Synthesis and properties of (6,7-dimethoxy-4-coumaryl)alanine: a fluorescent peptide label.

The synthesis of racemic and l-(6,7-dimethoxy-4-coumaryl)alanine (Dmca) is described and some spectral and physicochemical properties are reported. The utility of Dmca as a highly sensitive and specific label for the quantitative detection of synthetic peptides in HPLC and in minimal essential media (MEM) is also described.

Evidence for the location of a binding sequence for the alpha 2 beta 1 integrin of endothelial cells, in the beta 1 subunit of laminin.

To date no specific location on laminin 1 for the binding of alpha 2 beta 1 integrin has been described, although recent evidence supports a location in the E1XNd fragment of the cross region. We have identified a peptide sequence from this region, in the beta 1 chain of laminin 1, YGYYGDALR, which inhibits the adhesion of endothelial cells to laminin 1 and type-IV collagen. A structurally related sequence from the CNBr-cleaved fragment CB3 of the alpha 1 chain of collagen type IV, FYFDLR, inhibits endothelial cell adhesion to both collagen types I and IV and laminin 1. The CB3 fragment containing the FYFDLR sequence has been shown to contain binding sites for both alpha 1 beta 1 and alpha 2 beta 1 integrins. Present experiments with anti-integrin antibodies indicate that the alpha 2 beta 1 integrin on endothelial cells can account for all the cell binding to collagen types I and IV, and that this integrin makes a major contribution towards the adhesion of these cells to laminin 1. We therefore propose that the peptide FYFDLR participates in alpha 2 beta 1 binding to collagen type IV and that the putatively structurally similar peptide, YGYYGDALR, participates in alpha 2 beta 1 binding to laminin 1. This is the first account of structurally related peptide sequences from laminin 1 and type-IV collagen which show reciprocal inhibition of cell adhesion to either ligand and which might form part of a common integrin-binding site, as well as the first suggestion of a precise location contributing to the alpha 2 beta 1 integrin binding site on laminin 1.

Collagen-associated molecules in the cornea: localisation with monoclonal antibodies.

This paper describes the biochemical characteristics and immunostaining properties of four monoclonal antibodies (MAbs) raised to the extracellular matrix of bovine corneal endothelial cells in culture. All four antibodies labelled molecules in frozen sections of bovine cornea. MAb A15 labelled the collagen lamellae of the stroma. Immuno-electron microscopy located the label between rather than over the collagen fibrils. Antibody label was removed by collagenase treatment of the stroma. This antibody bound to several denatured collagen types in ELISAs and Western blots, the common epitope being located close to the collagenase binding site. A15 immunoprecipitated native polypeptides of 173 kDa and 150 kDa from the conditioned medium of corneal endothelial cells. Amino acid analysis of the 150 kDa molecule showed broad similarity to bovine type VI collagen although there was no immunological cross reactivity. The binding of this antibody in the corneal stroma may be to a collagen type VI-like molecule. MAb A70 bound to a collagenase sensitive molecule in corneal endothelial cell extracellular matrix in vitro, and to basement membranes in vivo. It showed staining typical of type IV collagen in frozen sections of cornea. MAbs A67 and A49 both labelled flexuous fibre-like structures in the cornea, which appeared to wrap around the fibrillar collagen lamellae. The molecule labelled by A67, of 86 kDa was sensitive to collagenase treatment of endothelial cell conditioned medium, yet totally resistant to collagenase treatment of the cornea, which completely removed the fibrillar collagen from the stroma. In the endothelial extracellular matrix, this antigen was resistant to all proteases tested and required severe denaturing conditions to remove antigenic activity. Amino acid analysis did not yield the high proportion of glycine typical of collagens, so if collagenous sequences occur they must be relatively short. The molecule labelled by A49 was a 51-kDa protein, of similar amino acid composition to antigen 67, and showed a similar distribution in frozen sections of bovine cornea. There was, however, no immunological cross reactivity between the two. Unlike antigen 67, antigen 49 was not sensitive to collagenase under any conditions and was very sensitive to protease treatment of the endothelial extracellular matrix. Immuno-electron microscopy showed labelling with both these antibodies between the collagen fibrils in the stroma. We postulate that these two molecules may be involved in stabilising the lamella structure of the corneal stroma.

The effect of extracellular matrix molecules on the in vitro behavior of bovine endothelial cells.

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.

Anti-fibronectin antibodies that modify heparin binding and cell adhesion: evidence for a new cell binding site in the heparin binding region.

A panel of monoclonal antibodies (mAbs) to bovine fibronectin (FN) is described which modulates either heparin binding or cell adhesion to FN, or both. A combination of competitive exclusion and binding to proteolytic fragments identified epitopes in the Hep II, Hep III/I and CBF (cell binding fragment) regions of FN. mAb A17, which bound to the CBF region, strongly inhibited the cell adhesion of BHK-21 fibroblasts, primary corneal fibroblasts and endothelial cells, and NM4 mammary adenocarcinoma cells, to FN at mAb concentrations as low as 1 microgram/ml. This mAb was not so effective at inhibiting the adhesion of B16 mouse melanoma cells. Adhesion of B16 cells to FN was more sensitive to inhibition by mAbs binding to Hep II (A2, A9, A32, A35). Of these, A32 and A35 significantly increased the binding of 35S-heparin to FN, whereas A2 and A9 did not affect it. mAbs A2, A9 and A32 showed good binding to HBF, the 40 kDa proteolytic fragment of human FN which contains both Hep II and IIICS (type III connecting segment). These mAbs inhibited B16 cell adhesion to the HBF (heparin binding fragment) by 30-50%, the greatest inhibition being shown by mAb A32. Two synthetic peptides from the HBF, CS1 (peptide 1) from the IIICS region and peptide I from the Hep II region, also inhibited B16 cell adhesion to HBF by approximately 70 and 30%, respectively. These results suggest that maximal cell adhesion to the HBF involves both CS1 and Hep II. The inhibitory effects of the two peptides were linearly additive in combination, whereas the inhibitory mAbs A2, A9 and A32 showed synergistic additive effects with each of the peptides. This points to the existence of an additional important cell binding site in Hep II, other than peptide I. Recent independent evidence for an additional cell binding site in Hep II supports this view. Melanoma cellular receptor(s) for the Hep II region may be cell surface proteoglycans but do not appear to bind to areas of Hep II with high affinity for soluble heparin, as the latter was not an inhibitor of B16 cell adhesion to the HBF. The increased effectiveness of A32 in inhibiting cell adhesion, compared to A2 and A9, may be due to conformational effects which increase the binding of soluble heparin, but reduce affinity for the cellular receptor. These results are discussed in context with other reports in the literature.

Solid-phase monoclonal antibodies. A novel method of directing the function of biologically active molecules by presenting a specific orientation.

Monoclonal antibodies were produced against fibronectin and vitronectin, using the extracellular matrix from cultured bovine corneal endothelial cells as immunogen. Some antibodies inhibited the adhesion of BHK-21 cells to these molecules, in a standard assay with fibronectin or vitronectin coated on a plastic surface. By first coating the plastic surface with the monoclonal antibodies and subsequently binding vitronectin, the inhibitory effect was markedly increased. Divalent fibronectin required the presence of antibody in the medium as well as coated on the surface for maximal inhibition of cell adhesion. The inhibitory effect of surface-coated antibodies was stable in the presence of cells up to at least 24 h in vitro. Using this antibody coating method the surface could either be made compatible for cell adhesion (using non-inhibitory antibodies) or refractory. Assays performed by this method have considerable potential for quantifying particular functions of extracellular matrix molecules.

A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin.

The effects of vitronectin and fibronectin upon the attachment and growth of bovine corneal endothelial cells (BCE) and BHK-21 cells were compared. Similar dose-response curves for cell attachment to the substratum were obtained for both molecules and both cell types, although BCE cells exhibited a slight preference for vitronectin, and BHK cells for fibronectin. When, however, cells were plated in medium containing bovine serum stripped of fibronectin, they attached and grew normally, whereas in medium containing serum stripped of vitronectin, cells either failed to attach (BHK-21) or attached but exhibited poor cell spreading and growth. This dependence of cells upon vitronectin, rather than fibronectin, in serum for cell attachment, was shown to be due to a failure of fibronectin to coat the substratum in the presence of other serum proteins. Vitronectin was able to coat the substratum efficiently in the presence of other serum proteins. Although dependent upon vitronectin for adhesion to the substratum, bovine endothelial cells were unable to synthesize endogenous vitronectin.

Evidence that sulphated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases.

Recent studies in this laboratory demonstrated that several sulphated polysaccharides can inhibit metastasis of the rat mammary adenocarcinoma 13762 MAT, probably by preventing the passage of tumour cells through the walls of blood vessels. In order to directly test this possibility, 13762 MAT cells were cultured with (35S)O4(=)-labelled subendothelial extracellular matrices (ECM) and ECM degradation was monitored in either the presence or absence of different sulphated polysaccharides. Degradation products were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent autoradiography. The 5 sulphated polysaccharides that had previously been shown to possess anti-metastatic activity were potent inhibitors of the degradation of subendothelial ECM by 13762 MAT cells. In contrast, of the 4 polysaccharides tested that failed to inhibit metastasis, 3 had no effect on ECM breakdown and one (carrageenan-kappa) was substantially less effective at inhibiting ECM degradation than the anti-metastatic preparations. It was also shown that 13762 MAT cells produce a heparan sulphate-specific glycosidase (heparanase) that degrades the heparan sulphate side-chains of the ECM, the action of this enzyme rather than that of other ECM-solubilizing enzymes being inhibited by the antimetastatic sulphated polysaccharides. Additional experiments indicated that the anti-coagulant activity of the polysaccharides probably plays a minor role in their anti-metastatic effects since heparin, almost completely depleted (98-99.5%) of heparin molecules with anti-coagulant activity by passage over an anti-thrombin III column, retained its ability to inhibit 13762 MAT heparanases and was almost as effective as unfractionated heparin at inhibiting tumour-cell metastasis. Collectively, these data suggest that sulphated polysaccharides inhibit the metastasis of 13762 MAT cells by inhibiting tumour-cell-derived heparanases involved in the penetration of the vascular endothelium and its underlying basement membrane by tumour cells.

Onset of neoplastic phenotype in an epithelial cell strain from adult BALB/c mouse lung alveolus.

Prolonged culture of NAL1A in 1 microM dexamethasone (CAS: 50-02-2) at low passage numbers resulted in the emergence of a morphologically altered and malignant cell strain, NAL1AM. (NAL1A was derived from lungs of normal adult female inbred BALB/c mice and exhibits several characteristics of epithelial cells.) A clone of NAL1A, B5, was shown to undergo a spontaneous morphologic alteration during culture in normal medium to resemble NAL1AM and cells of NUL1, a strain cultured directly from urethane (CAS: 51-79-6)-induced adenomas of mouse lung. Whereas NAL1A did not form colonies in soft agar, the clone B5 of NAL1A as well as NAL1AM and NUL1 showed quite high anchorage-independent growth (colony-forming efficiency, 6.3-7.8%). Compared with NAL1A cells, clone B5, NAL1AM, and NUL1 each exhibited a ninefold-reduced level of cellular binding of epidermal growth factor.

Monensin inhibits initial spreading of cultured human fibroblasts.

The monovalent ionophore, monensin, inhibits the secretion of both pro-collagen and fibronectin in cultured human fibroblasts and other cell types. The block to secretion is due to the ability of monensin to suppress the export of these secretory proteins from the Golgi apparatus. As such proteins are known to be implicated in the adhesion, spreading and movement of cultured fibroblasts, it might be expected that monensin treatment would interfere with these processes. However, it has recently been reported that monensin-treated human embryonal fibroblasts attached and spread onto glass substrata to the same extent as untreated cells, although at later stages they fail to develop focal adhesion sites. However, these experiments were performed using medium supplemented with fetal calf serum (FCS). We now demonstrate that in the absence of FCS, while monensin has little or no effect on the initial adhesion of fibroblasts to the substratum, subsequent spreading is much reduced. The inhibition of spreading is noticeable within 30 min of plating and is maintained for at least 100 min in monensin-free medium following prolonged pre-incubation of the cells with monensin.

Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase.

Liver of rat foetuses from 14 to 19 days of gestation and cultured hepatocytes derived from foetuses of 14 or 15 days gestation show a limited capacity to transaminate tyrosine. This low tyrosine transamination activity can be ascribed to aspartate aminotransferase. Definitive tyrosine aminotransferase can be demonstrated in 1-day-old cultures of hepatocytes taken from 19-day foetuses, but not from 15-day foetuses. However, after 3 days of culture hepatocytes from 15-day foetuses are able to synthesize tyrosine aminotransferase. Induction studies reveal that dexamethasone is capable of increasing tyrosine aminotransferase activity once it is detectable in culture.

The relationship of paroxysmal ventricular tachycardia complicating the acute phase and ventricular arrhythmia during the late hospital phase of myocardial infarction to long-term survival.

The long-term prognosis of paroxysmal ventricular tachycardia (PVT) complicating acute myocardial infarction remains unevaluated. Significant ventricular arrhythmia in the patient after infarction is said to carry a poor prognosis with regard to survival. To evaluate these two important aspects of myocardial infarction in man, 56 patients with documented myocardial infarction had Holter monitoring performed during the initial 24 hours and prior to hospital discharge. In 38 of the 45 survivors, Holter monitoring was repeated an average of 19 months after infarction. There were eight cardiac deaths during follow-up. Data analysis revealed that of 18 patients with PVT during the acute phase, one died during follow-up and 17 survived long-term. Even though the incidence of complex PVCs prior to hospital discharge and at long-term follow-up was higher in patients with PVT during the acute phase than in those without PVT, survival appeared unaffected. Thus, PVT during the acute phase of myocardial infarction and complex PVCs at the time of hospital discharge are not incompatible with long-term survival.