gels containing highly polymerized DNA and by isoelectric focusing techniques. The nucleases were characterized with respect to their pH optimum. An acid DNase at pH 5.0 was detected as a single distinct band after the electrophoretic separation. After isoelectric focussing also, only one acid DNase activity with an isoelectric point (IP) of 6.2 was detectable. Neutral DNases at pH 7.4 were demonstrated as major and minor bands by their different electrophoretic mobilities. In the isoelectric focusing system also, two neutral DNases, a major one (IP, 4.6) and a minor one (IP, 6.4), were found. Characterization with respect to their histologic location showed acid and neutral DNases across the epidermal layers with the highest activities in the upper layers, where DNA concentration had been shown to be lowest. In correlation with their subcellular distribution, the highest activities of both acid and neutral DNase were found in the 105,00 X g supernatant of the subcellular fractions.     

Intracellular distribution of Hg applied to epidermis in mice. Analysis of subcellular fractions

To analyze the permeation of Hg²⁺ in the epidermis as contact dermatitis allergen, the carrier substances from the HgCl₂-treated epidermis were isolated. The peak amount of Hg²⁺ in the epidermis of mouse tail skin was observed at 12 h after the painting. Thereafter, it remained almost stationary. In the analysis of subcellular fractions, the nuclei fraction showed the highest Hg²⁺ incorporation, while the soluble protein fraction showed the lowest.     

Comparative study with two polar types of murine leprosy: an involvement of plasminogen activator and its possible regulating factor in the granulomatous tissue reaction

Enzymatic activities in a saline-extractable fraction from two polar types of murine lepromas were investigated using pyroglutamyl-glycyl-arginine-p-nitroanilide and plasminogen-rich, as well as plasminogen-free, fibrin plates. An inhibitor activity for urokinase was also measured. C57BL/6NJcl (immunologically high responder strain) mice inoculated with 2 X 10(8) Mycobacterium lepraemurium developed a localized lepromatous lesion after 4 weeks. The tissue extracts obtained after 4-6 weeks exhibited inhibition for urokinase (8.8 IU/mg protein), but no enzymatic activity. After 8-11 weeks, when the lepromas showed an ulcerative change, prominent peptide hydrolytic activity (84.8 nmol/mg/protein/ min) was demonstrated. The fibrin plate assay confirmed that plasminogen activator is predominantly involved (26.4 IU/mg protein). The proteolytic activation was apparently correlated with discharge of purulent materials containing the bacilli and subsequent limitation of leproma development. However, similar modulation of the fibrinolytic enzyme-inhibitor system was not shown in CBA/N mice (immunologically low responders). The tissue extracts showed a low level of urokinase inhibitor activity (1.9 IU/mg protein), but no peptidolytic or plasminogen activator activity. Consequently, lepromas were developed progressively until 25 weeks after infection and dissemination from the lepromatous lesion took place thereafter. In comparison with histologic findings, which revealed accumulation of lymphocytes and mononuclear cells in the peripheral zone of lepromatous lesions in the C57BL/ 6NJcl, but not in the CBA/N mice, a controlling mechanism of plasminogen activator in tissue is assumed to be involved in the development of the granulomatous tissue reaction.     

Properties of acid phosphatase in human stratum corneum

Sheets of stratum corneum were prepared by a trypsinization procedure from human skin samples, homogenized with a freeze press and then fractionated into a soluble fraction and a sediment by centrifugation at 50,000 g. Acid phosphatase (AcP) activity was found in both fractions but the bulk of the activity was detected in the supernatant. Highest activities were observed after treatment with Triton X-100. The bulk of the AcP activity remained bound to the pellet, if suspension and fractionation of the homogenized stratum corneum were performed in acetate buffer in the range between pH 4.0-5.0, probably due to ionic or hydrophobic interactions. AcP activity was totally lost if homogenates or fractions were stored frozen at -20 degrees C in buffers with pH values lower than 4.0. Triton X-100 extracts from whole skin, epidermis, stratum corneum, cultured skin fibroblasts and leukocytes were compared by isoelectric focusing. Extracts from whole skin, epidermis and stratum corneum yielded almost identical patterns with one main AcP activity band at pI of 5.65, whereas a second pronounced band from whole skin behaved similarly to one band from cultured skin fibroblasts and leukocytes (pI 6.1). The prominent band from extracts of stratum corneum and epidermis was not observed in extracts of skin fibroblasts and leukocytes. Hence, we conclude that stratum corneum and epidermis contain a tissue-specific AcP.     

The fibrinolytic activity of human oral epithelium and epidermis

The fibrinolytic activity of normal human oral epithelium and abdominal epidermis was studied by two techniques. With fibrinolytic autography, dorsal tongue, ventral tongue, cheek, palate and gingival sulcus epithelium showed fibrinolytic activity but oral gingival epithelium and the epidermis showed no activity. With a fibrin plate technique, potassium thiocyanate extracts of epidermis and oral epithelium all showed strong fibrinolytic activity, but activity in tris buffer extracts was demonstrable only with oral epithelium, and the activity was much weaker. The fact that the tris buffer extractable fibrinolytic activator was demonstrated only in oral epithelium suggests that the presence of this activator in epithelium is related to the type of keratinization and/or its rate of cell turnover.     

Blood urokinase plasminogen activator system in chronic urticaria

The evidence gathered has pointed to the fibrinolytic system, which apart from its major role in hemostasis may also be involved in inflammatory and immune processes. To understand better the role of fibrinolysis in urticaria, we measured plasma levels of the urokinase system associated molecules such as urokinase-type plasminogen activator (uPA), its soluble receptor (suPAR; CD87) and an inhibitor, plasminogen activator inhibitor type 1 (PAI-1) activity in chronic urticaria (CU) patients. Plasma was obtained from symptomatic sixteen CU patients (12 females and 4 males) showing positive response to autologous serum skin test (ASST), 28 CU patients with negative ASST (20 females and 8 males) as well as from healthy subjects matched by sex and age. The plasma level of uPA and suPAR antigens, PAI-1 activity did not differ significantly among the three subjects groups. The data obtained suggest that CU patients showing positive response to ASST have plasma profile of the urokinase system-associated proteins, which is not markedly different as compared with CU patients with negative ASST as well as healthy subjects. Our findings have also confirmed the earlier studies, suggesting that systemic fibrinolysis may not be involved in chronic urticaria.This study was supported by a research grant from the Committee for Scientific Research (NN-1-246/03).     

Tissue plasminogen activator inhibitor in the epidermis

The presence of a tissue plasminogen activator inhibitor in the epidermis was investigated. A partially purified tissue plasminogen activator from a Bowes melanoma cell line medium was used as a tissue plasminogen activator. Extracts of epidermis dissolved in a 10 mM phosphate buffer, pH 7.0, were found to contain a tissue plasminogen activator inhibitor. The same extracts also contained a urokinase inhibitor. It is not clear whether these inhibitors are the same. This study is the first to show the existence of a tissue plasminogen activator inhibitor in the epidermis.     

Free radical reduction mechanisms in mouse epidermis skin homogenates

Scavenging mechanisms for persistent free radicals were investigated using nitroxide-type radicals as model compounds. The free radical reducing activity of a) isolated thioredoxin reductase, a flavin containing oxidoreductase, b) skin homogenates, and c) the epidermis of hairless mice was studied by electron spin resonance spectroscopy. In all three systems, reduction rates of different classes of nitroxide free radicals exhibited the following order: oxazolidinoxy greater than piperidinoxy greater than dihydropyrroloxy. The main reductant for piperidinoxy radicals in mouse skin homogenate is ascorbic acid. Other reducing activities were stimulated by NAD(P)H and could be inhibited by N-ethyl maleimide, suggesting involvement of thiol-dependent processes. Mammalian thioredoxin, a competitive inhibitor of nitroxide reduction by thioredoxin reductase, significantly stimulates nitroxide scavenging in skin homogenate. Thioredoxin reductase did not significantly participate in nitroxide reduction in skin homogenates. At the surface of mouse epidermis a cationic dihydropyrroloxy nitroxide, which was stable in the presence of mammalian thioredoxin reductase was readily reduced. The epidermal reduction was inhibited by zinc, N-ethyl maleimide, and by heat (70 degrees C, 5 min). At least for mouse epidermis, reduction of a variety of nitroxides is a complex phenomenon involving enzymatic and nonenzymatic mechanisms and cannot be used as a specific assay for an enzyme, e.g., thioredoxin reductase. The study indicates the epidermis contains an effective antioxidant system that scavenges ascorbate-sensitive piperidinoxy nitroxides as well as more reducing radicals exemplified by dihydropyrroloxy nitroxides.     

Dynamic aspects of protein deimination in developing mouse epidermis

The cornified layer of mammalian epidermis contains deiminated keratins and filaggrin whose arginine residues are partly converted to citrulline residues by peptidylarginine deiminase (EC 3.5.3.15). We have attempted to study dynamic aspects of protein deimination using late embryonic to early postnatal mouse skin. The epidermis was separated from the dermis by brief immersion of skin into a weakly alkaline ammonium chloride solution. The total homogenate of the epidermis was subjected to western blotting analyses for quantitative densitometry of major keratins, deiminated proteins and immunoreactive filaggrin. We found marked increases in both deiminated keratins and deiminated filaggrin from the 18th day of gestation to 2 h after birth followed by rapid decreases to minimum levels at 6 h and subsequent gradual increases surpassing the earlier levels by 72 h after birth. Such variations were associated with consistent changes of the intensity of deiminated proteins stained immunocytochemically. These results suggest that the protein deimination might play a role in dealing with the drastic environmental change after birth. Furthermore, we found compartmentalization of both total and deiminated filaggrins into soluble and particulate fractions. The soluble compartment contained relatively more deiminated filaggrin than the particulate fraction.     

Acid hydrolases of the epidermis: subcellular localization and relationship to cornification

Three lysosomal-type acid hydrolases were examined in subcellular fractions of the developing epidermis of fetal rats to assess the relationship of degradative enzymes to cornification. As the granular layer developed and cornified between 18 and 20 days (D) of gestation, epidermal acid phosphatase increased, acid phospholipase A remained constant, and beta-glucuronidase activity declined. The enzymes were present in 3,000, 17,000, and 100,000 g particulate fractions and soluble cytoplasm. However distribution differed: acid phosphatase and phospholipase A were more preferentially localized than was glucuronidase in the 17,000 g fraction which excluded mitochondria and ribosomes and was enriched in lamellar granules. The findings suggested that acid phosphatase and phospholipase were present in membrane-bound organelles (e.g., lamellar granules) in the granular layer. Particulate acid phosphatase increased with granular layers on days 19 and 20 while a 7-fold increase in soluble enzyme coincided with cornification on day 20. As shown by isoelectric focusing, the enzyme became more heterogeneous at day 20 than at day 18, suggesting increased glycosylation. The particulate fraction displayed lysosomal characteristics with respect to release of acid phosphatase, which was inhibited by hydrocortisone and enhanced by retinol. When fetal epidermis was allowed to cornify in organ cultures, similar increases in acid phosphatase occurred. The presence of hydrocortisone did not affect increase in total enzyme but a greater proportion remained in the particulate fraction. The findings suggest that particulate acid phosphatase and phospholipase are compartmentalized in organelles with lysosomal characteristics during development of granular cells and that release of phosphatase is coincident with cornification. This may reflect not only exocytosis of lamellar granules but also intracellular release of the hydrolytic enzyme.     

Properties of partially purified mouse epidermal transglutaminase

Mouse epidermal transglutaminase was partially purified from the epidermis of newborn BALB/c mice. The epidermal homogenate was subjected to CM-cellulose chromatography and then to gel filtration on Sephadex G-150. Two separate peaks with enzyme activity were reproducibly observed in CM-cellulose chromatography. Gel filtration on Sephadex G-150 of the peak fractions in CM-cellulose chromatography gave a single enzyme activity peak (molecular weight about 55,000). Native polyacrylamide gel electrophoresis at pH 4.5 showed two major bands with enzyme activity. The final enzyme preparation (Sephadex G-150 fraction) had a pH optimum of 10.0 in glycine-NaOH buffer. Calcium ion and SH-protecting agent (dithiothreitol) were essential for enzyme activity. The enzyme was not inactivated by heating at 56°C for 45 min in the presence of calcium. Trypsin treatment of the enzyme enhanced the activity about threefold; this enhancement was blocked by trypsin inhibitor. The enzyme bound to lysine-Sepharose 4B in the presence of casein in Tris-acetate buffer, pH 6.0, at 37°C; 40% of the enzyme activity was eluted from this enzyme-lysine-Sepharose complex with 1 M NaCl.     

Urokinase-type plasminogen activator is activated in stratum corneum after barrier disruption

The plasminogen/plasmin system in epidermis is thought to be the major protease involved in the delay of barrier recovery. However, little is known about the mechanism through which this system is activated. In order to clarify this mechanism, we first determined the distribution of proteolytic activity by using in situ zymography. As a result, plasminogen-activator activity was found to be present in the stratum corneum (SC) after barrier disruption. Next, SC subjected to repeated barrier disruption was collected to identify the protease. The protease was identified as urokinase-type plasminogen activator, because flybrinolytic activity of the collected SC was abolished by addition of anti-urokinase antibody. Urokinase activation in SC was confirmed by means of an in vitro assay, in which the precursor of urokinase (pro-uPA) became active after incubation with the insoluble component of SC homogenate. These findings indicated that urokinase-type plasminogen activator is activated in SC after barrier disruption and this activation might trigger the plasminogen/plasmin system in the epidermis.     

Fibrinolytic therapy for progressive systemic sclerosis and tissue fibrinolytic activity in affected skin.

In our present study, impairment of tissue fibrinolytic activity was observed in the affected skin of patients with progressive systemic sclerosis (PSS) by fibrinolytic autography. We treated PSS with a fibrinolytic agent, urokinase, based on the idea that the impairment of tissue fibrinolytic activity is implicated in the development of PSS. Two patients with PSS have responded favorably well to administration of Urokinase.     

Distribution pattern of DNA polymerases in epidermis

DNA polymerases are known to play important roles in DNA replication and repair processes. The present study revealed that the DNA polymerase α and β participate in the cell differentiation of normal and n-hexadecane-induced hyperplastic epidermis of the guinea pigs. The epidermal cells were separated into three layers; high (HDCL), middle (MDCL), and low (LDCL) density cell layer, respectively, by Percoll gradient centrifugation. In epidermal homogenate, the activity of DNA polymerase β was higher than that of DNA polymerase α. DNA polymerase α activity was higher in the HDCL than in the other layers; however, DNA polymerase β was higher in the LDCL than in the other layers. In hyperplastic epidermis, distribution of DNA polymerase α activity was similar to the normal pattern, but DNA polymerase β was lower in the LDCL than in the other layers. The distribution of DNA polymerase α activity in epidermal nuclei was similar to that of the whole epidermal cell pattern; however, DNA polymerase β activity differed from the enzyme distribution of whole epidermal cell homogenate. Its activity was higher in the HDCL than in the other layers. In hyperplastic epidermis, both nuclear DNA polymerase α and β activities were similar to the distribution patterns of polymerase activities in hyperplastic epidermal cells. From these results, it is concluded that the distribution pattern of DNA polymerases in n-hexadecane induced-hyperplastic epidermis is not a simple augmentation of the distribution pattern in normal epidermis.     

The Role of Plasminogen Activators in Alopecia Areata1

Plasminogen activators are serine proteinases which transform the serum zymogen, plasminogen, into plasmin, a broad-spectrum protease with fibrinolytic effect. Two main plasminogen activators have been described in humans: urokinase (UK; molecular weight, 55,000) and tissue-type plasminogen activator (tPA; molecular weight, 74,000). Thirteen subjects were studied who had alopecia areata (AA), nine in the active phase and four in remission. There were alterations in the perivascular and peribulbar fibrinolytic activity in the nine subjects in the active phase of disease, suggesting a possible role of plasminogen activators in AA. A modified Todd's autohistographic method was used to evaluate cutaneous fibrinolytic activity (which depended on the activity of plasminogen activators) in the 13 AA subjects and five volunteer controls. Cutaneous fibrinolytic activity was reduced in perivascular areas, but increased in peribulbar areas, in the nine subjects in the active phase of disease. Tests with monoclonal antibodies directed against the catalytic sites of tPA and UK showed that the perivascular fibrinolytic activity was tPA dependent, and the peribulbar fibrinolytic activity was UK dependent.     

Effect of coal tar fractions on guinea-pig and human skin

In this study, the effect of topical applications of coal tar and of fractions derived from it were investigated in guinea-pigs and humans. Coal tar and its fractions produced thickening of guinea-pig epidermis in various degrees which were accurately measured. There was great variability in the epidermal thickening caused by different batches of coal tar. Separation into ether soluble and insoluble fractions yielded products which caused greater epidermal thickening than the parent substance. The acid soluble and alkali soluble extracts caused only minimal skin changes. The acid soluble fraction appeared to have similar activity to that of coal tar in the treatment of psoriasis, whereas the alkali soluble extract was without activity in patients with psoriasis. Coal tar incorporated in emulsifying ointment as well as a mixture of bases known to be present in coal tar and incorporated into emulsifying ointment produced suppression of epidermal thickening in guinea-pigs when compared with appropriate controls. It must be concluded that coal tar contains both irritant (inflammation producing) and anti-inflammatory substsnces. Their precise relationship to therapeutic effectiveness is not clear.     

Retinol esterification by mouse epidermal microsomes: evidence for acyl-CoA:retinol acyltransferase activity

In an attempt to characterize the enzyme(s) responsible for retinol esterification in hairless mouse epidermis, various subcellular fractions were incubated with [3H]retinol and the reaction products (retinyl esters) isolated by high-performance liquid chromatography. The microsomal fraction exhibited the highest esterifying activity and was stimulated by the addition of palmitoyl-CoA and dithiothreitol, but not by palmitic acid. Saturation kinetics with an apparent Km of about 6 microM for retinol were noted. Experiments with competitive and noncompetitive inhibitors of [3H]retinol esterification established that the epidermal enzyme was an acyl-CoA:retinol acyltransferase (ARAT; EC 2.3.1.76). The specificity for retinol was not absolute; a few closely related vitamin A alcohols were equally good substrates. The ARAT activity was not significantly altered by physiologic variations in the epidermal vitamin A content. In conclusion, mouse epidermis expresses ARAT activity which may be of importance for the regulation of vitamin A metabolism at the cellular level.     

Patterns of activation and inhibition of fibrinolysis in the normal skin of rat, guinea pig, and rabbit.

The distribution and intensity of fibrinolytic activity and of inhibitors of fibrinolysis in the normal skin of the rat, guinea pig and rabbit were studied with histochemical techniques. Rat skin exhibited the highest overall fibrinolytic activity and rabbit skin the lowest, with guinea-pig intermediate. The distribution of fibrinolytic areas differed in the different species. The fibrinolytic activity was caused by an activator of plasminogen related to the blood vessels or in some instances (mainly in the rabbit) to the epidermis. The ability to inhibit plasmin was highest in guinea pig skin and lowest in rat skin, with rabbit skin intermediate. In all 3 species the inhibition was related chiefly to the muscular layer. Epidermis was an additional source of inhibition.