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Biomedical subjects

E A Bauer

Publications and source records attributed to E A Bauer.

At least 73 records · Page 4Linked to original sources
PubMed1985

Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts.

Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.

Aging↗
PubMed1985

Stimulation of in vitro human skin collagenase expression by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is both chemoattractant and mitogenic for stromal cells. Here, we examined the effects of PDGF on collagenase expression by normal human skin fibroblasts. Culturing cells for 24 hr in the presence of PDGF at 0-180 ng/ml resulted in a dose-dependent, saturable increase in collagenase activity in the culture medium that was paralleled by equal increases in immunoreactive collagenase protein, suggesting enhanced synthesis of a catalytically unaltered enzyme. The specificity of this effect was demonstrated by comparing the collagenase-stimulatory effect with that on total protein synthesis and DNA synthesis. Under in vitro conditions that produced a 2.5-fold increase in collagenase synthesis, there was an approximately equal to 20% increase in total protein synthesis and no change in DNA synthesis. In addition, platelet factor 4, another platelet-derived protein, caused a less than 20% increase in collagenase expression. In time-course studies, stimulation of collagenase synthesis was first observed 8-10 hr after exposure to the growth factor. Conversely, when cells were primed with PDGF for approximately equal to 24 hr and the stimulator was then removed, an increased rate of synthesis was seen for an additional approximately equal to 6 hr, after which the rate reverted to control levels. Since the kinetic data suggested a possible pretranslational effect, fibroblasts cultured with PDGF were used to prepare mRNA. In cell-free translation, total protein synthesis was essentially unaltered; however, the growth factor caused a greater than 2-fold increase in translatable collagenase mRNA. The data suggest that PDGF specifically modulates collagenase synthesis, possibly through a series of events that lead to increased transcription or preferential translation of collagenase mRNA.

Cells, Cultured↗
PubMed1985

Stimulation of skin fibroblast collagenase production by a cytokine derived from basal cell carcinomas.

Our previous studies of human basal cell carcinomas (BCC) revealed increased skin collagenase in vivo. Immunocytochemically the collagenase was localized to adjacent stroma, not to the tumor cells. When grown in culture, skin fibroblasts derived from tumor stroma showed a 3- to 4-fold increase in collagenase for the first 10-14 mean population doublings, after which collagenase expression reverted to control levels. These studies suggested that tumors stimulated adjacent fibroblasts to produce more collagenase. In the present study we sought direct evidence for epithelial-stromal interaction in this neoplasm. Under dissecting microscopy tumor islands were freed of stroma, homogenized, sonicated, and centrifuged to remove insoluble tissue. Tumor extracts were incubated with monolayer cultures of normal human skin fibroblasts to assess their effect on collagenase synthesis in these target cells. Culturing the fibroblasts for 24 h in the presence of individual BCC extracts resulted in a 1.6- to 3-fold increase in trypsin-activatable collagenase in the culture medium. This was paralleled by an equal increase in immunoreactive protein, suggesting enhanced enzyme synthesis. There was no change in the activity per immunoreactive protein, indicating a catalytically unaltered enzyme. Gel filtration of pooled BCC extracts showed that the stimulatory activity was contained in eluent fractions of Mr approximately 19Kd. These data suggest that BCCs elaborate a macromolecular cytokine that induces collagenase synthesis in skin fibroblasts and emphasize the importance of epithelial-stromal interactions in cutaneous tumor invasion.

Biological Products↗
PubMed1984

Epidermolysis bullosa: a review.

Epidermolysis bullosa is a group of inherited disorders characterized by blistering of the skin as a result of minor trauma. The disease can be divided into three anatomical categories: Epidermolytic, where blister cleavage occurs within the epidermis; Junctional, which has blister cleavage within the lamina lucida; and Dermolytic, where blister cleavage occurs below the basal lamina in the upper papillary dermis. Each of these three categories can be divided into several distinct entities based on clinical and histologic criteria. Basic biochemical studies have increased our understanding of several of these diseases, most notably recessive dystrophic epidermolysis bullosa. Although therapy for patients with EB is largely supportive, increased knowledge of the biochemistry of these disorders is making direct therapeutic interventions possible.

Adult↗
PubMed1984

Therapeutic efficacy of phenytoin in recessive dystrophic epidermolysis. A comparison of short- and long-term treatment.

To assess the potential short- and long-term efficacy of phenytoin in treating recessive dystrophic epidermolysis bullosa (RDEB), we treated 22 patients with therapeutic doses of oral phenytoin (blood level, 8 to 10 micrograms/mL) for periods ranging from eight to 99 weeks. Fourteen (64%) of the 22 patients had greater than a 40% mean percentage decrease in blistering of the skin. Nine (41%) of the 22 patients were treated for longer than 75 weeks, and seven (78%) of these nine patients had a mean decrease in blistering of at least 40% during phenytoin therapy. These data, along with previous reports, suggest that phenytoin has therapeutic efficacy in RDEB.

Adolescent↗
PubMed1983

Enzyme-linked immunosorbent assay for human skin collagenase.

A specific and sensitive indirect inhibition enzyme-linked immunosorbent assay (ELISA) was develop for human skin collagenase. Using an antiserum dilution of 1: 8000, the ELISA could detect 0.2 ng of enzyme, which was approximately 10 times more sensitive than the previously described radioimmunoassay for human skin collagenase. The assay was also highly reproducible. In comparative studies, bacterial, tadpole and crab collagenases did not react in the ELISA. Rat uterine collagenase and collagenases from bovine gingival explants and fibroblasts displayed approximately 0.1% of the reactivity found with human skin collagenase. Human synovial and gingival collagenases and collagenase from skin fibroblasts from patients with recessive dystrophic epidermolysis bullosa showed almost complete identity with the normal human skin fibroblast enzyme.

Antibody Specificity↗
PubMed1983

Human skin collagenase: assignment of the structural gene to chromosome 11 in both normal and recessive dystrophic epidermolysis bullosa cells using human-mouse somatic cell hybrids.

Somatic cell hybrids between mouse cells and human normal skin and corneal fibroblasts and recessive dystrophic epidermolysis bullosa (RDEB) skin fibroblasts have been used to assign the structural gene for collagenase to its human chromosome. A total of 46 hybrid subclones from several hybridization events were isolated in hypoxanthine-aminopterin thymidine (HAT) selection medium and used to measure the production of human collagenase by a specific radioimmunoassay. We have found that both the normal and RDEB human collagenase gene maps to human chromosome 11. This indicates that the abnormal collagenase produced by RDEB cells is probably not a totally distinct form of the enzyme, but is a structural mutation of the normal collagenase enzyme.

Animals↗
PubMed1983

Verruciform xanthoma. Occurrence in eroded skin in a patient with recessive dystrophic epidermolysis bullosa.

Verruciform xanthoma is a rare, granular, plaque-like lesion which occurs on mucosal surfaces and is characterized by acanthosis with xanthoma cells in the dermis between rete pegs. A case of verruciform xanthoma is reported which occurred on the sacral region in a patient with recessive dystrophic epidermolysis bullosa (RDEB). Electron microscopic studies suggested that many of the xanthoma cells were fibroblasts whose vacuolated appearance was expressed in cell culture and persisted for several passages. The occurrence of a verruciform xanthoma in a patient with severe recessive dystrophic epidermolysis bullosa suggests that repeated epidermal and/or dermal damage gave rise to a reactive process involving the formation of lipid-laden cells.

Adolescent↗
PubMed1983

Generalized dominant epidermolysis bullosa simplex: decreased activity of a gelatinolytic protease in cultured fibroblasts as a phenotypic marker.

To characterize biochemical traits associated with various forms of epidermolysis bullosa (EB), we used skin fibroblast cultures to measure a gelatin-specific neutral metalloprotease. Compared to normal cultures, levels of this gelatinase were 7-fold decreased in cell cultures from 3 patients from 3 kindreds with generalized dominant EB simplex of the Koebner type (DEBS-K) (p less than 0.001). The specificity of this trait was shown in several ways. The growth kinetics and total protein synthesis of the cells were unaltered. The activity of lactic dehydrogenase, a cytoplasmic enzyme, was also unaltered, indicating the integrity of the cells was not compromised. The decrease in gelatinase activity is specific for generalized DEBS-K, since cultures from recessive dystrophic EB, recessive junctional EB, dominant dystrophic EB, and a second genetic type of DEBS all fail to show this defect. However, since it has been suggested that DEBS-K and the localized form of DEBS, the Weber-Cockayne type (DEBS-WC), may represent allelic mutations of varying severity, we measured gelatinase activity in cell cultures of 13 patients of this type. The levels displayed a biphasic segregation in which 7 of 13 values were equal to, or greater than, the mean activity of control cells. Cultures from the remaining 6 patients were greater than 1 SD below the mean control value and approximated those seen in the generalized DEBS-K patients. These studies suggest that the decrease in gelatinolytic activity is a marker for DEBS-K and that DEBS-K and DEBS-WC may be closely related genetic disorders in which the defect in gelatinolytic protease represents a pleiotropic effect of the gene for DEBS or is genetically linked to the DEBS gene.

Adolescent↗
PubMed1983

Retinoic acid inhibition of collagenase and gelatinase expression in human skin fibroblast cultures. Evidence for a dual mechanism.

Human skin fibroblast cultures have been employed to study the effects of a variety of vitamin A analogues (retinoids) on the expression of two enzymes involved in collagen degradation in the skin, collagenase and a gelatinolytic protease. In normal and recessive dystrophic epidermolysis bullosa fibroblast cultures, retinoic acid compounds were effective inhibitors of the accumulation of both enzymes in the culture medium with half-maximal inhibitions occurring at 0.25-1 microM for collagenase and at 3-6 microM for the gelatinolytic protease. Various retinoids exhibited differing degrees of inhibitory actions, so that at a 1 microM concentration, relative inhibitions were: 13-cis-retinoic acid greater than all-trans-retinoic acid greater than aromatic retinoid (Ro 10-9359) much greater than retinol. The retinoic acid-mediated decrease in collagenase activity was accompanied by a parallel decrease in immunoreactive collagenase protein, suggesting that the retinoic acids were acting to inhibit synthesis of the enzyme. However, an additional effect of these agents was encountered. Although the retinoids themselves had no direct collagenase inhibitory action, medium derived from cultures maintained in these retinoids showed direct inhibitory capacity which was dependent both on the concentration of retinoic acid and on the length of time in culture. The results suggest that the retinoic acids modulate collagenase in vitro by two mechanisms: by decreasing the synthesis of enzyme protein and by modulating the expression of an inhibitory molecule.

Cells, Cultured↗
PubMed1982

Inhibition of collagen degradative enzymes by retinoic acid in vitro.

The effects of a variety of retinoids on collagenase and gelatinase expression have been examined in skin fibroblast cultures derived from normal volunteers and from patients with the hereditary blistering disorder, recessive dystrophic epidermolysis bullosa. Both 13-cis- and all-trans-retinoic acid were effective inhibitors of collagenase production in both cell types. In the case of collagenase, the inhibition of collagenase activity was paralleled by a reduction in immunoreactive enzyme protein, suggesting that these retinoids act by inhibiting synthesis and/or secretion of the enzyme. Retinoic acid also inhibited production of the second enzyme in the collagen degradative pathway, gelatinase. In this case, the decrease in gelatinase activity was equal to or slightly greater than the achieved in collagenase expression. The observation that certain retinoids modulate the two crucial enzymes in the degradation of collagen in the skin suggests that they might be useful therapeutic agents in recessive dystrophic epidermolysis bullosa, a disease in which the pathogenesis of blistering is in part related to connective tissue destruction.

Cells, Cultured↗
PubMed1982

Enhanced cell-free translation of human skin collagenase in recessive dystrophic epidermolysis bullosa.

The regulatory mechanisms for collagenase synthesis in recessive dystrophic epidermolysis bullosa (RDEB) have been studied using messenger RNA (mRNA) harvested from normal and RDEB skin fibroblasts to direct protein synthesis in a rabbit reticulocyte lysate translation system. Fibroblast mRNA encoded the synthesis of approximately 60,000 and approximately 55,000 dalton forms of procollagenase in cell-free translation. In contrast to biosynthesis by intact cells, there was preferential translation of the approximately 60,000 dalton specie. For quantitative comparisons with mRNA from normal cells, mRNA was harvested from fibroblasts of 2 RDEB patents whose intact cells have been documented to have increased synthesis of collagenase. Although total translational activity was equal in normal and RDEB mRNA preparations, translatable collagenase mRNA was increased 3.5- to 10-fold, suggesting that the enhanced collagenase synthesis characteristic of RDEB is due to increased concentrations or preferential translation of collagenase mRNA.

Animals↗
PubMed1982

Decreased collagenase production by regional fibroblasts cultured from skin of a patient with connective tissue nevi of the collagen type.

A 10-yr-old female presented with cerebriform tumors covering the plantar surfaces of both feet. Histologically, the lesions consisted of thick collagen fibers and the content of collagen per surface area of skin was increased about 8-fold. Examination of the collagen by SDS-polyacrylamide gel electrophoresis, after limited pepsin proteolysis, showed that the lesions consisted almost exclusively of type I collagen, the predominant collagen type in human skin. Thus, a diagnosis of connective tissue nevi of the collagen type was made. Fibroblast cultures were established from the affected and normal-appearing areas of the skin, and examined for the rate of collagen synthesis, production of collagenase and growth kinetics of the cells. Cell cultures derived from the lesion and from control skin synthesized procollagen at the same rate and in a normal type I/type III procollagen ratio. However, the production of enzymatically active and immunologically detectable collagenase was reduced by 70-82% in the cultures derived from the lesion as compared to controls (p less than 0.005). Fibroblasts derived from the lesions also displayed a mean population doubling time of 1.17 +/- 0.08 days compared to 1.83 +/- 0.24 and 1.92 +/- 0.09 days for control cell strains and cells derived from normal skin of the patient, respectively (p less than 0.025). These results suggest that the excessive deposition of collagen in this case may have resulted from decreased local degradation of collagen. Enhanced proliferative capacity of the regional fibroblasts may have contributed to the accumulation of collagen in these lesions.

Cells, Cultured↗
PubMed1982

Colchicine-induced modulation of collagenase in human skin fibroblast cultures. I. Stimulation of enzyme synthesis in normal cells.

Microtubule-active agents affect the secretion of a variety of proteins, including collagenase. To gain insight into the mechanisms involved in this process, we examined the effects of colchicine on the synthesis, secretion, and activity of human skin collagenase. When added to monolayer cultures of human skin fibroblasts, 10(-6) M colchicine produced a mean 3-fold increase in trypsin-activatable collagenase in the culture medium. Stimulation was not observed with lumicolchicine. The enhanced accumulation of collagenase was dose-dependent with 10(-9), 10(-8), 10(-7), and 10(-6) M colchicine giving collagenase activities/mg protein that were 100 +/- 6%, 165 +/- 20%, 186 +/- 34%, and 297 +/- 62% of control, respectively. Although the effect on collagenase was seen under conditions independent of cellular growth (i.e., in serum-free medium), maximum stimulation occurred in subconfluent cultures. The colchicine-induced increase in activity was paralleled by an increase in immunoreactive enzyme protein, suggesting stimulation of enzyme synthesis. The catalytic efficiency of the enzyme (activity per unit immunoreactive protein) was unchanged, however, indicating that a structurally normal enzyme was being synthesized. To examine the process in more detail, the biosynthesis of 3H-labeled collagenase was quantitated in these cultures by specific immunoprecipitation. Although 10(-6) M colchicine produced no increase in total protein synthesis, an increased rate of collagenase synthesis was seen after only 1.5 hr. These data suggest that colchicine has a specific effect on the synthesis of collagenase and may be a useful probe for studying its regulation.

Cells, Cultured↗