Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies

Abstract

Here, we report a method, adapted to the human epidermis, allowing isolation of desmosomes in small tissue fractions. The methods previously developed for animal skin did not work efficiently with human tissue. Enrichment of desmosomes was performed by the association of two incubation steps in acidic solutions containing detergent NP-40 at two different concentrations followed by a sonication step. The suspension was centrifuged twice: first to remove the heavy cell fragments and then at 16 000 g on a discontinuous sucrose gradient. A desmosome-enriched fraction (DsF) was collected at the 30-50% sucrose interface. We demonstrate by immunoelectron microscopy and by western blotting that the central part of the desmosome structure is preserved as well as the antigenicity of its components. Our approach, allowing a significant enrichment of the cell fractions containing desmosomes, can be used to immunize animals and create new antibodies directed against desmosomal components. Using this strategy, new and so far poorly studied molecules incorporated into the desmosome cores could be targeted more easily.

Improvement of Bone Strength and Dermal Thickness Due to Dietary Edible Bird’s Nest Extract in Ovariectomized Rats

“…The phosphorus concentration was determined by the molybdovanadate method. 12) The hydroxyproline concentration was determined by a colorimetric method, in which the hydrolyzed solution was oxidized with chloramine-T and applied to colorimetry by Ehrlich reaction at 558 nm. 13) The maximum breaking force of the left femoral diaphysis (the center of the femur) was measured as the bone strength.…”

Cell surface glycans in the human stratum corneum: distribution and depth‐related changes

“…Mouse monoclonal anti‐CD44 HCAM (clone DF1485, diluted 1:10; Progen Biotechnik GmbH, Heidelberg, Germany); rabbit polyclonal anti‐syndecan 1 (diluted 1:40; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); mouse monoclonal IgM from KM48 hybridoma cultures recognizing a highly glycosylated differentiation‐dependent epidermal antigen (undiluted culture supernatant; Our Laboratory, Lyon, France); mouse monoclonal anti‐corneodesmosin (diluted 1:100; Abnova, Jhongli City, Taiwan); mouse monoclonal anti‐DSG1 p23 directed to the extracellular part of desmoglein 1 (undiluted; Amersham Biosciences, Little Chalfont, UK); mouse monoclonal anti‐heparin/heparan sulphate (diluted 1:50; Bio‐Rad, Kidlington, UK); mouse monoclonal anti‐chondroitin sulphate (clone CS‐56, diluted 1:50; Sigma‐Aldrich, Saint Louis, MO, USA).…”

“…Despite the rich literature concerning glycan expression on the surface of viable keratinocytes, the presence and distribution of glycans on the SC corneocytes are scarcely studied . Yet, sugar moieties may play an important functional role in the SC formation, cohesion and desquamation . On the other hand, deglycosylation of glucosyl ceramides is essential for the SC lipid maturation and permeability barrier formation …”

“…Studies dealing with the identification of epidermal glycans used lectin‐labelling technique and showed the presence of N ‐acetyl‐ d ‐glucosamine, sialic acid, α‐ d ‐mannosyl, α‐ d ‐glucosyl and d ‐galactosyl terminal sugars in the basal layer of the epidermis and N ‐acetylgalactosamine in the upper epidermal layers . Several epidermal proteoglycans bearing heparan sulphate glycosaminoglycans (GAGs), such as syndecans, epican, perlecan and glypican, many of them showing differentiation‐dependent expression, were detected in human epidermis . In their vast majority, these studies indicate a drastic reduction of these labellings within SC.…”