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Biomedical subjects

E A Bauer

Publications and source records attributed to E A Bauer.

At least 55 records · Page 3Linked to original sources
PubMed1988

Tissue stress and tumor promotion. Possible relevance to epidermolysis bullosa.

Cutaneous carcinomas often arise in patients with severe epidermolysis bullosa (or other cutaneous ulcers) at multiple primary sites. Chronic tissue stress thus appears to promote carcinogenesis in preexisting somatic mutants in a stem cell population. Altered contractile properties of fibroblasts cultured from skin with epidermolysis bullosa may result from an altered interaction of these cells with their surrounding, chronically stressed, connective tissue matrix.

Adult↗
PubMed1987

Eriochrome black T inhibition of human skin collagenase, but not gelatinase, using both protein and synthetic substrates.

The intracellular degradation of interstitial collagen is accomplished by two neutral metalloproteases, collagenase and gelatinase. Both enzymes are inhibited by metal chelating agents, by certain sulfhydryl reagents, and by similar protein inhibitors. Here, we demonstrate that the dye eriochrome black T (EBT) appears to be unique in its capacity to inhibit collagenase but not gelatinase. Using native reconstituted helical collagen in gel form at 37 degrees C, half-maximal inhibition of collagenase activity by EBT occurs at approximately 45 microM. EBT more effectively inhibits the breakdown of native collagen in solution, with a KI of approximately 8 microM. Using a newly-developed spectrophotometric substrate, AcProLeuGly-S-LeuLeuGly-OC2H5, a KI of 1.4 microM was calculated for EBT on collagenase. Although this same thiopeptolide serves as a substrate for gelatinase with kinetics similar to those of collagenase, no inhibition by EBT was observed. EBT also did not inhibit the gelatinase-mediated breakdown of the natural substrate, gelatin. The data suggest that EBT may have significant potential for allowing the differentiation in biological fluids of two metalloproteases with similar cleavage site specificities.

Azo Compounds↗
PubMed1987

Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells.

We have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO4/PAGE of 60 and 57 kDa. The minor 60-kDa polypeptide is a glycosylated form of the major 57-kDa protein containing N-linked complex oligosaccharides. Zymogen activation by trypsin results in the removal of 84 amino acids from the amino terminus of the enzyme generating a 45-kDa active enzyme species. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively (1-2 micrograms per 10(6) cells per 24 hr). Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. Our data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.

Amino Acid Sequence↗
PubMed1987

Regulation of collagen gene expression in cutaneous diseases with dermal fibrosis: evidence for pretranslational control.

Dermal fibrosis, characterized by collagen accumulation, is the hallmark of several cutaneous diseases. To examine the mechanisms of collagen deposition in fibrotic skin diseases, fibroblast cultures were established from the skin of patients with progressive systemic sclerosis, morphea, scleredema, familial cutaneous collagenoma, connective tissue nevi of the collagen type, or keloids; these patients served as prototypes of fibrotic skin diseases with varying clinical features and potentially different etiologic factors. Collagen production was assayed by the synthesis of [3H]hydroxyproline, and types I and III procollagen messenger RNA (mRNA) levels were determined by dot blot hybridizations using human type I and type III procollagen-specific cDNA probes. The collagen production in fibroblast cultures from the fibrotic diseases was increased up to 6-fold over the controls, and a relatively good correlation between the collagen production and type I collagen mRNA levels was noted. The type I/III procollagen mRNA ratio in control fibroblast cultures was 5.9 +/- 1.6 (mean +/- SD). The corresponding ratio in keloid cell culture was markedly increased, while slightly decreased values were noted in the case of morphea and familial cutaneous collagenoma; the values in other cultures were within the normal range. The results suggest that procollagen production in fibroblast cultures derived from fibrotic skin diseases reflects elevated levels of the corresponding procollagen mRNA. The increased mRNA abundance, suggesting pretranslational control, may result from enhanced transcriptional activity of the corresponding gene or alternatively reflects increased stability of the mRNA molecule.

Collagen↗
PubMed1987

Behavior of epidermolysis bullosa fibroblasts in a hydrated collagen lattice.

To determine if an altered ability to contract a hydrated collagen lattice is characteristic of fibroblasts from patients with recessive dystrophic epidermolysis bullosa (RDEB), we examined contraction by fibroblasts from normal subjects and patients with RDEB, dominant dystrophic epidermolysis bullosa (DDEB), and dominant epidermolysis bullosa simplex (DEBS). An extremely broad range of contractility (normal, poor, and hypercontraction) was observed in all types of epidermolysis bullosa (EB). When contraction in control fibroblasts was defined as the mean +/- 2 SD, (all control values were within this range) and the data were analyzed by the chi-square test, only 32% of EB cells fell within this range, with 47% poorly contractile and 21% hypercontractile. These data, derived from 34 patients, indicate that no single genetic defect resulting in altered contractility in the 3 distinct types of EB is likely. Neither cell viability, collagenase expression, nor PGE2 synthesis as correlated with gel contraction in any group. Indomethacin had no effect on contraction in RDEB. It is possible that the genetic defects in EB cause blister formation in vivo and may lead in some way to an abnormal interaction of fibroblasts with the extracellular matrix resulting in an altered collagen lattice contraction in vitro.

Cells, Cultured↗
PubMed1987

The mechanobullous diseases.

The mechanobullous diseases exhibit extensive skin fragility such that minimal trauma results in blistering. Clinical manifestations range from mild to severe generalized involvement with significant morbidity and mortality. Ultrastructural and biochemical abnormalities have been described and provide insight into these disease processes.

Epidermis↗
PubMed1986

Diminished response of Werner's syndrome fibroblasts to growth factors PDGF and FGF.

Patients with Werner's syndrome, an autosomal recessive disorder, undergo an accelerated aging process that leads to premature death. Fibroblasts from such patients typically grow poorly in culture. Here it is shown that fibroblasts from a patient with Werner's syndrome have a markedly attenuated mitogenic response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In contrast, they have a full mitogenic response to fetal bovine serum. Both PDGF binding and receptor numbers per cell are unaltered. The Werner's syndrome cells express high constitutive levels of collagenase in vitro. Although PDGF enhances collagenase expression through increased levels of hybridizable collagenase messenger RNA in normal skin fibroblasts, no induction of collagenase occurs in the Werner's syndrome fibroblasts. Moreover, the failure to respond to this agonist effect of PDGF is not restored by fetal bovine serum. The data suggest that failure of one or more PDGF-mediated pathways in Werner's syndrome cells may contribute to the phenotypic expression of the disorder.

Cell Division↗
PubMed1986

Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein.

We have determined the complete sequence of the cDNA clone representing the full size human skin collagenase mRNA. Collagenase is synthesized in preproenzyme form, Mr 54,092, with a 19 amino acid long signal peptide. The primary secretion products of the enzyme consist of a minor glycosylated form, Mr 57,000, and a major unmodified polypeptide of predicted Mr 51,929. Proteolytic activation of human skin procollagenase results in removal of 81 amino acid residues from the amino-terminal portion of the proenzyme. Both potential N-glycosylation sites are contained within the proteolytically activated form of the enzyme. The primary structure of the coding region of the presented clone is homologous to an oncogene-induced rat protein whose function is still unknown, although preliminary observations suggest that it is not rat skin collagenase.

Amino Acid Sequence↗
PubMed1986

Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology.

Blister fluids from a variety of bullous disorders were examined for the presence of human collagenase inhibitor. A protein immunologically identical to the collagenase inhibitor produced by human skin fibroblasts was found in high concentrations within bullae of diverse etiologies. Levels of collagenase inhibitor in blister fluids ranged from 0.9-12.5 micrograms/ml, averaging 4.9 micrograms/ml. The mean values were 3- to 4-fold greater than those present in the sera of corresponding patients and exceeded plasma levels by 6- to 8-fold. The time course of collagenase inhibitor accumulation in blister fluid was studied using heat- and suction-induced bullae. The concentration in newly formed blisters was approximately 0.5 micrograms/ml, virtually identical to plasma inhibitor levels, and remained constant for approximately 4 h. Inhibitor concentrations then rose rapidly, reaching peak values of approximately 6 micrograms/ml after 48 h. We speculate that the role of this inhibitor in blister fluid involves the inhibitions of active proteinases within the bulla cavity and may occur to limit the extent of blister formation or to assist in wound repair.

Adult↗
PubMed1986

Antenatal diagnosis of recessive dystrophic epidermolysis bullosa: collagenase expression in cultured fibroblasts as a biochemical marker.

We performed fetoscopy and skin biopsy on a 19-week fetus at risk for recessive dystrophic epidermolysis bullosa (RDEB). Ultrastructural analysis of the tissue revealed dermolytic blister formation in the skin characteristic of the disease. To develop a biochemical test for use in antenatal diagnosis of RDEB, we established skin fibroblast cultures from the 20-week aborted fetus. The collagenase production by fetal RDEB fibroblast cultures was greater than seen in normal fetal fibroblast cultures. The concentration in culture medium from fetal RDEB cultures was 5.42 +/- 0.74 micrograms/ml (mean +/- SE) compared with 2.24 +/- 1.11 micrograms/ml in normal adult control cultures and 2.05 +/- 0.61 micrograms/ml in cultures from patients with other genetic forms of epidermolysis bullosa (p less than 0.025). In contrast, the concentration of collagenase in the fetal RDEB culture medium was not different from that seen in cell cultures from known patients with RDEB (5.34 +/- 1.12 micrograms/ml). Collagenase activity of the fetal RDEB medium was also increased approximately 3.5-fold. These data indicate that enhanced expression of collagenase by fetal RDEB skin fibroblasts can serve as a biochemical adjunct, and possibly an alternative, to morphologic examination of tissue for antenatal diagnosis.

Biopsy↗
PubMed1986

Progressive nodular fibrosis of the skin: altered procollagen and collagenase expression by cultured fibroblasts.

A 33-year-old man presented with a spontaneous progressive cutaneous tumor-like fibrosis involving the right leg and buttock. Histologically the deep dermis was composed of numerous fibroblasts and dense bands of collagen, suggesting that the lesion might be related to an abnormality in collagen metabolism. Fibroblast cultures were established from the affected and normal-appearing skin. The growth rate of the lesional cells was essentially equal to that of control cells. The synthesis of procollagen was approximately 3.5-fold increased in the cells derived from the nodules when compared with control fibroblasts (p less than 0.001). The increase in procollagen synthesis was reflected by an approximate 6-fold increase in both type I and type III procollagen mRNA abundance in the lesional fibroblasts (p less than 0.001), thus suggesting an aberration in the pretranslational level of procollagen gene expression. In contrast, the synthesis of collagenase, the enzyme required for the initiation of collagen degradation, was decreased to approximately 25% of control values (p less than 0.0025), although the enzyme was catalytically normal. The data indicate that these cells are characterized by an increased synthesis of procollagen and decreased synthesis of collagenase, 2 phenotypic characteristics that could account pathophysiologically for the lesions. The unusual reciprocal nature of these biochemical parameters in 2 proteins important in connective tissue homeostasis suggests that this progressive tumor-like condition may have resulted from the expansion of a clonal population of cells.

Adult↗
PubMed1986

Basal cell carcinoma and collagenase.

The interaction of connective tissue stroma and epithelial cutaneous cancer is an active area of investigation in dermatology. Studies summarized here explore the role of collagenase in basal cell carcinoma (BCC) invasiveness. Evidence is presented to support the role of a cytokine or cytokines secreted by BCCs that stimulate collagenase production by surrounding stromal fibroblasts. Prospects for further research in this area are proposed.

Biological Products↗
PubMed1986

Severe infantile epidermolysis bullosa simplex. Dowling-Meara type.

We encountered eight patients with epidermolysis bullosa (EB) simplex of the Dowling-Meara type, who presented in infancy with severe blistering and were originally clinically thought to have recessive dystrophic EB. One infant died in the neonatal period, and the others have had reduced blistering with advancing age. However, in two of the three older patients, the development of severe disabling hyperkeratosis of the palms and soles has been a prominent feature. The correct diagnosis of EB simplex was initially not made in five patients, because, on routine histologic examination, the blister was apparently subepidermal. Electron microscopy confirmed the correct diagnosis of EB simplex by demonstration of basal cell cytolysis. There was clumping of tonofilaments in seven patients. Immunofluorescence demonstrated a cleft above the basal layer in three cases. The findings of severe extensive blistering at birth that improves with age, milia formation, acral distribution with herpetiform groups of blisters in older children, intraoral lesions, absence of scarring, and intraepidermal clefting due to basal cell cytolysis and clumping of tonofilaments within these basal cells as seen on electron microscopic examination present a subtype of EB simplex similar to that described by Dowling and Meara. This has been recognized in the European but not in the American literature and is probably more frequent than has been previously reported.

Adolescent↗
PubMed1985

Glucocorticoid modulation of collagenase expression in human skin fibroblast cultures. Evidence for pre-translational inhibition.

Glucocorticoids inhibit collagenase accumulation in the medium of human skin explant cultures. To examine the mechanism for this process, skin fibroblasts were placed in serum-free medium containing various steroids. Dexamethasone produced a dose-dependent inhibition of trypsin-activatable collagenase in the culture medium with maximal inhibition of approx. 85% at 10(-6) M. Dexamethasone failed to inhibit collagenase activity directly. The decrease in activity in the medium was paralleled by a decrease in immunoreactive protein, suggesting inhibition of enzyme synthesis. The specificity of the effect was shown in two ways. At 10(-6) M steroid, only dexamethasone and hydrocortisone were inhibitory; estradiol, progesterone and testosterone produced less than 10% inhibition. In biosynthetic studies, exposure to 10(-7) M dexamethasone for 24 h produced approx. 50% inhibition of collagenase synthesis but caused no greater than 10% inhibition of total protein synthesis. The T1/2 for achieving the effect was approx. 16 h after initial exposure to dexamethasone. These kinetics were parallel to the inhibition caused by actinomycin D and cordycepin, two inhibitors of transcription, but were longer than that caused by cycloheximide (T 1/2 less than 3 h). To examine this process, cells were cultured in the presence or absence of 10(-6) M dexamethasone prior to harvesting mRNA for cell-free translation. In each case the inhibition or enzyme activity in the intact cells was paralleled by a reduction in translatable collagenase mRNA from the same cells. At the same time, there was no significant inhibition of total protein translation by the steroid. These data suggest that glucocorticoids regulate collagenase synthesis at a pre-translational level, possibly through inhibition of transcription.

Cells, Cultured↗
PubMed1985

Stimulation of collagenase synthesis by a 20,000-dalton epithelial cytokine. Evidence for pretranslational regulation.

Certain products of cultured rabbit corneal epithelium or epidermis regulate collagenase production by rabbit corneal stromal cells. Here, we examined the effects of a 20-kDa cytokine derived from cultured epithelium on collagenase expression by normal human skin fibroblasts and by cells from recessive dystrophic epidermolysis bullosa, a disease characterized by over-production of a structurally altered collagenase. Culturing cells for 24 h in the presence of the cytokine resulted in an approximately 2-fold increase in trypsin-activatable collagenase activity paralleled by an increase in immunoreactive protein, suggesting enhanced synthesis. There was no change in the activity/immunoreactive protein, indicating a catalytically unaltered enzyme. In kinetic studies, stimulation of collagenase synthesis was first observed approximately 8 h after exposure to the epidermal cytokine. Conversely, when cells were primed with cytokine for 24 h and the stimulator was then removed, an increased rate of synthesis was seen for an additional approximately 9 h, after which the rate reverted to control levels. Since the kinetic data suggested an effect at a pretranslational level, fibroblasts cultured with 20-kDa stimulator were used to prepare mRNA. In cell-free translation total protein synthesis was unaltered; however, the cytokine caused a greater than 2-fold increase in translatable collagenase mRNA. The data suggest that this epithelial cytokine specifically modulates collagenase synthesis through transcription and that epithelial-stromal interactions are important to cutaneous connective tissue metabolism.

Animals↗