Fibrosarcoma of superficial soft tissue origin.
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Antibodies against intermediate-sized filaments, of the prekeratin or vimentin type, were used to investigate the presence of these filaments by indirect immunofluorescence microscopy in cultured and non-cultured amniotic fluid cells, in frozen sections of the placenta and in isolated cells of the amniotic epithelium. Two major classes of cells can be cultured from amniotic fluids, namely cells of epithelial origin containing filaments of the prekeratin type and cells of different origin which contain filaments of the vimentin type but are negative when tested with antibodies to epidermal prekeratin. The presence of prekeratin type filaments correlates with the morphology of colonies of amniotic fluid cell cultures in vitro as classified by Hoehn et al. (1974). Cells of E-type colonies are shown to be of epithelial origin. In contrast our data indicate a different origin of almost all cells of F-type colonies and of the large majority of cells of AF-type colonies. Cells of epithelial origin and positively stained with antibodies to epidermal prekeratin are occasionally scattered in F-type colonies and in variable percentages (up to 30%) in AF-type colonies. Surprisingly, cryostat sections of the amniotic epithelium and isolated groups of amniotic cells showed positive reactions with both antibodies to vimentin and prekeratin. The possibility that amniotic cells may be different from other epithelial cells in that they contain both types of filaments simultaneously already in situ is presently under investigation.
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Cell fusion was induced by inactivated Sendai virus in different human diploid cell lines. These were derived from the kidney, lung or skin originating from embryos or adult donors and representing predominantly epithelioid cells (kidney) and fibroblastic cells (lung and skin). The fusion sensitivity (FS) potentials of these cell lines were determined and related to various aspects of cell ageing. In case of the fibroblastic lines, an inverse relation was demonstrated both to the culture age in vitro and to the donor age. The FS potential of embryonal fibroblasts decreased some 40-50% during the in vitro cultivation. In comparison to exponentially growing fibroblasts, the FS potentials were higher in cells in the stationary phase of growth. This was shown to correlate well with the fact that the life-span in calendar time also increased in cultures predominantly grown in the stationary phase. In the case of the kidney cells, the FS potentials were some 50% higher than those of fibroblasts. Since the cellular manifestation of the FS potential most likely primarily resides in the cell membrane-cytoskeleton structure, the results emphasize the importance of this system in relation to ageing.
We studied cytoskeletal proteins and other markers for embryologic origin in the outflow pathways of the aqueous humor, cornea, sclera, and ciliary muscle of the cynomolgus monkey. The corneal endothelium and trabecular cells stained with markers for vimentin, smooth muscle cell alpha-actin, F-actin, spectrin, vinculin, and talin. The endothelium of Schlemm's canal stained with markers for vimentin, spectrin, and F-actin. These results suggest that trabecular cells are a kind of myofibroblast and support the belief that the endothelial cells of Schlemm's canal are vascular in origin. Fibrillary staining with antibodies to vimentin, spectrin, neurofilament protein, and glial acid fibrillary protein was observed along and between the ciliary muscle cells. Cells in the deep sclera adjacent to the supraciliary space stained with antibodies to smooth muscle alpha-actin, alpha-vinculin, talin, and desmin. These cells may anchor ciliary muscle cells into the sclera or may be developmental remnants of ciliary muscle cells. Leu 19 immunoreactivity was found in the corneal endothelium, in all trabecular cells, in ciliary muscle cells, and in keratocytes and fibroblasts in the superficial part of the cornea and sclera. All of these cells are therefore likely to express neural cell adhesion molecules indicating neuroectodermal origin.
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Lipoxygenases (LOXs) have been implicated in carcinogenesis in various cancer types. In the current study, three structurally different lichen metabolites, protolichesterinic acid (1), lobaric acid (2) and baeomycesic acid (3) were tested for anti-proliferative effects against 12 different human cancer cell lines. All compounds have known in vitro 5-LOX inhibitory activity, and 1 and 2 also inhibit 12-LOX. The activity of the lichen metabolites was compared to that of a specific 5-LOX inhibitor, zileuton (4). The following cancer cell lines were tested: Capan-1, Capan-2 and PANC-1 (all from pancreas), T47-D (breast), PC-3 (prostate), NCI-H1417 (small cell lung), NIH:OVCAR-3 (ovary), AGS (stomach), WiDr (colorectal), HL-60, K-562 and JURKAT (acute promyelocytic, erythro- and T-cell leukemia, respectively). Compound 1 showed the greatest inhibitory effect against all cell lines, with EC50 ranging from 2.4-18.1 microg mL(-1) (7.4-55.8 microM), followed by 2, with EC50 of 15.2 - 65.5 microg mL(-1) (33.2-143.6 microM). The effects of 3 and 4 were of similar orders of magnitude, with EC50 of 28.7 - >80 microg mL(-1) (76.8 - > 213.9 microM) and 12.9 - > 80 microg mL(-1) (50.4 - > 313.7 microM). The dual 5- and 12-LOX inhibitors 1 and to some extent 2 thus exert significant anti-proliferative effects against a variety of human cancer cell lines, while the selective 5-LOX inhibitors 3 and 4 are considerably less active.
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One of the challenges of manipulating genes in primary cells is that the cells have a finite proliferation capacity. This, combined with the lower gene targeting efficiency of somatic cells, makes identification of targeted clones very difficult. The objective of this study was to establish a system that allows porcine foetal fibroblasts to reach their maximal proliferation capacity in vitro. The influence of fibroblast origin, stage of foetal development, cell seeding densities and concentration of foetal bovine serum (FBS) on the population doublings, the percentage of beta-galactosidase-activity-positive cells and the genome stability of foetal fibroblasts during in vitro culture was investigated. It was found that porcine foetal fibroblasts could be cultured for over 80 population doublings in the appropriate culture system. Fibroblasts from earlier stages of foetal development were better candidate cells than those from the later stages. Cells from the heart were more actively proliferative and more resistant to replicative senescence than those from the liver. Compared to 10% FBS content, 15% FBS provided better homeostatic support, not only to proliferative performance, but also in maintaining a normal karyotype. In addition, the proliferative life span of porcine foetal fibroblasts is also dependent on seeding density of the culture.
Different layers of rabbit large and small intestine display different peptide sensitivity and different profiles of eicosanoid release. Isolated perfused mesenteric pedicle alone, with muscularis/submucosa or with muscularis and mucosa from normal small bowel, normal colon, or inflamed colon were stimulated with bradykinin (BK) or n-formyl-methionyl-leucyl-phenylalanine (fMLP). Released prostaglandin (PG)E2, thromboxane (Tx)B2, and leukotriene (LT)B4 were assayed using extensively validated radioimmunoassays. In rabbit colon, PGE2 arises primarily from the mesentery, while in small intestine the muscularis/mucosa releases 70-80% of the total PGE2. BK releases no significant thromboxane from healthy colon, although both muscularis/submucosa and mucosa respond in inflamed colon. In contrast, fMLP stimulates thromboxane from muscularis/submucosa and mucosa of even healthy colon, while release is greatly potentiated in inflammation. Lipoxygenase in the colon is regulated differently than cyclooxygenase; it is not stimulated by BK in either healthy or inflamed colon. fMLP releases equal amounts of LTB4 from healthy and inflamed colon, but release was primarily from healthy colonic mucosa, whereas it was distributed throughout mesenteric pedicle, muscularis, and mucosa in inflamed colon. The ability of normal colonic mucosa to release proinflammatory LTB4 in response to a chemotactic factor (fMLP) produced by enteric bacteria suggests a possible role for these compounds as a stimulus for inflammation in some patients with inflammatory bowel disease.
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