RESUMO
A rabbit antiserum prepared against the serum-stimulated lipase (lipoprotein lipase) from bovine milk crossreacted with serum-stimulated lipases from human milk and from human postheparin plasma, but not with bile salt-stimulated lipase from human milk or with salt-resistant lipase from human postheparin plasma. Thus, the serum-stimulated lipase in bovine milk has immunological determinants in common with the serum-stimulated lipases in human milk and in human postheparin plasma. The time-courses for the appearance of serum-stimulated lipase and salt-resistant lipase activities in human plasma after heparin injection were different. The two activities were separated by heparin-Sepharose chromatography. After treatment of postheparin plasma with the antiserum only the salt-resistant lipase activity could be eluted from the column. Thus, these two enzyme activities in postheparin plasma reside in two different enzyme molecules.
Assuntos
Lipase/imunologia , Lipase Lipoproteica/imunologia , Leite Humano/enzimologia , Leite/enzimologia , Animais , Sangue , Bovinos , Reações Cruzadas , Feminino , Heparina/farmacologia , Humanos , Lipase/metabolismo , Lipase Lipoproteica/metabolismo , Gravidez , gama-GlobulinasRESUMO
The effects of lipoprotein lipase, phospholipase A2 and phospholipase C on chylomicron phosphatidylcholine and triacylglycerol were studied with rat lymph chylomicrons containing phosphatidylcholine labeled with [14C]oleic acid. Lipoprotein lipase purified from bovine milk readily hydrolyzed chylomicron phosphatidylcholine to lysophosphatidylcholine and fatty acid, and triacylglycerol to monoacylglycerol, fatty acid and glycerol. The rates of hydrolysis of phosphatidylcholine and triacylglycerol increased with enzyme concentration, and both decreased when fatty-acid binding sites on albumin in the incubation medium were limited. The proportion and amount of phosphatidylcholine hydrolyzed was always less than that of triacylglycerol. Analyses of hydrolytic products showed that lipoprotein lipase cleaved the 1-acyl ester bond of phosphatidylcholine. The findings indicate that lipoprotein lipase can account for some of the phospholipase A1 activity found in postheparin plasma. Phospholipase A2 and phospholipase C hydrolyzed chylomicron phosphatidylcholine, greater than 92% in 10 min, but not triacylglycerol. The resultant phosphatidylcholine-deficient chylomicrons, which could be concentrated by ultra-centrifugation and resuspended in incubation medium, were readily depleted of triacylglycerol when incubated with lipoprotein lipase. The findings indicate that phosphatidylcholine can be removed from the surface film of chylomicrons without disrupting the particles or blocking the action of lipoprotein lipase on the core triacylglycerol.
Assuntos
Quilomícrons , Lipase Lipoproteica/metabolismo , Fosfatidilcolinas , Fosfolipases/metabolismo , Animais , Ácidos Graxos/análise , Isoenzimas/metabolismo , Cinética , Ácidos Palmíticos/análise , Ratos , TriglicerídeosRESUMO
Palmar and plantar epidermis is characterized by specific features such as the development of a striking lucidum, a very thick stratum corneum, prominent rete ridges and the unique expression of keratin K9. Using organotypic cocultures of keratinocytes and fibroblasts, we investigated to which extent the specific phenotype of palmar keratinocytes is maintained in vitro and under systemic host influences after transplantation onto nude mice. In vitro, palmar keratinocytes developed a thick epithelium with a prominent, although parakeratotic stratum corneum showing no significant differences in proliferation and differentiation in coculture with either palmar or nonpalmoplantar fibroblasts. All differentiation markers including keratohyaline and membrane coating granules as well as keratin K9 were also found, but at reduced levels and with slightly altered localization. In transplants, substantial normalization towards the palmar phenotype occurred. In 3-week-old grafts, a homeostatic state was reached, as illustrated by a constant thickness of the stratum Malpighii, presence of keratin K10 throughout the entire suprabasal compartment, increased numbers of K9- and filaggrin-positive cells, and reduction of keratins K16 and K17. At the ultrastructural level, numerous membrane coating granules and an enlargement of keratohyaline granules were seen accordingly, and immunofluorescence showed intense continuous lining of the dermo-epidermal junction by laminin, type IV collagen and integrin alpha 6. The high percentage of bromodesoxyuridine-positive cells, mainly in the basal compartment, underlined the hyproproliferative state, comparable to palmoplantar epidermis. In conclusion, (i) palmar keratinocytes can preserve the potential to express their specific phenotype upon transfer to culture conditions, and (ii) this intrinsic property is not significantly modulated by the type of cocultured fibroblasts. This suggests that fibroblasts act primarily by sustaining keratinocyte proliferation which is permissive for the fully differentiated phenotype.
Assuntos
Queratinócitos/metabolismo , Queratinas/metabolismo , Pele/metabolismo , Adolescente , Adulto , Idoso , Animais , Diferenciação Celular , Transplante de Células , Células Cultivadas , Criança , Proteínas Filagrinas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Pele/citologiaRESUMO
In recent work we have shown that a serine proteinase, stratum corneum chymotryptic enzyme, with properties compatible with a role in desquamation in vitro as well as in vivo, is generally present in human stratum corneum. The enzymologic properties of the stratum corneum chymotryptic enzyme in a KCl extract of dissociated plantar corneocytes were compared with those of other known chymotryptic serine proteinases. Stratum corneum chymotryptic enzyme was found to differ significantly from bovine chymotrypsin, human cathepsin G, and human mast cell chymases in regard to inhibitor profile and substrate specificity. Stratum corneum chymotryptic enzyme was further purified from KCl extracts of dissociated plantar corneocytes by affinity chromatography on gels with covalently linked soybean trypsin inhibitor. The purified preparation contained one major component with apparent molecular weight 25 kD and one minor component with slightly higher apparent molecular weight as revealed by Coomassie staining after electrophoresis in polyacrylamide gels with sodium dodecyl sulphate of samples that had not been reduced. Both these components were associated with chymotrypsin-like activity as revealed by zymography in polyacrylamide gels with co-polymerized casein. On zymography gels, the purified preparation was also found to contain minor amounts of components with trypsin-like activity. The major purified protein had an apparent molecular weight of around 28 kD after reduction and full denaturation and was shown to contain carbohydrate.
Assuntos
Ceratolíticos/isolamento & purificação , Serina Endopeptidases/isolamento & purificação , Animais , Carboidratos/análise , Bovinos , Cromatografia de Afinidade , Humanos , Calicreínas , Ceratolíticos/química , Peso Molecular , Oxirredução , Ácido Periódico/metabolismo , Desnaturação Proteica , Serina Endopeptidases/químicaRESUMO
Granular deposits of IgA in radioactively labeled thin slices of papillary dermis from 4-mm punch biopsies of clinically normal skin from patients with dermatitis herpetiformis were solubilized with sodium dodecyl sulfate (SDS) or peptic digestion at pH 4.5. Solubilized IgA-like material was isolated by immunoprecipitation and analyzed by electrophoresis in one and two dimensions in polyacrylamide gels containing SDS followed by autoradiography. SDS extracts contained IgA-like components corresponding to monomers, dimers, as well as higher polymers of IgA. A fraction of the SDS-soluble material behaved like IgA upon immunoprecipitation but could not be deaggregated to alpha- and light chains by reduction and alkylation, suggesting that it was present as irreversible aggregates with itself or with other proteins. Peptic digestion at pH 4.5 released fragments which were precipitated by antibodies to human alpha-chains and had molecular weights similar to the proteolytic fragments corresponding to F(ab)2 and Fab formed by peptic digestion of human monomeric IgA under the same conditions.
Assuntos
Dermatite Herpetiforme/imunologia , Imunoglobulina A/análise , Pepsina A/farmacologia , Pele/imunologia , Antígenos/imunologia , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina A/imunologia , Peso Molecular , Dodecilsulfato de Sódio/farmacologiaRESUMO
We have recently described a process leading to a unipolar cell shedding from pieces of plantar stratum corneum incubated in vitro, which seems to be dependent on the activity of a serine proteinase. This process has been studied further. Electron microscopy studies suggest that cell dissociation is preceded by a degradation of the intercellular parts of desmosomes. An antiserum was raised against the transmembrane protein desmoglein I (DG I) of bovine desmosomes. In extracts of layers of plantar stratum corneum with strong intercellular cohesion, this antiserum reacted with a protein of the same apparent molecular weight as bovine DG I. In dissociated cells this DG I-like protein could not be detected; instead components with molecular weights lower than DG I which reacted with the antiserum were found. During incubation of pieces of plantar stratum corneum, under conditions leading to unipolar cell shedding, there was a progressive decrease in the amounts of the DG I-like protein, and the appearance of the lower molecular weight components with DG I-like immunoreactivity. This apparent degradation of the DG I-like protein was inhibited by aprotinin, chymostatin, and zinc ion, but not by leupeptin. The results suggest that proteolytic degradation of desmosomes may be an important part of the process leading to cell dissociation in plantar stratum corneum in vitro, and that desmosomes may play an important role in plantar stratum corneum cell cohesion.
Assuntos
Proteínas do Citoesqueleto , Glicoproteínas de Membrana/análise , Pele/análise , Animais , Aprotinina/farmacologia , Desmogleína 1 , Desmogleínas , Desmoplaquinas , Humanos , Soros Imunes/análise , Imunoquímica/métodos , Glicoproteínas de Membrana/imunologia , Microscopia Eletrônica , Coelhos/imunologiaRESUMO
Cell shedding from plantar stratum corneum was studied in vitro. Cells were shed only from the surface that had faced outwards in vivo. A quantitative measure of the cell release was obtained by determining the amount of protein that could be extracted from released and sedimented cells with 1 M sodium hydroxide. The cell release was optimal at pH 7-9 but was significant also at pH 6. The rate of cell release increased with increasing temperature, but was decreased abruptly at temperatures above 50 degrees C. The cell dissociation could be inhibited by aprotinin (Trasylol) and soybean trypsin inhibitor. Thus, it is evident that the unipolar cell dissociation in this system is mediated by an enzymatically catalyzed process, most likely with the involvement of a serine protease with an alkaline pH-optimum. The in vitro cell release shows properties indicating that it may be mediated by mechanisms also active in vivo.
Assuntos
Peptídeo Hidrolases/fisiologia , Fenômenos Fisiológicos da Pele , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Calcanhar , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Inibidores de Proteases/farmacologia , Pele/enzimologia , Pele/metabolismoRESUMO
A technique has been devised to isolate the thin papillary part of the dermis from punch biopsies. Papillary dermis has been treated with various proteolytic enzymes in order to release or solubilize the granular IgA deposits from the papillary dermis of patients with dermatitis herpetiformis. Incubation of the thin skin preparations with pepsin caused a disappearance of the specific fluorescence with antibodies to human IgA. After peptic digestion small amounts of IgA could be detected in the supernatants. There was some evidence that this amount was larger for preparations from patients with dermatitis herpetiformis than from controls. Corresponding procedures with trypsin, collagenase, or elastase had no detectable effect on the IgA deposits. The experiments with elastase seemed to give support for previous reports on association between the granular IgA deposits and the microfibrils of elastic fibers.
Assuntos
Dermatite Herpetiforme/imunologia , Imunoglobulina A/isolamento & purificação , Peptídeo Hidrolases/farmacologia , Pele/imunologia , Biópsia por Agulha , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Imunoglobulina A/análise , Microscopia de Fluorescência , Pele/efeitos dos fármacosRESUMO
We have recently shown that cell cohesion in plantar stratum corneum is mediated to a significant extent by protein structures, and that endogenous proteolysis plays an important role in desquamation in this tissue. This paper is a report of our investigations into whether similar mechanisms for cell cohesion and desquamation can be found in non-palmo-plantar stratum corneum. Biopsies of non-palmo-plantar human skin were incubated at 37 degrees C, pH 8, in a buffer with and without additions of detergents (a mixture of N,N-dimethyldodecylamine oxide and sodium dodecyl sulphate), ethylene diamine tetraacetate (EDTA), and the proteinase inhibitor aprotinin. Released cells were examined by phase contrast microscopy and counted. The incubated biopsies were examined by light microscopy. As has been previously shown by others, we found that in the presence of detergents there was a dissociation of stratum corneum cells. This dissociation was stimulated by EDTA and inhibited by aprotinin. After 36 h of incubation the entire stratum corneum and, on some parts of the biopsies, the stratum granulosum had dissociated. There was no evidence of cell dissociation in the spinous or basal epidermal layers. We conclude that the detergent-induced cell dissociation in non-palmo-plantar human stratum corneum is dependent on the action of proteinases present in the tissue on protein structures. These structures may be of significant importance for non-palmo-plantar stratum corneum cell cohesion.
Assuntos
Detergentes/farmacologia , Pele/citologia , Aprotinina/farmacologia , Biópsia , Adesão Celular/efeitos dos fármacos , Contagem de Células , Ácido Edético/farmacologia , Técnicas Histológicas , Humanos , Valores de Referência , Pele/efeitos dos fármacosRESUMO
Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered human serine proteinase that may be specific for keratinizing squamous epithelia. SCCE has properties compatible with a function in the degradation of intercellular cohesive structures during stratum corneum turnover and desquamation. SCCE is expressed in suprabasal keratinocytes. In this study, we demonstrate the subcellular localization of SCCE in the upper granular layer, in the stratum corneum of normal non-palmoplantar skin, and in cohesive parts of hypertrophic plantar stratum corneum, using immunoelectron microscopy of ultrathin cryosections labeled with SCCE-specific monoclonal antibodies detected with gold-labeled secondary antibodies. A narrow zone close to the transition between the granular and cornified layers showed positive SCCE staining after fixation. By means of immunoelectron microscopy, SCCE was found in association with structures resembling intracellular lamellar bodies in the uppermost granular cells and in similar structures undergoing extrusion to the extracellular space between the uppermost granular cells and the lowermost cornified cells. In the stratum corneum, the detected SCCE was confined to the extracellular space and was found in association with intact and partially degraded desmosomes, as well as in the parts of the extracellular space devoid of desmosomes. We conclude that SCCE may be stored in lamellar bodies in the stratum granulosum and transported via these structures to the stratum corneum extracellular space. The results further support the idea that the physiologic function of SCCE may be to catalyze the degradation of desmosomes in the stratum corneum during remodeling of the deeper layers of this tissue, and at a later stage serve as a prerequisite for desquamation.
Assuntos
Serina Endopeptidases/análise , Serina Endopeptidases/farmacocinética , Adulto , Especificidade de Anticorpos , Transporte Biológico , Desmossomos/enzimologia , Estudos de Avaliação como Assunto , Feminino , Fixadores/farmacologia , Humanos , Imuno-Histoquímica , Calicreínas , Masculino , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Concentração Osmolar , Serina Endopeptidases/imunologia , Pele/metabolismo , Pele/ultraestrutura , Coloração e Rotulagem/normas , Frações Subcelulares/enzimologiaRESUMO
Stratum corneum chymotryptic enzyme may be important in desquamation. It has also been suggested that other proteases, especially stratum corneum tryptic enzyme, may be involved. Stratum corneum tryptic enzyme has been purified and its cDNA has been cloned. Results from expression analyses indicate that stratum corneum tryptic enzyme is as skin specific as stratum corneum chymotryptic enzyme. In this work we have produced and characterized antibodies specific for stratum corneum tryptic enzyme. We have also by means of biochemical, immunochemical, and immunohistochemical methods performed studies on stratum corneum tryptic enzyme in normal human epidermis. Antibodies against bacterial recombinant stratum corneum tryptic enzyme were produced and purified by affinity chromatography. Two types of antibodies were obtained: one reacting only with pro-stratum corneum tryptic enzyme and one specific for the catalytically active part of stratum corneum tryptic enzyme. Immunohistochemistry with the antibodies reacting with pro-stratum corneum tryptic enzyme showed a staining pattern similar to stratum corneum chymotryptic enzyme-specific antibodies, i.e., the expression was confined to cornifying epithelia with a need of desquamation-like processes. Extracts of tape strips with superficial human stratum corneum were found to contain precursors as well as active forms of stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme. The enzymes had maximal activity at pH 8, but both had considerable activity also at pH 5.5. The results were compatible for a role of stratum corneum tryptic enzyme in desquamation. Stratum corneum tryptic enzyme may act in concert with stratum corneum chymotryptic enzyme and/or function as a stratum corneum chymotryptic enzyme-activating enzyme. The presence in normal superficial stratum corneum of precursors as well as of active forms of stratum corneum chymotryptic enzyme and stratum corneum tryptic enzyme, and the activity of both enzymes over a broad range of pH-values, suggest some possible ways by which the desquamation may be regulated.
Assuntos
Epiderme/enzimologia , Serina Endopeptidases/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Calicreínas , Valores de Referência , Serina Endopeptidases/fisiologia , Fenômenos Fisiológicos da PeleRESUMO
Human stratum corneum chymotryptic enzyme (SCCE) may play a central part in epidermal homeostasis. Its proposed function is to catalyze the degradation of intercellular structures, including desmosomes, in the stratum corneum as part of the desquamation process. In order to facilitate physiologic and pathophysiologic studies on SCCE we have looked for the corresponding murine enzyme. A cDNA obtained by reverse transcription-polymerase chain reaction with total RNA prepared from mouse tails as starting material was cloned, and the expression of the corresponding mRNA studied. The murine cDNA showed 77% homology to human SCCE cDNA. It had an open-reading frame encoding a protein comprising 249 amino acids with 82% amino acid sequence homology to human SCCE including the conserved sequences of the catalytic traid of mammalian serine proteases. The murine protein was deduced to have a 21 amino acid signal peptide and a four amino acid propeptide ending with a tryptic cleavage site, followed by a sequence motif identical to the N-terminal amino acid sequence of native active human SCCE. As in human SCCE the P2 position of the propeptide was occupied by an acidic amino acid residue, and the position corresponding to the suggested bottom of the primary substrate specificity pouch occupied by an asparagine residue. Analyses of mouse tissues by reverse transcriptase-polymerase chain reaction showed high expression in the skin, low expression in lung, kidney, brain, heart, and spleen, and no expression in liver or skeletal muscle. In situ hybridization of mouse skin showed expression in high suprabasal keratinocytes and in the luminal parts of hair follicles. Our results strongly suggest that we have cloned the murine analog of human SCCE cDNA.
Assuntos
Quimotripsina/genética , Pele/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , Desmossomos/enzimologia , Humanos , Camundongos , Dados de Sequência Molecular , Pele/metabolismoRESUMO
Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.
Assuntos
Cálcio/metabolismo , Epiderme/metabolismo , Adulto , Diferenciação Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Epiderme/ultraestrutura , Humanos , Tretinoína/farmacologiaRESUMO
Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered serine proteinase, which has been purified from human plantar stratum corneum. Evidence has been presented that it may play a role in the terminal stages of epidermal turnover, especially in desquamation. Two mouse monoclonal antibodies (MAb) were raised, TE4b and TE9b, that reacted specifically with SCCE in immunoprecipitation, immunoblotting, and gel-exclusion chromatography. When used in immunohistochemical experiments with the peroxidase-anti-peroxidase method, both MAb detected an antigen located in high suprabasal keratinocytes of the epidermis in normal human skin and at the vermilion border of the lip, with maximal staining of the stratum granulosum. In the hair follicles the MAb reacted with the inner root sheet only. In human oral mucosa the MAb stained the high suprabasal epithelial cells of the hard palate. This is a site where the epithelium forms an orthokeratotic stratum corneum. There was no specific staining of the epithelium of the lip mucosa or the buccal mucosa, where the epithelium does not form a stratum corneum under non-pathological conditions. A correlation therefore seems to exist between the presence of SCCE in high suprabasal cells and the ability of the epithelium to form an orthokeratotic cornified layer. We suggest that SCCE is specifically expressed in keratinizing squamous epithelia and that its expression may be part of the terminal differentiation program of this type of epithelium. These results also give further support to the idea that SCCE may play a role in the turnover and/or formation of the stratum corneum.
Assuntos
Queratinas/biossíntese , Mucosa Bucal/enzimologia , Serina Endopeptidases/isolamento & purificação , Pele/enzimologia , Diferenciação Celular , Epitélio/enzimologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Calicreínas , Queratinócitos/enzimologia , Masculino , Serina Endopeptidases/imunologia , Distribuição TecidualRESUMO
We have recently presented evidence that human plantar stratum corneum and psoriatic scales contain biologically active interleukin-1beta (IL-1beta) which has been activated in a process not involving interleukin-1beta-converting-enzyme. The aim of the present study was to compare this form of native IL-1beta with recombinant mature human IL-1beta as regards activity and the effects of inhibitors. In an assay based on the ability of IL-1 to induce the expression of E-selectin in cultured endothelial cells, the maximal activity of IL-1beta partially purified from plantar stratum corneum and recombinant IL-1beta was approximately the same. The specific activity was slightly higher for recombinant IL-1beta, although this difference was within one order of magnitude. An antibody to IL-1beta caused total inhibition of both forms of IL-1beta with no significant differences in the dose-response curves for the antibody. Immunochemical analyses and experiments with neutralising antibodies specific for IL-1alpha and tumor necrosis factor-alpha (TNF-alpha) verified that the observed activity in the partially purified preparation was due to IL-1beta, and not to IL-1alpha or TNF-alpha. There were no significant differences between the two forms of IL-1beta as regards the inhibitory effects of recombinant IL-1 receptor antagonist. Partially purified IL-1beta from plantar stratum corneum and from psoriatic scales were both highly active in the D10 proliferation assay. This activity could be totally inhibited with an IL-1beta specific antibody. This work thus confirms the presence of biologically active IL-1beta in plantar stratum corneum and psoriatic scales. Alternatively activated IL-1beta in the epidermis should be considered in future studies on skin biology and pathophysiology.
Assuntos
Interleucina-1/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta Imunológica , Selectina E/biossíntese , Selectina E/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/administração & dosagem , Interleucina-1/análise , Interleucina-1/imunologia , Psoríase/metabolismo , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/administração & dosagem , Sialoglicoproteínas/análise , Sialoglicoproteínas/farmacologia , Pele/química , Fator de Necrose Tumoral alfa/análiseRESUMO
Stratum corneum chymotryptic enzyme (SCCE) is a serine protease which may function in the turnover of the stratum corneum by means of degradation of intercellular adhesive structures between corneocytes. It is also potentially an epidermal activating enzyme for cytokines such as interleukin-1beta. The aim of this work was to study the expression of SCCE in psoriatic epidermis by means of immunohistochemistry, and to elucidate the nature of the SCCE present in psoriatic scales by means of biochemical analyses. In comparison to normal skin the number of cell layers expressing SCCE in psoriatic lesions was consistently increased. In nonlesional psoriatic skin the pattern of SCCE expression varied. It was similar to the pattern in normal skin in some biopsies, whereas in other biopsies evidence of an increased expression of SCCE was found. By means of zymography and immunoblotting, extracts of psoriatic scales were found to contain active SCCE as well as enzymatically inactive SCCE precursor. Also the effects of inhibitors on the activity towards a chromogenic protease substrate in the extracts after partial purification by gel exclusion chromatography were compatible with the presence of enzymatically active SCCE. We conclude that the expression of SCCE in psoriasis may be upregulated, and that the conversion of inactive SCCE-precursor to active SCCE occurs in the psoriatic lesion. The possible role of SCCE in the pathophysiology of psoriasis remains to be elucidated, but should be considered in future studies.
Assuntos
Psoríase/enzimologia , Serina Endopeptidases/análise , Adulto , Idoso , Especificidade de Anticorpos , Aprotinina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Calicreínas , Masculino , Pessoa de Meia-Idade , Psoríase/patologia , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/imunologia , Especificidade por Substrato , Sulfato de Zinco/farmacologiaRESUMO
We have recently reported that unipolar cell shedding from plantar stratum corneum incubated in vitro, and the associated degradation of the desmosomal protein desmoglein I, are dependent on the activity of a proteinase that can be inhibited by aprotinin, chymostatin and zinc ion. The aim of this work was to find a proteinase in plantar stratum corneum that fulfils the criteria for being the responsible enzyme. Dissociated plantar corneocytes were incubated with the chymotrypsin substrate 3-carbomethoxypropionyl-L-Arg-L-Pro-L-Tyr-p-nitroanilide hydrochloride (S-2586) and H-D-Ile-Pro-Arg-p-nitroanilide dihydrochloride (S-2288), a substrate for a wide range of serine proteinases with arginine specificity. There was a significant rate of hydrolysis of S-2586, but S-2288 was hydrolysed only very slowly. Extraction of dissociated corneocytes with buffers containing KCl or sodium dodecyl sulphate released one major proteinase that could be detected by electrophoresis in polyacrylamide gels with copolymerized casein and subsequent incubations of the gels. Both the caseinolytic activity and the S-2586-hydrolysing activities were inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. The S-2586-hydrolysing activity was also inhibited by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride. Both activities were optimal at pH 7-8 but were also significant at pH 5.5. On gel exclusion chromatography, the S-2586-hydrolysing and caseinolytic activities were eluted with an apparent molecular weight of around 18 kDa. When analyzed by electrophoresis in the presence of sodium dodecyl sulphate under non-reducing conditions the caseinolytic enzyme had an apparent molecular weight of around 25 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Quimotripsina/fisiologia , Epiderme/fisiologia , Aprotinina/farmacologia , Comunicação Celular/fisiologia , Cromatografia em Gel , Quimotripsina/antagonistas & inibidores , Eletroforese em Gel de Poliacrilamida , Células Epidérmicas , Humanos , Ceratolíticos/farmacologia , Oligopeptídeos/farmacologia , Zinco/farmacologiaRESUMO
In preparations of papillary dermis taken from clinically normal skin of patients with dermatitis herpetiformis (DH), the disappearance of granular IgA deposits induced by peptic digestion was partial at pH 4.5 and total at pH 4.1, as visualized by immunofluorescence microscopy. In a model system in which human monomeric IgA was digested by pepsin using a pepsin-IgA ratio of 100:0.3, an IgA concentration of 3 lambda g/ml and a pH range of 5.1-4.1, the degradation of IgA molecules did not appear to proceed beyond the formation of fragments corresponding to F (ab)'. When supernatants after peptic digestion of papillary dermis at pH 4.1 were analysed by sodium-dodecylsulphate (SDS)-polyacrylamide gel electrophoresis after reduction and alkylation followed by electrophoretic transfer to nitrocellulose membranes and subsequent immunochemical detection, IgA-like material corresponding to the alpha-chain part of F (ab)' fragments was found in preparations from DH patients but not in those from controls.
Assuntos
Dermatite Herpetiforme/imunologia , Imunoglobulina A/análise , Fragmentos Fab das Imunoglobulinas/análise , Pele/imunologia , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Pepsina A/farmacologiaRESUMO
A recently described endogenous proteolytic process in pieces of human plantar stratum corneum incubated in vitro has been further studied. This process leads to a decrease in cohesion between the cells that had been facing outwards in vivo. Using two methods, that differed with respect to efficiency, to detach surface cells with decreased cohesion, the process could be divided into two steps. The first step took place irrespective of the presence of ethylenediaminetetraacetate (EDTA) and led to a moderate decrease in cohesion between surface cells. The second step occurred only in the presence of EDTA and advanced to a point where the surface cells could be separated from the remaining cohesive tissue pieces by simple agitation. Both degradation steps could be inhibited by aprotinin and chymostatin but not by leupeptin. Zinc sulfate inhibited the first step. The results indicate that there are two different types of protein structures being degraded during the process of cell shedding in vitro. A chymotrypsin-like enzyme may be involved in the process.
Assuntos
Proteínas do Citoesqueleto , Proteínas/metabolismo , Serina Endopeptidases/fisiologia , Pele/citologia , Aprotinina/farmacologia , Quimases , Técnicas de Cultura , Desmoplaquinas , Ácido Edético/farmacologia , Pé , Humanos , Glicoproteínas de Membrana/metabolismo , Pele/metabolismoRESUMO
Corneodesmosin, defined as the protein recognized by the monoclonal antibody G36-19, is a recently described late differentiation protein of human cornified epithelium. In the stratum corneum it is localized in the extracellular parts of modified desmosomes (corneodesmosomes) and adjacent parts of the cornified cell envelope. The aim of the present study was to investigate whether corneodesmosin undergoes changes in the stratum corneum which can be related to the cohesive state of the tissue and to desquamation. Extracts of plantar stratum corneum from various tissue levels and tape-stripped non-palmoplantar stratum corneum were analysed by immunoblotting with G36-19. In addition, the fate of corneodesmosin during shedding of surface cells in a recently described in vitro model of desquamation in plantar stratum corneum was investigated and compared with the degradation of the desmosomal protein desmoglein I in this system. The apparent molecular weights of the major G36-19-positive components in plantar stratum corneum ranged between 33 and 48 kDa. The components with the highest molecular weights were predominant in the deepest tissue layers. In the intermediate tissue layers G36-19-positive components of molecular weight 33-36, 39 and 44-48 kDa were found. There seemed to be a further degradation of the 33 to 36-kDa components in the most superficial parts of the tissue. In surface cells dissociated in vivo as well as in vitro no G36-19-positive components with molecular weights above 36 kDa were detected. Results from analyses of nonpalmoplantar stratum corneum suggested that corneodesmosin is degraded in this tissue in a way that may be similar to that in plantar stratum corneum.(ABSTRACT TRUNCATED AT 250 WORDS)