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.

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 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 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.

Monoamine oxidase activity in cultured human skin fibroblasts

1977 ◽  
Vol 80 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Regina Groshong ◽  
D.Ann Gibson ◽  
Ross J. Baldessarini
Keyword(s):  
Monoamine Oxidase ◽  
Human Skin ◽  
Oxidase Activity ◽  
Skin Fibroblasts ◽  
Monoamine Oxidase Activity ◽  
Human Skin Fibroblasts

scholarly journals Purification and properties of four species of lysyl oxidase from bovine aorta

1979 ◽  
Vol 177 (1) ◽  
pp. 203-214 ◽  
Author(s):  
Herbert M. Kagan ◽  
Kathleen A. Sullivan ◽  
Theodore A. Olsson ◽  
Anne L. Cronlund
Keyword(s):  
Metal Ion ◽  
Lysyl Oxidase ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Active Species ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Apparent Homogeneity ◽  
Multiple Species

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49–60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000–33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of α-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu2+, and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.

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

Protective Effects of Green Tea Seed Extract against UVB-irradiated Human Skin Fibroblasts

2014 ◽  
Vol 43 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Ok Kyung Kim ◽  
Da-Eun Nam ◽  
Min-Jae Lee ◽  
Namgil Kang ◽  
Jae-Youn Lim ◽  
...  
Keyword(s):  
Human Skin ◽  
Green Tea ◽  
Seed Extract ◽  
Skin Fibroblasts ◽  
Protective Effects ◽  
Human Skin Fibroblasts
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