SUMMARY Human extensor tendons of the hand were pulled to rupture, and the torn ends, when observed in the scanning electron microscope, appeared to be knotted, possibly owing to the denaturation of the collagen. This was confirmed by fluorimetry assays of both the ruptured ends and the middle unbroken sections of the same tendons. The use of a proteolytic enzyme, trypsin, to remove the denatured material and enhance the repair of organised collagen fibres from both ends is suggested if the ruptured ends have the denatured knotted appearance commonly observed clinically.Tendons that have become ruptured in vivo have usually failed because of 2 conditions: because an unusually large force has been applied through the tendon before muscular control has resisted the load, or because the tendon, in particular the collagen fibre network, which resists tensile loads transmitted through the tendon, has undergone biochemical attack substantially weakening the collagen. Tendons that have become divided as a result of trauma show a lesion at the rupture in which the ends have become atrophic and rounded producing a blunt amputation stump. After several months of repair histological evidence shows the junction of the new and old tendon and no regeneration from the proximal stump (Peacock, 1977).Clinical evidence and our interests in degeneration of collagenous tissue led to us conduct a preliminary study on the morphology of tendon ends after mechanical rupture (Steven et al., 1975). A hypothesis we put forward during that study was that the appearance of the ruptured end was a result of denaturation at the fibre ends, so the ends of the distal portions were exposed to trypsin for 24 hours. The tapered and knotted ends were not evident on the trypsin-exposed preparations, suggesting that the ruptured ends contained a substantial amount of denatured collagen.
Guanidinobenzoatase is a trypsin-like protease associated with tumour cells (Steven et al., 1985) which may be assayed in solution by the cleavage of methylumbelliferone from the fluorogenic substrate 4-methylumbelliferyl-p-guanidinobenzoate (Steven & Al-Ahmad, 1983). This protease has been shown to cleave the tetrapeptide GlyArgGlyAsp which is thought to link cell surfaces to fibronectin (Pierschbacher & Ruoslahti, 1984). Guanidinobenzoatase is competitively inhibited by 9-aminoacridine (Steven et al., 1985), and this observation was used to locate tumour cells containing guanidinobenzoatase by fluorescent microscopy of formaldehyde fixed wax embedded sections. It was later established that most host tissues contained extractable proteins which were non-competitive inhibitors of guanidinobenzoatase in solution (Steven et al., 1988a). These protein inhibitors prevented the binding of 9-aminoacridine to the protease of most tumour cells in fresh frozen sections (Steven et al., 1988b), whilst some tumour cells possessed uninhibited enzyme in these frozen sections. In the present paper we have studied those tumour cells in frozen sections which possess uninhibited guanidinobenzoatase. When treated with 9-aminoacridine and examined by fluorescent microscopy these uninhibited cells exhibit cytoplasmic and cell surface yellow fluorescence; whilst other morphologically similar tumour cells with fully inhibited guanidinobenzoatase appear to be non-fluorescent with blue-green cytoplasm and cell surfaces. Those tumour cells which possess uninhibited guanidinobenzoatase were located at the advancing edge of the tumour mass or were detected as small groups of individual tumour cells outside the main tumour mass. This paper is concerned with the inhibition of the guanidinobenzoatase on these cells at the advancing edge of the tumour mass, since these can be more easily located than single cells in sequential sections. The role of host tissue protein inhibitors in the control of cell migration will be discussed.BZAR [bis-(N-benzyloxycarbonyl-L-argininamido)-Rhodamine] was first described by Leytus et al. (1983). BZAR inhibits guanidinobenzoatase in solution and on the surface of tumour cells (Steven et al., 1988), but is cleaved by other trypsin-like enzymes (Leytus et al., 1983). As both BZAR and 9-aminoacridine bind to the active centre of guanidinobenzoatase we used these inhibitors, one after the other, on Materials and methods 9-Aminoacridine was purchased from Sigma Chemical Company, St Louis, MO, USA. A stock aqueous solution, 10-3 M, of 9-aminoacridine was used for fluorescent staining. We employed 1O6 M BZAR dissolved in isotonic saline for the inhibition of cell bound guanidinobenzoatase. Frozen sections of human tumours taken from the head and neck region were kindly provided by the Pathology Department of the Justus-Liebig University, Giessen, West Germany. In all, over 500 frozen sections were provided from 55 subjects; these sections contained normal tissue as well as tumour cells, and the fluorescent examinat...
Departments of' IBiocheimistry' anCd 2Pharniacolo!gu, Stop! l-rd This study demonstrates the use of a fluorescent probe for the active centre of a cell surface protease (Steven et al., 1985) to locate the malignant cells of a rat T-cell leukaemia, (Diblev et a!., 1975;Jackson et al., 1984) in the host tissues. The enzyme, guanidinobenzoatasc, degrades fibronectin and has been shown to be associated with the surface of cells capable of migration (Steven et al., 1985). Using the fluorescent probe leukaemic cells can readily be demonstrated in sections of kidney, liver, testis, and epididymis in this experimental model; even individual leukaemia cells can be so located. We illustrate the application of the fluorescent probes in both wax and resin embedded sections, but frozen tissue sections can also be used.Fluorescent labelling is achieved by treating sections with aqueous 9-aminoacridine which is known to be a competitive inhibitor at the active centre of the protease. Cells possessing this protease 'stack' 9-aminoacridine and exhibit a yellow surface fluorescence on a blue background. A second staining procedure using an aqueous solution of propidium iodide following the 9-aminoacridine staining, enhances the colour contrast for photography. This combined staining procedure relies on the fact that both 9-amino acridine and propidium iodide are planar molecules which are capable of intercalating in DNA. If a cell surface possesses guanidinobenzoatase, then it is possible to stack 9-aminoacridine and subsequently co-stack propidium iodide on the cell surface (Steven et al., 1986). The co-stacking of propidium iodide leads to a change in fluorescent emission from yellow to pink. The structures of these fluorescent molecules are shown diagrammatically in Figure 1. Materials and methodsAnaial.s T-Leukaemia cell suspensions were prepared for intramuscular transmission of the disease in the inbred hooded Oxford strain of rats following the procedure previously described (Jackson et al., 1984). Chenmic als 9-Aminoacridine, propidium iodide and N-tosyl-lysyl-chloromethylketone were purchased from Sigma Chemical Company, St Louis, Mo., USA.Flulorescent staining Dewaxed sections (5 pm) were placed in an aqueous solution containing 9-aminoacridine (10 3 M) and N-tosyl-lysy-lchloromethylketone (10 -M) for 2min. Excess reagent was removed by 2 min washing in each of a series of 3 tanks containing isotonic sodium chloride. Resin sections (1I im) were stained in the same mixture for 5 min then washed with isotonic saline for 30 sec prior to microscopic examination. The combined staining procedure involved placing the 9-aminoacridine-stained slide in an aqueous solution of propidium iodide (6 x 10-I M) for 1 min followed by washing with water for 10 sec prior to microscopic analysis.The combined staining had 3 results; (a) nuclei of all cellsshowed an overall red fluorescence as would be expected (DNA reaction) (b) the plasma membranes of those cells possessing guanidinobenzoatase now appeared pink and (c) mast cells exhibite...
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