Biomedical subjects

M M Brysk

Publications and source records attributed to M M Brysk.

At least 55 records · Page 3Linked to original sources

PubMed1988

Purification of proteins from polyacrylamide gels, free of detergent or dye.

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.

Chromatography, Ion Exchange↗

PubMed1987

Aberrant expression of a chemokinetic glycoprotein in psoriatic skin.

Clinically involved and uninvolved skin samples of 6 psoriatic patients, 4 samples each of normal skin specimens, basal cell carcinoma and keratoacanthoma were studied by an indirect immunofluorescence technique. The monospecific antibody used in this study was directed against a 30 kD glycoprotein, normally expressed by the terminally differentiated corneocytes. Functional characterization of this glycoprotein was evaluated by neutrophil cell movement assays. The involved and uninvolved skin of psoriatic patients expressed the 30 kD glycoprotein not only in the stratum corneum but in all the viable epidermal layers as well. Functional studies revealed this glycoprotein to be a potent chemokinetic molecule. These results suggest that the 30 kD glycoprotein is an intrinsic chemokinetic molecule of the terminally differentiated corneocytes, and its precocious and aberrant expression in psoriatic epidermis is potentially responsible for some of the pathophysiologic aspects of psoriasis.

Chemotactic Factors↗

PubMed1986

Isolation and characterization of a 30 kDa membrane glycoprotein from human stratum corneum.

Using iodinated concanavalin A in conjunction with gel electrophoresis, we have identified a 30 kDa glycoprotein in the stratum corneum of human skin. We isolated this glycoprotein by extraction in nonionic detergent, affinity chromatography and preparative gel electrophoresis. It binds to concanavalin A but not to three other lectins. The purified glycoprotein migrates at 30 kDa whether or not reducing agents are present. It is rich in histidine and lysine, but lacks arginine, proline, tyrosine and methionine. It is clearly distinct from filaggrin. We prepared a monospecific polyclonal antibody to this glycoprotein and localized it by immunohistochemistry exclusively to the cell membrane of corneocytes. We postulate that the glycoprotein may play a role in the cohesion and desquamation of corneocytes.

Adult↗

PubMed1986

Response of malignant and nonmalignant epidermal cell lines to tunicamycin.

Exposure of fibroblasts to tunicamycin has been found to be cytotoxic for transformed cells, but not for nontransformed cells. With two mouse epidermal cell lines of common origin, we observe a contrary pattern: The malignant cells are more resistant to tunicamycin than their nonmalignant counterparts, as measured by growth and viability. With respect to the glycosylation of sugar precursors, the incorporation of mannose is more inhibited than that of glucosamine, while fucose is least impacted. Sugar incorporation is less reduced for the malignant cells, by a factor of two for fucose and more modestly for the other two sugars. There are no significant morphological changes; in particular, the desmosomal junctions are not affected. On polyacrylamide gels, we note intensity variations in several protein bands in response to tunicamycin, but little difference between malignant and nonmalignant cells when using either Coomassie stains or Concanavalin A overlays.

Animals↗

PubMed1986

Endogenous lectin from terminally differentiated epidermal cells.

We isolated a concanavalin A (Con-A)-binding glycoprotein from human stratum corneum by nonionic detergent extraction, lectin affinity chromatography, and preparative gel electrophoresis. This glycoprotein migrates as a single band at 40 kilodaltons at sodium-dodecyl-sulfate gel electrophoresis with or without the presence of 2-mercaptoethanol. It was shown to have a heterogeneous distribution between pH 5.6 and 7.6 by isoelectric focusing. The glycoprotein is histidine rich (10.4%) but is distinct from other histidine-rich proteins (epidermal filaggrin and the histidine-rich glycoprotein from serum). It does not bind to lectins specific for L-fucose or alpha-D-galactose. We prepared a monospecific polyclonal antibody to the 40-kilodalton glycoprotein; at the ultrastructural level, it cytoimmunolocalizes exclusively to the membranes of the stratum corneum. A unique feature of the glycoprotein is that it is an endogenous lectin: it hemagglutinates trypsinized and gluteraldehyde-fixed rabbit erythrocytes. The inhibition of its hemagglutination was found to be greatest with amino sugars, down to a saccharide concentration of 10(-5) mM. Such a high affinity of binding at the cell surface suggests that this glycoprotein is a major carbohydrate-binding, cross-linking molecule that holds adjacent corneocytes together in the stratum corneum. We hypothesize that this lectin plays a role in the adhesion and desquamation of the stratum corneum.

Amino Acids↗

PubMed1986

Modified distribution of epidermal glycoproteins in the nude mouse.

The nude mouse is an athymic mutant whose immunological deficiency has been exploited for transplantation of normal and diseased xenogeneic tissue. Histologically, its skin has no unusual features apart from the absence of hair. We report here a biochemical study of its epidermis, with comparison to the hairless mouse (which is devoid of hair but otherwise functionally normal). The epidermal glycoproteins were probed with the lectin, concanavalin A (Con A). Fluorescein isothiocyanate (FITC)-Con A overlays of cryostat skin sections gave a similar fluorescent pattern for both mouse strains: all the viable epidermal cell layers were labeled but not the stratum corneum. In contrast, when different populations of keratinocytes that were separated on Percoll gradients were analyzed by gel electrophoresis, and the gels then overlaid with iodinated Con A, all the epidermal layers, including the stratum corneum, were labeled. For all the epidermal cell layers there are substantial differences between the two mouse strains. We observe changes in the glycoprotein distribution with the stage of differentiation. Comparison with our earlier data for human epidermis indicates that the discrepancies between the nude mouse and the hairless mouse are much greater than those between the latter and man. The most striking difference is the absence in the stratum corneum of the nude mouse of a 40 K glycoprotein which is the dominant feature for the hairless mouse and for man. The gel patterns point to functional discrepancies in the epidermis of the nude mouse, particularly in the stratum corneum, not evident histologically or with FITC-Con A.

Animals↗

PubMed1984

Pachyonychia congenita. Electron microscopic and epidermal glycoprotein assessment before and during isotretinoin treatment.

Two patients, a father and son, with pachyonychia congenita were treated with orally administered isotretinoin because the extreme deformity and discomfort associated with their massive keratoderma interfered with their work and school, respectively. While clinical benefits could not be sustained, electron microscopic findings compatible with suppression of abnormal keratinization were observed. In addition, skin biopsy samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels were then subjected to a lectin overlay technique with concanavalin A labeled with iodine 125. The distribution of specific glycoproteins was found to be different for lesional as against normal epidermis. The procedure was repeated after oral treatment with isotretinoin. The labeled glycoprotein pattern of the lesional epidermis was clearly distinguishable from both the pretreatment lesional and the normal epidermis; it was mostly intermediate between the two. The normal epidermis was virtually unaffected by the retinoid treatment.

Adult↗

PubMed1984

Characteristics of a human epidermal squamous carcinoma cell line at different extracellular calcium concentrations.

A continuous line derived from a human skin squamous cell carcinoma has been grown in media of high, normal and low Ca2+ concentrations. The growth rate was unaffected by the Ca2+ levels even though morphological changes were observed. Desmosomes were absent at low Ca2+ and areas of cell piling were observed at high Ca2+. Cell protein staining patterns on polyacrylamide gels were identical for cells grown at the three Ca2+ levels. The variations were minor for the glycoproteins reacted with 125I-conA. Lactoperoxidase iodination revealed changes in cell surface proteins, most markedly in the emergence of new proteins at high Ca2+.

Calcium↗

PubMed1984

Concanavalin A distinguishes among diseases of altered epidermal differentiation.

Mannose-containing of glycoproteins from lesional tissue of several diseases of aberrant epidermal differentiation (palmar-plantar keratoderma, pachyonychia congenita, psoriasis, and epidermolytic hyperkeratosis) were analyzed by overlaying iodinated concanavalin A onto molecules separated by polyacrylamide gel electrophoresis. Gel autoradiograms showed that biopsy samples from patients with the same disease were very similar. The radioactivity profiles were different for each disease and were distinguishable from each other and from normal epidermis and callus. The resolution and sensitivity of this technique may be of diagnostic significance.

Cell Differentiation↗

PubMed1984

Concanavalin A binding glycoprotein in human stratum corneum.

A mannose-containing 40K glycoprotein has been identified in the stratum corneum of normal human epidermis. It is apparently membrane-bound and in the intact epidermis it is inaccessible to either concanavalin A or to trypsin. After it is detergent-solubilized, it can be labeled with concanavalin A or destroyed with trypsin. There is little or none of this glycoprotein in the viable cells of the epidermis.

Autoradiography↗

PubMed1984

Discrimination between nonmalignant and malignant cells by combinations of IFN-gamma and IFN-alpha/beta.

Combination treatment with interferon (IFN)-gamma and either IFN-alpha or IFN-beta synergistically enhances (potentiates) the anticellular effects of the IFNs. IFNs have been shown to have anticellular activity against normal as well as malignant cells. To study the relative effectiveness of IFN's anticellular activity against nonmalignant and malignant cells in the mouse system, two paired sets of nonmalignant/malignant cells (JB-1/JB-8; D-1/D-11a) were studied using 3-day kinetics of cell growth experiments. Treatment of the nonmalignant cells with IFN-gamma or IFN-alpha/beta employed separately had little or no effect on the growth of the cells at the IFN concentrations employed. Combination IFN treatment had only a slight effect on the growth of these cells. Treatment of the malignant cells with IFN-gamma or IFN-alpha/beta employed separately had low levels of effect on the growth of the cells. In contrast, treatment of these malignant cells with combined IFN-gamma and IFN-alpha/beta had a very marked anticellular effect, dramatically showing the growth of, or even killing, the malignant cells. These observations appeared to be independent of the growth status of the cells, since stationary-phase and log-phase malignant cells maintained their relatively greater sensitivity to the IFNs. Thus, IFN treatment, particularly combination IFN treatment, had a differential effect on the growth of the paired sets of nonmalignant and malignant cells and appeared to sharply differentiate between nonmalignant and malignant cells.

Animals↗

PubMed1983

Calcium-mediated changes in related malignant and nonmalignant mouse epidermal cell lines.

Two epidermal cell lines of common origin, one tumorigenic and the other not, have been grown in media of different Ca2+ concentrations (low: 0.07 mM; normal: 1.4 mM; high: 10 mM). At normal Ca2+ levels, both cell lines had similar growth rates. The growth rates were reduced at both higher and lower Ca2+ concentrations, with greater inhibition of the tumorigenic than the nontumorigenic cells. At a given level of Ca2+, the 2 cell lines could not be distinguished in their polyacrylamide gel patterns by protein staining, by [125I]concanavalin A reactivity, and by lactoperoxidase iodination. Changes in surface membrane proteins with Ca2+ concentrations were observed by lactoperoxidase iodination, whereas the other techniques, which measure total cell proteins or glycoproteins, showed no substantial variation.

Animals↗

PubMed1982

Lactoperoxidase-catalyzed iodination of membrane proteins in normal and neoplastic epidermal cells.

Cell surface proteins of normal human, mouse, and rat cells in primary culture, of human basal cell carcinoma, and of carcinogen-transformed cell lines were examined by lactoperoxidase-catalyzed iodination. Autoradiography was used to record the distribution of label in the polypeptide subunits separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was no significant difference in the results for normal cells of human, mouse, and rat. On the other hand, carcinogen-transformed mouse cells had many more labeled polypeptide bands of widely distributed molecular weights. The iodination profiles from human basal cell carcinoma cells were much more akin to those from normal cells than to those from carcinogen-transformed cells. Treatment of iodinated cells with proteolytic enzymes visibly altered the polypeptide bands.

Adult↗

PubMed1982

Concanavalin A binding glycoproteins of epidermal cells.

The Concanavalin A reactive glycoproteins of epidermal cells were analyzed by the application of the iodinated lectin to molecules separated by SDS-PAGE. Normal epidermal cells were maintained as undifferentiated or differentiated by controlling the Ca++ concentration of the growth medium. Some 20 labeled bands could be resolved. Their relative intensities changed dramatically with the stage of differentiation. Fresh tissue gave a radioactive profile similar to that for cultured differentiated cells, except for evidence of damage from the techniques used to separate the epidermis from the dermis (the damage being progressively more severe going from heat to ammonium chloride to trypsin separation). The labeling patterns for three carcinogen-transformed cell lines were markedly different from those of the normal cells. The least tumorigenic cell line had a profile in many respects intermediate between those of the normal differentiated and undifferentiated cells, while the other 2 lines showed greater deviation.

Animals↗

PubMed1982

The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins.

Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with 125I-lectins, and by the metabolic incorporation of L-(3H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells (and also for neoplastic cells) but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with 125I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells.

Agglutinins↗

PubMed1981

The activity of interferon on ultraviolet light-induced squamous cell carcinomas in mice.

The activity of interferons was tested in ultraviolet light-induced skin tumors in mice. After the tumors were well established, they were injected and measured daily for 19 days. Mouse virus type (IF-alpha + IF-beta) and immune (IF-gamma) interferons were injected intralesionally into three groups of test animals and compared with a fourth group which received mock interferon (control). When used separately, virus type and immune interferons did not affect tumor growth; however, we observed regression in tumor size when the two interferons were used in combination.

Animals↗

PubMed1981

Immunity and skin cancer.

Observations in humans and animal studies support the theory that immunologic surveillance plays an important role in limiting the development of skin malignancies. These immune responses undergo progressive diminution with age. In addition, other factors, such as bereavement, poor nutrition, and acute and chronic exposure to ultraviolet light, can further diminish immune mechanisms. We believe these decreases in immune function in combination with the carcinogenic effect of ultraviolet light are important in the production of skin cancers.

Adult↗

PubMed1981

Separation of newborn rat epidermal cells on discontinuous isokinetic gradients of PERCOLL.

A one-step procedure has been developed for the separation of epidermal cells using PERCOLL (a new colloidal silica medium of low viscosity, osmolarity, and toxicity) for density gradient centrifugation. Newborn rat epidermal cells were dispersed with trypsin-EDTA and separated into 4 fractions in discontinuous isokinetic gradients. The cell fractions were characterized by their appearance in photomicrographs and their distribution by number and size. Preferential incorporation of 3H-thymidine and 14C-glycine, by basal and granular cells respectively, confirmed the identification of cell types. The basal cells, which were collected in the densest fraction (1.090), were the most homogeneous population with a mean diameter between 7-8 mum and showed 98% viability. The granular cells predominated in the least dense fraction (1.023). The intermediate fractions contained spinous cells admixed with the other cell types.

Animals↗