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Biomedical subjects

M M Brysk

Publications and source records attributed to M M Brysk.

69 records · Page 4Linked to original sources
PubMed1977

Tonofilament protein from newborn rat epidermis. Isolation, localization, and biosynthesis of marker of epidermal differentiation.

A novel extraction procedure, previously used on the cell walls of dermatophytes, has been applied to the epidermis of newborn rat. A leucine-rich fraction was isolated which contained over 60% of the total epidermal radioactivity from [3H]leucine in 15 to 20% of the total protein. This fraction was further purified by chromatography in DEAE-cellulose and Sephadex G-200. The protein with the highest specific activity from [3H]leucine was isolated and gave a single band in sodium dodecyl sulfate polyacrylamide gels of molecular weight = 58,000. Antibody to this protein gave a single precipitin band by immunodiffusion and immunoelectrophoresis in agar with the purified protein. This antibody ultrastructurally immunolocalized specifically over tonofilaments in all layers of the epidermis, but showed no reaction in the dermis. The synthesis of this protein in vitro was inhibited by puromycin but not by actinomycin D, suggesting ribosomal synthesis involving a relatively long lived messenger.

Amino Acids↗
PubMed1969

Beta-cyanoalanine formation by Chromobacterium violaceum.

Nonproliferating cells of Chromobacterium violaceum incubated with glycine, methionine, and succinate as substrates accumulated beta-cyanoalanine in the culture fluid. Tracer experiments showed that carbons-2, -3, and -4 of beta-cyanoalanine are derived from the 2-carbon of glycine. When methionine-methyl-(14)C, succinate-1,4-(14)C, or succinate-2,3-(14)C was used as substrate, beta-cyanoalanine did not become labeled. If K(14)CN and serine were used as substrates, the cyano group of beta-cyanoalanine was labeled. Radioactive beta-cyanoalanine, labeled in the 3-carbon, was formed when glycine and H(14)CHO were used as substrates. (14)C-formic acid did not replace formaldehyde. Asparagine also accumulated in the incubated mixture and was found to be labeled in the amide carbon. Incubation of cells with beta-cyanoalanine-4-(14)C produced labeled aspartic acid in cell hydrolysates.

Alanine↗
PubMedDate not supplied

Repigmentation of vitiliginous skin by cultured cells.

Epidermal cells from pigmented areas of a patient with vitiligo were cultured in MCDB-153 medium, which supports the clonal growth of undifferentiated keratinocytes and melanocytes. The cells were grown on collagen-coated substrata. After the cells reached semiconfluence, the composite of substratum and cells was emplaced onto dermabraded vitiliginous areas as a graft. Re-epithelialization of the grafted areas was complete after 2 weeks. Repigmentation was evident after 1 month and continued over the observation period of several months. There was complete and normal differentiation of the graft, including a normal distribution of melanocytes in the basal layer. Ultrastructural studies showed a normal distribution of melanosomes in the melanocytes and showed keratinocytes that were indistinguishable from the uninvolved skin.

Antibodies↗
PubMedDate not supplied

Local inflammation may influence oral tumor cell differentiation.

The mRNA levels of keratin K13, involucrin, protein kinase C alpha and epsilon, and interferon-gamma and its receptors were examined in biopsies from human oral squamous cell carcinomas. Expression of all the genes was elevated in the histologically more differentiated tumors, but it was at or below normal (perilesional control) levels in the poorly differentiated ones. For the same set of biopsies, we had previously shown that the well differentiated tumors expressed higher levels of T cell markers. As interferon-gamma stimulates differentiation, its secretion by inflammatory cells at the tumor site may influence the differentiation status of the tumor.

Cell Differentiation↗
PubMedDate not supplied

Differentiation-dependent expression of growth regulatory cytokines and their receptors in squamous cell carcinomas of the head and neck.

Cytokines, such as IFN-alpha and TNF-alpha are capable of affecting keratinocyte proliferation in the microenvironment of the tumor. Their elevated expression along with high levels of their receptor mRNAs was determined by a semiquantitative reverse transcription- polymerase chain reaction (RT-PCR) method in biopsies of head and neck squamous cell carcinomas that were established as histologically well or moderately differentiated. In contrast, tumors with poor differentiation exhibited low levels of these growth suppressive factors, although levels of their receptors were elevated. In fact, expression of these growth suppressive cytokines highly correlated with the histological status of tumors suggesting a role of these agents in growth regulation of those tumors. Apparently, growth signaling in these tumors differs in the availability of either the ligand or the receptor.

Antigens, CD↗
PubMedDate not supplied

Differentiation and IFN gamma regulate WAF1/CIP1 transcription in p53-independent and p53-dependent pathways in epithelial cells.

In normal epithelia differentiation and proliferation are regulated by factors involving p53 and related pathways. WAF1/CIP1 protein is believed to inhibit cell growth as a downstream mediator of p53 function. Also, WAF1/CIP1 is identified as a candidate gene linking differentiation signals to G1 arrest. Different epithelia with different keratinizing profiles respond differently to differentiation/proliferation signals, such as calcium or interferon gamma (IFNgamma). Other factors, such as p53 mutation or presence of human papillomaviruses (HPVs) also affect responses to those signals. Since WAF1/CIP1 can be regulated via a p53-dependent or an independent manner, our study aimed to determine: a.) differentiation-related expression of WAF1/CIP1 in keratinocytes, b.) involvement of WAF1/CIP1 in IFNgamma action, and c.) p53-dependence of WAF1/CIP1 transcription under these conditions. The results demonstrated that differentiation increases WAF1/CIP1 transcription in keratinizing but not in nonkeratinizing keratinocytes in a p53-independent manner. IFNgamma upregulated WAF1/CIP1 in keratinizing keratinocytes in a differentiation and p53 dependent manner. Inactivation of wild type p53 by mutation or by presence of HPVs uncouples IFNgamma-mediated WAF1/CIP1 transcription from p53 and from differentiation. Our data suggest a mechanism which operates via WAF1/CIP1 in keratinocytes and regulates epithelial differentiation/proliferation under different physiological conditions.

Cell Differentiation↗
PubMedDate not supplied

Response of cultured cells from the epidermis and the buccal mucosa to TGF-beta 1 and comparison to interferon-gamma.

Normal human cells from epidermis and from buccal mucosa were cultured to confluence in three media with graded differentiation potential (at low Ca2+, high Ca2+, and supplemented with serum) and treated with transforming growth factor beta 1 (TGF-beta 1), as had been done previously with interferon-gamma (IFN-gamma). The response of the cells to TGF-beta 1 was monitored in terms of the expression of regulatory genes associated with proliferation and differentiation (cdc2, c-myc, p53) and of genes for structural proteins expressed at varying stages of maturation (keratins K5 and K10, involucrin, flaggrin). For both tissues, the results obtained with both agents were very similar for those genes expressed in the basal cells (cdc2, c-myc, p53, K5), regardless of their function, but diverged for the other genes, which are expressed in the suprabasal cells. Another related contrast is that, although IFN-gamma induced apoptosis in epidermal keratinocytes cultured in the serum containing medium, TGF-beta 1 did not. Thus, the two agents appear to affect the earlier stages of cell differentiation in the same way but to differ at the later stages, particularly in that IFN-gamma pushes maturation further than does TGF-beta 1).

CDC2 Protein Kinase↗
PubMedDate not supplied

Interferons alpha, beta and gamma induce different patterns of gene expression in cultured human epidermal keratinocytes.

Differences in the effects on confluent epidermal keratinocytes of treatment with interferons (IFNs) alpha, beta, and gamma were observed in their modulation of the mRNA levels of representative structural and functional cellular proteins. Comparisons of the responses in culture media with varying cellular maturation potential indicated the dependence of the modulation on the stage of differentiation. Differentiation was correlated with upregulation of all the genes by interferon gamma, but this effect was not seen with the other interferons. Even though IFNs alpha and beta share the same cell surface receptor, their effects on gene expression were clearly distinguishable and varied with the culture medium. These findings might have relevance in the treatment of skin lesions with varying degrees of differentiation.

CDC2 Protein Kinase↗
PubMedDate not supplied

Gene expression of zinc-alpha 2-glycoprotein in normal human epidermal and buccal epithelia.

Zinc-alpha 2-glycoprotein (Zn alpha 2gp) is almost ubiquitous in body fluids. We have found it to be also present in stratified epithelia. We compare its mRNA expression in cells from human epidermis and buccal mucosa cultured in media of graded differentiation potential (attained by varying calcium ion concentration and adding serum). The Zn alpha 2gp gene is upregulated in both epithelia with differentiation and further with exposure to interferon gamma or transforming growth factor beta 1. The upregulation by these agents increases with differentiation in epidermal cells, but peaks in the low-differentiation medium in buccal epithelia. We compared gene expression levels of Zn alpha 2gp with those of characteristic cytokeratins of stratified epithelia (k5 for basal cells, K10 for epidermal suprabasal cells, and K13 for mucosal suprabasal cells). This pattern correlation associates Zn alpha 2gp cell-type dependently with late differentiation, i.e. with keratin K10 in epidermis and with K13 in buccal epithelium.

Cells, Cultured↗
PubMedDate not supplied

Modulation by interferon-gamma of zinc-alpha 2-glycoprotein gene expression in human epithelial cell lines.

Zinc-alpha 2-glycoprotein has been detected in most body fluids, and its antibody labels the corresponding glandular epithelia. We have also detected it in human stratified epithelia (epidermis and buccal mucosa). In this study, the mRNA levels of zinc-alpha 2-glycoprotein were found to be about twice as high in epithelial cells of mucosal origin (whether normal primaries or neoplastic cell lines) as in epidermoid cells (normal epidermal primary cultures, an immortalized but non-tumorigenic epidermal cell line, and neoplastic vulvar and cervical cell lines). Interferon-gamma strongly upregulated gene expression, but substantially less in mucosal than epidermoid cells. To compare responses as a clue to the function of zinc-alpha 2-glycoprotein, we ran parallel experiments with three markers of distinct properties, all known to be induced by interferon-gamma. There was the least resemblance for involucrin, a qualitative similarity for HLA-DR, and a rather better match for 2'-5' oligoadenylate synthetase.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

Differentiation markers in oral carcinoma cell lines and tumors.

Cell lines are useful as models if they retain the relevant characteristics of the tissue of origin. We compared two human squamous carcinoma cell lines derived from tumors of the tongue that vary in their extent of differentiation, with human biopsies of carcinomas of the tongue that were either poorly or well-differentiated. The mRNA levels of suprabasal cell proteins (keratin K13, involucrin, transglutaminase) and of protein kinase C (PKC) isozymes were measured by RT-PCR. Apart from PKC beta and PKC delta (mostly expressed by Langerhans cells and missing in culture), qualitatively similar patterns were found in vitro and in vivo. The more differentiated cells had expression levels moderately lower to higher than the normal controls. The poorly differentiated cells generally had substantially lower levels.

Biomarkers↗
PubMedDate not supplied

Proteasomal RNase activity in human epidermis.

The proteasome is a cytoplasmic high-molecular-weight structure composed of several smaller protein and RNA subunits. It has been associated with non-lysosomal pathways of intracellular degradation, expressing multicatalytic proteinase activities and specific RNase activity. By standard methods, we have isolated andpartially purified proteasomes from human epidermis. We obtained the expected multiple 24-32 kDa subunits by SDS-PAGE, and evidence of RNA. Proteasomes degraded casein, as well as chromogens for t-PA and trypsin but not for chymotrypsin, these proteolytic activities overlap, but do not coincide with those observed in other organs. We found that human epidermal 28 S and 18 S rRNAs were degraded, but yeast RNA was not. By means of zymography, we demonstrated, for the first time, that RNase activity persists after dissociation of the proteasome on the gel and that it co-localizes to the same range of molecular weight subunits as the proteinase activity.

Chromatography, High Pressure Liquid↗