scholarly journals Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

1977 ◽  
Vol 168 (1) ◽  
pp. 91-103 ◽  
Author(s):  
Christopher H. J. Sear ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Human Skin ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Cultured Fibroblasts ◽  
Cscl Density Gradient ◽  
Before And After

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.

scholarly journals Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts

1985 ◽  
Vol 79 (1) ◽  
pp. 67-81 ◽  
Author(s):  
C. Guidry ◽  
F. Grinnell
Keyword(s):  
Human Skin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Collagen Fibrils ◽  
Skin Fibroblasts ◽  
Electron Microscopic Level ◽  
Total Protein Content ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Electron Microscopic

During reorganization of collagen gels by human skin fibroblasts the total protein content of the gels remained approximately constant. Only 5% of the collagen was degraded, although the volume of the gels decreased by 85% or more. It could be concluded, therefore, that gel reorganization required physical rearrangement of pre-existing collagen fibrils rather than degradation of the original collagen and resynthesis of a new matrix. Collagen molecules in the gels were not covalently crosslinked or otherwise modified enzymically during gel reorganization, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and collagen repolymerization studies. Serum was required for gel reorganization and, in the absence of serum, cell spreading was predominantly filipodial, i.e. there was little cytoplasmic reorganization. At the electron-microscopic level it was found that many more collagen fibrils became associated with the cells in the presence of serum than in its absence. Serum was also found to promote the synthesis and secretion of proteins by the cells, and conditioned medium could take the place of serum in promoting gel reorganization. The involvement of cell-secreted factors was also demonstrated by the ability of cycloheximide to inhibit gel reorganization. Finally, when gel reorganization was stopped by adding cytochalasin D to the incubations or removing cells by detergent treatment, a small but significant re-expansion of the collagen fibrils was observed. Consequently, a portion of the collagen that had been physically reorganized by the gels was unstable and could not hold its position without continued force exerted by the cells.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals The rate of sphingomyelin synthesis de novo is influenced by the level of cholesterol in cultured human skin fibroblasts

1998 ◽  
Vol 335 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Petra LEPPIMÄKI ◽  
Robert KRONQVIST ◽  
J. Peter SLOTTE
Keyword(s):  
Palmitic Acid ◽  
Human Skin ◽  
De Novo ◽  
Cholesterol Content ◽  
Skin Fibroblasts ◽  
Cholesterol Depletion ◽  
Human Skin Fibroblasts ◽  
Cellular Cholesterol ◽  
Cholesterol Levels ◽  
A Cell

Plasma membrane sphingomyelin (SM) is known to affect the cellular distribution of cholesterol. The aim of this work was to examine how SM homoeostasis in human skin fibroblasts is affected by alterations in the level of cholesterol in the cell. The cellular cholesterol level was decreased by exposing cells to 2-hydroxypropyl-β-cyclodextrin, and increased by exposing cells to cholesterol–methyl-β-cyclodextrin inclusion complexes. A lowering of the cellular unesterified cholesterol content by 20% was shown to increase the incorporation of [14C]palmitic acid into SM by 70%. Subsequently, the cellular SM mass was shown to be increased (24% increase after a 24 h period). Since l-cycloserine completely abolished the increased incorporation of [14C]palmitic acid into SM in cholesterol-depleted cells, we concluded that the de novo synthesis of the sphingosine backbone of SM was activated in cholesterol-depleted cells. This conclusion was further verified by performing a cell-free assay of serine C-palmitoyltransferase (SPT) in cholesterol-depleted cells, which showed that the activity of the enzyme was increased by 30% after cholesterol depletion. Most of the newly synthesized SM in cholesterol-depleted cells was susceptible to degradation by sphingomyelinase, indicating that it was transported efficiently to the cell surface. Loading of fibroblasts with cholesterol had essentially the opposite effects on SM homoeostasis to those of cholesterol depletion, i.e. 20–30% decreased incorporation of [14C]palmitic acid into SM and decreased activity of SPT. The results of this study show that cellular cholesterol levels have marked effects on the homoeostasis of SM.

scholarly journals Proteome Analysis of Cultured Fibroblasts from Type 1 Diabetic Patients and Normal Subjects

2006 ◽  
Vol 91 (9) ◽  
pp. 3507-3514 ◽  
Author(s):  
Lucia Puricelli ◽  
Elisabetta Iori ◽  
Renato Millioni ◽  
Giorgio Arrigoni ◽  
Peter James ◽  
...  
Keyword(s):  
Human Skin ◽  
Proteome Analysis ◽  
Normal Subjects ◽  
Skin Fibroblasts ◽  
Structural Proteins ◽  
Diabetic Patients ◽  
Human Skin Fibroblasts ◽  
Cultured Fibroblasts ◽  
Type 1 Diabetic Patients

Abstract Context: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. Objective: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. Design: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. Results: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and α-actinin 4 and a decrease of tubulin-β2 and splice isoform 2 of tropomyosin β-chain were detected. Conclusions: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

scholarly journals The biosynthesis of plasmodial myosin during starvation of Physarum polycephalum

1973 ◽  
Vol 135 (4) ◽  
pp. 639-647 ◽  
Author(s):  
F. Harry White ◽  
June Lascelles
Keyword(s):  
Life Cycle ◽  
Physarum Polycephalum ◽  
Adenosine Triphosphatase ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
Time Intervals

The actomyosin protein complex of Physarum polycephalum was prepared from vegetative and starved plasmodia. The yield of actomyosin per unit wet wt. was the same from both types of plasmodia. Myosin was resolved from the complex by gel filtration and purified by ion-exchange chromatography. The Ca2+-stimulated adenosine triphosphatase activities of myosin preparations from vegetative and starved plasmodia were not appreciably different. Synthesis of myosin de novo was shown to occur during the starvation phase of the life-cycle by the isolation of labelled myosin preparations from plasmodia starved in the presence of [2-14C]glycine. Fractionation of polyacrylamide gels after gel filtration of labelled myosin confirmed the presence of label in the adenosine triphosphatase-active myosin band. It is concluded that during starvation myosin synthesis continues although there is a net loss of approx. 50% of the total protein. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of Physarum myosin showed the presence of low-molecular-weight components of the molecule, similar to those of muscle myosins. The content and composition of the free amino acid pool of Physarum was measured at various time-intervals during the vegetative and starvation phases of the life-cycle.

scholarly journals Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts.

1986 ◽  
Vol 34 (8) ◽  
pp. 1063-1068 ◽  
Author(s):  
E Schwartz ◽  
R Fleischmajer
Keyword(s):  
Human Skin ◽  
Indirect Immunofluorescence ◽  
Normal Skin ◽  
Elastic Fiber ◽  
Sodium Dodecyl ◽  
Skin Fibroblasts ◽  
Elastic Fibers ◽  
Human Skin Fibroblasts ◽  
Fiber System ◽  
Oxytalan Fibers

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.

scholarly journals Partial characterization of lysyl oxidase from several human tissues

1985 ◽  
Vol 230 (3) ◽  
pp. 639-643 ◽  
Author(s):  
H Kuivaniemi
Keyword(s):  
Human Skin ◽  
Oxidase Activity ◽  
Lysyl Oxidase ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Active Species ◽  
Skin Fibroblasts ◽  
Human Tissues ◽  
Human Skin Fibroblasts ◽  
Tissue Samples

Lysyl oxidase activity was assayed in urea extracts of a number of human tissues, proving to be highest in skin. Antibodies to human placental lysyl oxidase completely inhibited the activity of crude lysyl oxidase from all the human tissues studied, with no significant differences in the amounts of antiserum required for 50% inhibition. By contrast, marked differences were found in this value between skin tissue samples from different species. The Mr of lysyl oxidase in crude extracts of human skin and in the medium of cultured human skin fibroblasts was 30 000 by gel filtration, no active species with a higher Mr being detectable. Four forms of lysyl oxidase activity were seen in DEAE-cellulose chromatography of urea extract from human skin, all having Mr 30 000. Antibodies to human placental lysyl oxidase stained a 30 000-Mr protein in urea extracts of all the human tissues studied and in the medium of cultured human skin fibroblasts when examined by immunoblotting after sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, but they also stained high-Mr material. The findings suggest that there are no immunologically distinct lysyl oxidase isoenzymes in the various human tissues and that the true Mr of lysyl oxidase in crude urea extracts is 30 000.

117-LB: Nonviral Direct Reprogramming of Human Skin Fibroblasts into Hormone-Expressing ß-Like Cell Clusters

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 117-LB
Author(s):  
LUKE R. LEMMERMAN ◽  
MARIA ANGELICA RINCON-BENAVIDES ◽  
SARAH A. TERSEY ◽  
BRITANI N. BLACKSTONE ◽  
HEATHER M. POWELL ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Fibroblasts ◽  
Direct Reprogramming ◽  
Human Skin Fibroblasts ◽  
Cell Clusters
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