RESUMEN
SKALP/elafin is an epithelial proteinase inhibitor with antimicrobial properties that is not normally expressed in human epidermis, but is induced under inflammatory conditions and in some types of skin cancer. SKALP is a member of the recently described trappin gene family, which encodes a new class of proteins, characterized by a four-disulphide core and a transglutaminase substrate domain. Polyclonal antisera against SKALP have been shown to be useful for monitoring disease activity in psoriasis and tumour differentiation in squamous cell carcinoma. We developed ten different mouse monoclonal antibodies (mAbs) against synthetic peptides corresponding to a hexapeptide epitope in the transglutaminase substrate domain and three mAbs recognizing an epitope in the proteinase-inhibiting domain. The antibodies could be used with high specificity by immunohistochemistry on formalin-fixed tissue, by affinity chromatography, by Western blotting, and by enzyme-linked immunoadsorbent assay (ELISA) for the detection of SKALP/elafin. These antibodies have several advantages over existing polyclonal antisera, such as a defined epitope, the detection of full-length SKALP/elafin and unlimited supply. An antibody against the hexapeptide epitope, which is common to all known human, simian, bovine and swine trappin family members, was used to immunolocalize bovine trappins expressed in trachea, that have recently been discovered. These mAbs will serve as important new tools to measure SKALP/elafin and trappin family members in research and diagnostics.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Técnicas Inmunológicas , Familia de Multigenes/inmunología , Proteínas/genética , Proteínas/inmunología , Proteínas/fisiología , Secuencias de Aminoácidos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/químicaRESUMEN
ICAM-1 protein in keratinocytes is thought to contribute to cutaneous inflammatory reactions. Its induction depends-among others-on cytokines such as TNF-alpha, IFN-gamma, IL-1 or on retinoic acid (RA), a key regulator of epidermal homeostasis. We investigated the effect of treatments with TNF-alpha, RA or their combination on ICAM-1 expression on proliferative or differentiating keratinocytes over an 8 day culture period. Basal ICAM-1 levels were undetectable at low (30 microM) and standard (88 microM) Ca2+ and RA alone did not induce ICAM-1. However, at high Ca2+ (1500 microM), ICAM-1 levels were augmented in response to RA-treatment. TNF-alpha induced a transient ICAM-1 increase in NHK, which reached peak-levels 2-4 days post cytokine stimulus. RA potentiated the TNF-alpha-induced ICAM-1 response in all Ca2+-concentrations. This potentiating effect of RA was confirmed at the mRNA level. In summary, our results establish retinoic acid as an enhancer of TNF-alpha-induced ICAM-1 levels in NHK.
Asunto(s)
Molécula 1 de Adhesión Intercelular/biosíntesis , Queratinocitos/metabolismo , Queratolíticos/farmacología , Tretinoina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Calcio/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Humanos , PielRESUMEN
The natural form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) decreases proliferation and promotes terminal differentiation of cultured human epidermal keratinocytes. The purpose of this study was to find out to what extent the culture conditions determine the sensitivity of keratinocytes to 1,25(OH)2D3 and synthetic vitamin D analogues. Human keratinocytes were grown in microplates. Cell proliferation (MTT-assay) and differentiation (quantity of transglutaminase type I) were measured consecutively in the same monolayer. When vitamin D3 analogues were added to 50-60% confluent keratinocytes grown in serum-free keratinocyte growth medium with 0.09 mM Ca2+, stimulation of the proliferation was either minimal or non-existent, while differentiation was unaffected or slightly inhibited. There was no difference in the sensitivity to 1,25(OH)2D3 and the vitamin D3 analogues. When 1,25(OH)2D3 was added to less confluent keratinocytes (30%) a marked antiproliferative effect was observed. Addition of 3% charcoal stripped foetal calf serum further enhanced the antiproliferative effect of 1,25(OH)2D3, and a difference in the sensitivity of the vitamin D3 analogues was noted. If, finally, the Ca2+ concentration was raised to 0.3 mM, 1,25(OH)2D3 and the vitamin D3 analogues stimulated differentiation. Also, a biphasic effect on proliferation occurred: stimulation at low vitamin D concentrations and inhibition at higher concentrations. Furthermore, keratinocytes became more sensitive to the synthetic vitamin D3 analogues than to 1,25(OH)2D3: KH1060 > EB1089 > GS1500 > or = EB1213 > calcipotriol > 1,25(OH)2D3. For all compounds tested differentiation occurred at concentrations 10 to 30 times lower than for proliferation. These results indicate that the sensitivity to vitamin D3 analogues as well as the direction of the response to vitamin D3 analogues is dependent on the keratinocyte density, the availability of serum and Ca2+ concentrations.
Asunto(s)
Calcitriol/análogos & derivados , Calcitriol/farmacología , Calcio/fisiología , Queratinocitos/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Carbón Orgánico/farmacología , Medio de Cultivo Libre de Suero , Ensayo de Inmunoadsorción Enzimática , Humanos , Sales de Tetrazolio , TiazolesRESUMEN
Liarozole showed antitumoral activity in the Dunning AT-6sq, an androgen-independent rat prostate carcinoma. To investigate its potential mechanism of action, the effects of the drug doses (ranging from 3.75 to 80 mg/kg b.i.d.) on endogenous plasma and tissue all-trans-retinoic acid levels and on the differentiation status of the tumor cells were evaluated. To follow modulation of differentiation, cytokeratins were localized in the (un)treated tumors by immunocytochemistry and quantitatively determined by immunoblotting. Results showed that liarozole statistically significantly reduced tumor weight from 30 mg/kg upwards and induced accumulation of all-trans-retinoic acid both in plasma and tumors. In the tumors, a statistically significant accumulation was already noted from 7.5 mg liarozole/kg upwards. Concomitantly, the differentiation status shifted from a keratinizing towards a non-keratinizing squamous carcinoma, which was further confirmed by the cytokeratin profile of the carcinoma (presence of CK 8, 10, 13, 14, 18, 19). Immunoblotting revealed an overall decrease in cytokeratin content, except for CK 8. These findings suggest that the antitumoral properties of liarozole might be related to an increase in the degree of tumor differentiation through accumulation of all-trans-retinoic acid.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/metabolismo , Imidazoles/farmacología , Queratinas/biosíntesis , Neoplasias de la Próstata/metabolismo , Tretinoina/metabolismo , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Imidazoles/uso terapéutico , Immunoblotting , Inmunohistoquímica , Queratinas/análisis , Queratinas/genética , Masculino , Trasplante de Neoplasias , Tamaño de los Órganos/efectos de los fármacos , Próstata/efectos de los fármacos , Próstata/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Distribución Aleatoria , Ratas , Ratas Endogámicas F344 , Tretinoina/sangre , Células Tumorales Cultivadas , Vimentina/análisis , Vimentina/genética , Vimentina/inmunologíaRESUMEN
The binding of the antiviral compound R 61837 to human rhinovirus 9 (HRV 9) was studied quantitatively and compared with binding of R 61837 to HRV 9H, a semiresistant variant. For both strains, radiolabelled R 61387 bound to native particles only. The Kd values obtained by Scatchard analysis of saturation binding data were 37 nM for HRV 9 and 172 nM for HRV 9H, whereas the concentrations resulting in a 50% reduction of cytopathic effect were 42 nM and 840 nM, respectively. Reversibility experiments showed that 65% of the compound could be extracted with chloroform from HRV 9H but less than 5% could be extracted from HRV 9. Dissociation studies demonstrated that in the presence of excess unlabelled compound, the half-lives of the virus compound complex HRV 9 and HRV 9H were 385 and 15 min, respectively. The effect of this antirhinoviral compound on the formation of subviral particles induced by low pH or heat was also investigated. Rate zonal centrifugation experiments using [35S]methionine-labelled HRV 9 showed that binding of R 61837 protected the virus against heat (56 degrees C) and acid (pH 5.0) and that at the same concentration of R 61837 the semiresistant strain was stabilized to a lesser extent. This observation was confirmed immunochemically with nonneutralizing and neutralizing monoclonal antibodies. Both 80S and 130S subviral particles have C antigenic determinants, whereas native particles (150S) have been designated D. R 61837 prevented the switch from D to C antigenicity which can be induced by exposure of rhinoviruses to mild denaturing conditions. These findings indicate that the compound is able to prevent a conformational change of the capsid which may be a prerequisite for infection.
Asunto(s)
Antivirales/metabolismo , Cápside/efectos de los fármacos , Piridazinas/metabolismo , Rhinovirus/metabolismo , Anticuerpos Monoclonales , Antígenos Virales/inmunología , Antivirales/farmacología , Cápside/inmunología , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Semivida , Células HeLa , Calor , Humanos , Concentración de Iones de Hidrógeno , Cinética , Metionina/metabolismo , Piridazinas/farmacología , Rhinovirus/inmunologíaRESUMEN
We have localized desmin in the quail ovary, by the unlabelled antibody peroxidase-antiperoxidase technique, using two monoclonal and one polyclonal antisera. Special attention has been paid to the influence of fixation and of proteolytic pretreatment of sections. It appeared that the immunostaining of desmin largely depends on the nature of the fixative. Carnoy fluid, Bouin's fixative, and a paraformaldehyde-acetic acid fixative preserved the histological structure very efficiently. However, trypsin pretreatment proved to be necessary to unmask the antigenic sites in the ovaries fixed in Bouin's fixative and the paraformaldehyde-acetic acid fixative. Desmin immunoreactivity was detected in the tunica albuginea and the chordae, a number of which surrounding the blood vessels, from the hilus to the thecal surface of the follicles. Small branches of chordae connected them with the tunica albuginea, forming a suspensory apparatus. Desmin was also localized in the smooth-muscle cells of the blood vessels. In the theca, immunoreactivity was detected in the wall of arterioles, of venules, and of capillaries. Further experimental and immunohistochemical research have to be performed to establish if the suspensory apparatus is a myoid tissue.
Asunto(s)
Coturnix/metabolismo , Desmina/análisis , Ovario/análisis , Codorniz/metabolismo , Animales , Western Blotting , Desmina/inmunología , Electroforesis en Gel de Poliacrilamida , Femenino , Fijadores , Inmunohistoquímica/métodos , Dodecil Sulfato de Sodio , Tripsina/metabolismoRESUMEN
A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern.
Asunto(s)
Oro , Técnicas de Inmunoadsorción , Proteínas/análisis , Plata , Coloración y Etiquetado , Animales , Electroforesis en Gel de Poliacrilamida , Punto Isoeléctrico , Neoplasias Hepáticas Experimentales/análisis , Peso Molecular , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/inmunología , Proteínas/inmunología , RatasRESUMEN
A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.
Asunto(s)
Proteínas/aislamiento & purificación , Animales , Ácido Cacodílico , Cationes , Pollos , Colodión , Coloides , Colorantes , Hierro , Membranas Artificiales , Nylons , Polisorbatos , Dodecil Sulfato de Sodio , Coloración y EtiquetadoRESUMEN
Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Citoesqueleto/ultraestructura , Miosinas/metabolismo , Actinina/metabolismo , Animales , Células Cultivadas , Pollos , Proteínas Contráctiles/metabolismo , Filaminas , Técnica del Anticuerpo Fluorescente , Oro , Pulmón/citología , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica/métodos , Miocardio/citologíaRESUMEN
A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.
Asunto(s)
Oro , Proteínas/análisis , Plata , Animales , Fenómenos Químicos , Química , Embrión de Pollo , Colodión , Coloides , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Pulmón/análisis , Peso Molecular , Coloración y EtiquetadoRESUMEN
The use of colloidal gold and colloidal gold followed by silver enhancement as marker for dot or blot immune overlays is described. Colloidal gold probes concentrating at the sites of immune reaction gradually develop a pinkish colour during incubation that can be seen with the naked eye. With a physical developer, very high sensitivity and contrast is obtained by silver precipitation on the gold marker. Comparison of the immunogold and immunogold/silver staining methods with the indirect, PAP and ABC immunoperoxidase methods demonstrates that colloidal gold probes are excellent markers for immune overlay assays.
Asunto(s)
Complejo Antígeno-Anticuerpo/análisis , Proteínas Contráctiles/análisis , Proteínas de Microfilamentos/análisis , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Células Epiteliales , Filaminas , Molleja de las Aves , Oro , Inmunoensayo/métodos , Pulmón/citología , Plata , Coloración y EtiquetadoRESUMEN
Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.