Biomedical subjects

L T Furcht

Publications and source records attributed to L T Furcht.

At least 91 records · Page 5Linked to original sources

PubMed1989

Novel synthetic heparin binding peptides of laminin and fibronectin which promote the adhesion of melanoma cells.

A synthetic peptide derived from the B1 chain of laminin, F-9, as well as two peptides derived from the 33-kilodalton fragment of fibronectin, I and II, directly promoted the adhesion of K-1735-M4 metastatic murine melanoma cells. In competition assays, adhesion of these cells to laminin was inhibited by excess soluble peptide F-9. Peptides F-9, I, and II specifically bound 3H-heparin, both by direct binding assays and indirect competition assays. 3H-heparin binding to peptide F-9 was specific as determined by competition with excess unlabeled heparin, dextran sulfate, and dermatan sulfate. These findings suggest that melanoma cell surface associated heparin-like molecules may act as receptors for these domains of laminin and fibronectin.

Animals↗

PubMed1989

Quantification of corneal organ culture migration: central and peripheral epithelium.

This report outlines a method for rapid quantification of corneal epithelial cell movement in a block organ culture system designed to model a penetrating or perforating wound of the cornea. Rabbit corneas were secured over a plastic conformer with a chalazion clamp. Two-millimeter, full-thickness corneal specimens were sampled from central and peripheral cornea and placed in tissue culture. The aim of the procedure was to produce full-thickness corneal specimens from well-defined geographic areas of the cornea of reproducible size and shape without undue trauma to the epithelium. The instruments used for measurements were simple, allowing for accumulation of a large number of observations which could be subjected to rigorous statistical evaluation. This method was used to determine if there was a detectable difference in the rate of epithelial cell movement over stromal cut surface of specimens from the periphery of the cornea, versus the rate over specimens from the central area of the cornea. In this block organ culture system the peripheral epithelial cells migrated more rapidly than samples of central epithelial cells (P less than 0.05). This study shows a simple, rapid method for producing and evaluating block corneal organ cultures and shows that the site of specimen sampling must be controlled in corneal epithelial cell studies employing block organ culture. This method will be useful for in vitro screening of novel pharmacologic agents which have the potential for influencing corneal wound healing prior to evaluating these agents in vivo.

Animals↗

PubMed1988

Heparin type IV collagen interactions: equilibrium binding and inhibition of type IV collagen self-assembly.

Interactions between type IV collagen and heparin were examined under equilibrium conditions with rotary shadowing, solid-phase binding assays, and affinity chromatography. With the technique of rotary shadowing and electron microscopy, heparin appeared as thin, short strands and bound to the following three sites: the NC1 domain, and in the helix, at 100 and 300 nm from the NC1 domain. By solid-phase binding assays the binding of [3H]heparin in solution to type IV collagen immobilized on a solid surface was found to be specific, since it was saturable and could be displaced by an excess of unlabeled heparin. Scatchard analysis indicated three classes of binding sites for heparin-type IV collagen interactions with dissociation constants of 3, 30, and 100 nM, respectively. Furthermore, by the solid-phase binding assays, the binding of tritiated heparin could be competed almost to the same extent by unlabeled heparin and chondroitin sulfate side chains. This finding indicates that chondroitin sulfate should also bind to type IV collagen. By affinity chromatography, [3H]heparin bound to a type IV collagen affinity column and was eluted with a linear salt gradient, with a profile exhibiting three distinct peaks at 0.18, 0.22, and 0.24 M KCl, respectively. This suggested that heparin-type IV collagen binding was of an electrostatic nature. Finally, the effect of the binding of heparin to type IV collagen on the process of self-assembly of this basement membrane glycoprotein was studied by turbidimetry and rotary shadowing. In turbidity experiments, the presence of heparin, even in small concentrations, drastically reduced maximal aggregation of type IV collagen which was prewarmed to 37 degrees C. By using the morphological approach of rotary shadowing, lateral associations and network formation by prewarmed type IV collagen were inhibited in the presence of heparin. Thus, the binding of heparin resulted in hindrance of assembly of type IV collagen, a process previously described for interactions between various glycosaminoglycans and interstitial collagens. Such regulation may influence the assembly of basement membranes and possibly modify functions. Furthermore, qualitative and quantitative changes of proteoglycans which occur in certain pathological conditions, such as diabetes mellitus, may alter molecular assembly and possibly permeability functions of several basement membranes.

Animals↗

PubMed1988

Localization of three distinct heparin-binding domains of laminin by monoclonal antibodies.

Monoclonal antibodies were utilized to localize novel heparin-binding domains of laminin. A solid-phase radioligand binding assay was designed such that [3H] heparin bound to laminin in a time- and concentration-dependent manner. Tritiated heparin binding to laminin was saturable and specific as determined by competition with unlabeled heparin, dextran sulfate, and dermatan sulfate. By Scatchard analysis, two distinct dissociation constants were calculated (Kd = 50 and 130 nM), suggesting the presence of at least two binding sites for heparin on laminin. Tritiated heparin bound to thrombin-resistant (600 kDa) and chymotrypsin-resistant (440 kDa) laminin fragments, both known to lack the terminal globular domain of the long arm. Sodium dodecyl sulfate-polyacrylamide gels of chymotrypsin- and thermolysin-digested laminin chromatographed on a heparin-Sepharose column showed multiple proteolytic fragments binding to the column. Monoclonal antibodies generated against laminin were tested for their ability to inhibit [3H]heparin binding to laminin. Four monoclonal antibodies significantly inhibited the binding of [3H]heparin to laminin in the range of 15-21% inhibition. Laminin-monoclonal antibody interactions examined by electron microscopy showed that one antibody reacted at the terminal globular domain of the long arm, domain Hep-1, while epitopes for two of these monoclonal antibodies were located on the lateral arms of laminin, domain Hep-2, and the fourth monoclonal antibody bound below the cross-region of laminin, domain Hep-3. When two monoclonal antibodies recognizing distinctly different regions of laminin were added concomitantly, the inhibition of [3H]heparin binding to laminin increased almost 2-fold. These results suggest that at least two novel heparin-binding domains of laminin may be located in domains distinct from the terminal globular domain of the long arm.

Antibodies↗

PubMed1988

The effect of nonenzymatic glucosylation on the binding of the main noncollagenous NC1 domain to type IV collagen.

Type IV collagen has the ability to self-assemble by amino end, carboxyl end, and lateral associations to complex network-like structures which can be visualized by rotary shadowing. The main noncollagenous NC1 domain which is located at the carboxyl end of type IV collagen molecules binds to itself to form dimers and also binds along the length of type IV collagen. The latter binding initiates lateral assembly. Following in vitro nonenzymatic glucosylation of the isolated NC1 domain, binding to the helix-rich domain of type IV collagen was impaired. In turbidity experiments, the nonenzymatically glucosylated NC1 domain minimally suppressed the development of turbidity of collagen solutions when compared to control NC1 domain. In rotary shadowing experiments nonenzymatically glucosylated NC1 domain did not significantly inhibit lateral associations or networks formed by type IV collagen, whereas control NC1 domain caused a drastic decrease in laterally assembled structures. These data suggest that nonenzymatic glucosylation of the NC1 domain may interfere with normal assembly of type IV collagen in diabetes mellitus and may be related to abnormal functions of basement membranes in this pathological condition.

Animals↗

PubMed1988

Metastasis inhibition of different tumor types by purified laminin fragments and a heparin-binding fragment of fibronectin.

Tumor cell metastasis is a complex process that depends in part on tumor cell adhesion to components of basement membranes and the extracellular matrix. Previous studies have indicated that the experimental metastasis of murine melanoma cells can be inhibited by ex vivo pretreatment of cells with purified adhesion-promoting fragments of laminin or the synthetic peptide arginyl-glycyl-aspartyl-serine (RGDS) prior to tail vein injection. This study extended the earlier reports to demonstrate that adhesion-promoting fragments of laminin and fibronectin can inhibit the metastasis of a tumor of different histologic origin, such as murine fibrosarcoma cells. Furthermore, ex vivo pretreatment of cells with a purified 33-kDa heparin-binding fragment of fibronectin, which promotes tumor cell adhesion by an RGDS-independent mechanism, was effective at inhibiting experimental melanoma and fibrosarcoma pulmonary metastases. The survival rate of animals receiving tumor cells pretreated with this fragment was significantly enhanced relative to control groups. As with previous studies, the mechanism of inhibition appeared to involve an increased clearance rate of tumor cells from the pulmonary microcirculation. These results suggest a role for cell surface proteoglycans in the adhesion and metastasis of certain malignant neoplasms. Furthermore, this study emphasizes the complexity of tumor metastasis and suggests that multiple strategies may be developed to inhibit hematogenous metastasis formation.

Animals↗

PubMed1988

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities.

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

C1q component of complement binds to fibrinogen and fibrin.

The interaction of complement component C1q with fibrinogen and fibrin was studied by using a solid-phase direct binding assay. Scatchard analysis of radioiodinated fibrinogen binding to C1q indicated at least two high-affinity binding constants (Kd) calculated as 8.5 and 120 nM. In contrast, binding of radioiodinated fibrin to C1q showed only a single class of binding sites with a calculated Kd of 600 nM. Fibrinogen-C1q binding was shown to decrease as a function of increasing salt concentrations, indicating either the presence of charged amino acids in the binding sites or an ionic strength induced conformational dependency of the binding. In direct binding studies using isolated fragments of C1q, both the collagen-like domain of C1q and the globular domains of C1q were shown to bind fibrinogen, indicating at least one binding site for fibrinogen is located in each of the major domains of C1q. Addition of the thrombin-generated peptides of fibrinogen, fibrinopeptides A and B, enhanced C1q-fibrinogen binding, again indicating a complex binding interaction. These results indicate that C1q and fibrinogen are capable of high-affinity interactions that may serve to sequester these complexes in areas of tumors, immune complex deposition, or wounds.

Binding Sites↗

PubMed1988

Immunoreactivity for laminin in the developing ventral longitudinal pathway of the brain.

The first long tract to form in the brain of a vertebrate embryo is the ventral longitudinal pathway. In order to investigate what chemical cues may guide nerve growth cones along this pathway, affinity-purified antibodies to laminin and collagen type IV were used to stain sections of mouse embryos from Embryonic Days 8 through 17. A monoclonal anti-neurofilament antibody was used to show the development of the ventral longitudinal pathway in relationship to immunoreactivity for laminin and collagen type IV. At Day 8 fluorescent immunoreactivity for laminin is bright in the external limiting membrane of the neural tube, but the neuroepithelium does not show bright laminin or neurofilament immunoreactivity. At E9 the ventral longitudinal pathway is forming and punctate immunoreactivity for laminin is present on the surfaces of neuroepithelial cells in the marginal zone, through which axons of the ventral pathway extend. Punctate immunofluorescence for laminin remains concentrated in the marginal zone on Days E10 through E14, but on E16 punctate immunofluorescence was much reduced, although immunoreactivity for laminin remained bright in the maturing pial and arachnoid membranes and on blood vessels in the brain. Immunoreactivity for collagen type IV was strong in the external limiting membrane and on blood vessels, but never showed concentrated punctate immunofluorescence in the marginal zone. These results indicate that laminin may be available on cell surfaces and in extracellular spaces as an adhesive ligand for growth cones during the formation of the ventral longitudinal pathway.

Animals↗

PubMed1988

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity.

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.

Animals↗

PubMed1988

Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration.

Laminin and type IV collagen were compared for the ability to promote aortic endothelial cell adhesion and directed migration in vitro. Substratum-adsorbed IV promoted aortic endothelial cell adhesion in a concentration dependent fashion attaining a maximum level 141-fold greater than controls within 30 min. Aortic endothelial cell adhesion to type IV collagen was not inhibited by high levels (10(-3) M) of arginyl-glycyl-aspartyl-serine. In contrast, adhesion of aortic endothelial cells on laminin was slower, attaining only 53% of the adhesion observed on type IV collagen by 90 min. Type IV collagen when added to the lower well of a Boyden chamber stimulated the directional migration of aortic endothelial cells in a concentration dependent manner with a maximal response 6.9-fold over control levels, whereas aortic endothelial cells did not migrate in response to laminin at any concentration (.01-2.0 X 10(-7) M). Triple helix-rich fragments of type IV collagen were nearly as active as intact type IV collagen in stimulating both adhesion and migration whereas the carboxy terminal globular domain was less active at promoting adhesion (36% of the adhesion promoted by intact type IV collagen) or migration. Importantly, aortic endothelial cells also migrate to substratum adsorbed gradients of type IV collagen suggesting that the mechanism of migration is haptotactic in nature. These results demonstrate that the aortic endothelial cell adhesion and migration is preferentially promoted by type IV collagen compared with laminin, and has a complex molecular basis which may be important in angiogenesis and large vessel repair.

Animals↗

PubMed1988

A novel synthetic peptide from the B1 chain of laminin with heparin-binding and cell adhesion-promoting activities.

Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]heparin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.

Amino Acid Sequence↗

PubMed1988

Epithelial and neural localization and heparin binding of the cell-substratum adhesion molecule, epinectin.

Epinectin, a cell-substratum adhesion promoting molecule, was first isolated from the extracellular matrix of A431 human squamous carcinoma cells. In order to determine the biologic significance of epinectin, we determined the distribution of epinectin in various rat epithelial tissues by indirect immunofluorescence microscopy. Polyclonal antibodies to epinectin stained basal cells and basilar regions of skin, urinary bladder, and vagina. There was predominantly cytoplasmic staining along with amorphous extracellular staining. Strong staining was also noted in sebaceous glands and hair follicles. The immunoreactivity for epinectin in the skin was distinct from that for fibronectin, laminin, and type IV collagen. Antibodies to epinectin also stained subpopulations of neurons in the cerebrum and cerebellum. Epinectin antibodies strongly stained the cytoplasm of some pineal cells and cells of the pars intermedia of the pituitary. The distribution of epinectin suggests a role not only in epithelial cell-substratum adhesion, but in neuronal cell function. Heparan sulfate is known to be involved in the binding of several adhesion promoting molecules to cell surfaces. In order to assess the mechanism of adhesion of epinectin to cells, we measured the binding of 3H-heparin to epinectin. Binding of 3H-heparin was concentration dependent and inhibitable with cold heparin.

Animals↗

PubMed1988

Molecular mechanisms in basement membrane complications of diabetes. Alterations in heparin, laminin, and type IV collagen association.

We report alterations in the ligand-binding properties of laminin, the major noncollagenous protein of basement membranes, resulting from nonenzymatic glycosylation in vitro. Mouse laminin was incubated in vitro with 500 mM glucose, and the level of nonenzymatically glycosylated amino acids increased 2-, 6.2-, and 12-fold after incubation for 1, 3, and 12 days, respectively. Ligand binding assays conducted in solution with varying concentrations of [3H]heparin and a constant amount of control or nonenzymatically glycosylated laminin showed a reduction in heparin binding proportional to laminin glycosylation. An analysis of the stoichiometry of [3H]heparin binding to control and nonenzymatically glycosylated laminin at saturating levels of heparin was performed. The results indicated that the total number of heparin binding sites on laminin decreased 4.7- and 14.7-fold due to nonenzymatic glycosylation of laminin for 1 and 3 days, respectively. [3H]heparin binding to 12-day nonenzymatically glycosylated laminin was abolished. Scatchard analyses of the binding data for heparin and laminin gave a dissociation constant (Kd) of 3.4 X 10(-8) M. The data for nonenzymatic glycosylation of laminin for 1 day in vitro resulted in a biphasic heparin binding curve. Both high- and low-affinity binding was observed (Kd values of 3.3 x 10(-8) and 2.6 x 10(-7) M, respectively). Similar high- and low-affinity binding sites were seen on 3-day nonenzymatically glycosylated laminin (Kd values of 2.1 x 10(-8) and 3.9 x 10(-7) M, respectively). [3H]heparin binding at a fixed concentration to a constant amount of control or 12-day nonenzymatically glycosylated laminin and type IV collagen was also studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Effects of matrix proteins on rabbit corneal epithelial cell adhesion and migration.

Extracellular matrix and basement membrane proteins are important in promoting the adhesion and migration of various cell types. Laminin and type IV collagen are found in basement membranes throughout the body while fibronectin is found associated with some basal lamina, in loose connective tissue, and in all body fluids. In the current study fibronectin, laminin, and type IV collagen were assessed to determine their effect on the adhesion and migration of dissociated rabbit epithelial cells. In a kinetic analysis fibronectin was found to be the most effective of the three proteins in promoting cell adhesion. Cell adhesion promoted by fibronectin could be modulated by an arg-gly-asp-ser (RGDS) peptide, a sequence which is found within the fibronectin molecule. In dose-response experiments type IV collagen effectively promoted cell adhesion at concentrations as low as 4 x 10(-16) moles per well in 96 well tissue culture plates. Type IV collagen was the most effective protein tested in promoting chemotactic and haptotactic migration. These proteins, proteolytic fragments, and peptides derived from them, could prove useful as therapeutic agents, modifying corneal epithelial phenotypic behavior in wound healing, corneal transplantation and following ocular surgery or trauma.

Animals↗

PubMed1988

Transient expression of laminin in the optic nerve of the developing rat.

The optic nerve of the developing rat was examined for the presence of laminin, an adhesive glycoprotein, to assess whether it might serve as a substrate for retinal axon growth in vivo. The optic stalk and nerve of developing rats were screened immunohistochemically for the presence of laminin before, during, and after the period of retinal axon growth. On embryonic day 14 (E14), laminin immunoreactivity was present in the ventral portion of the optic stalk, the same region in which the first retinal axons grow. Between E16 and postnatal day 10 (P10), cells positive for laminin were distributed throughout the cross-sectional area of the nerve. There was a progressive appearance of glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for astrocytes, from the optic nerve head towards the chiasm beginning on E20. At the advancing front of GFAP immunoreactivity, cells were positive for both laminin and GFAP. Behind the front, laminin immunoreactivity disappeared from the cells. By P12, the only laminin immunoreactivity that remained within the optic nerve surrounded the vasculature. This is a time after the last retinal axons grow through the optic nerve. Monolayer cell cultures were prepared from perinatal rat optic nerves and processed for immunohistochemistry to determine which astrocyte type was laminin-positive. Type 1 astrocytes, which primarily compose the immature nerve, are GFAP-positive, A2B5-negative, and laminin-positive. Type 2 astrocytes, a major component of the mature optic nerve, were GFAP-positive, A2B5-positive and laminin-negative. An extract of developing optic nerve was analyzed by immunoblot along with laminin purified from Engelbreth-Holm-Swarm (EHS) sarcoma. Purified laminin ran with SDS-PAGE under reducing conditions as 2 bands with Mrs of 200,000 and 4000,000. Both bands reacted with antibodies to laminin. A low-salt extraction of whole optic nerve from E18 rats resulted in 2 bands with the same Mr as seen with laminin from EHS sarcoma. When only the inside of the optic nerve (which lacked the basal lamina and meninges that surround the outside) was processed, there was a dark 200,000 D band, but the 400,000 D band was virtually absent. These results are consistent with the hypothesis that laminin, or a variant form of laminin, serves as a substrate for retinal axon growth in the developing rat optic nerve.

Animals↗

PubMed1987

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus.

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗

PubMed1987

Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody.

Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the binding of the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

Animals↗