Laminin Type Research Articles

Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulinbinding site of type V collagen was in a 30-kDa CNBr fragment of the α1(V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.

Immunohistochemical studies on the localization of fetal antigen 2 (FA2), laminin, and collagen type 4 in basal cell carcinoma

The localization of fetal antigen 2 (FA2), a recently described basement membrane (BM) associated antigen, was studied by immunohistochemical techniques in 21 basal cell carcinomas (BCC). In both superficial and infiltrating BCC, FA2 was located in a broad diffuse band around the tumor elements and in close contact with the BM. Compared to normal skin, a more extensive distribution of FA2 was seen in BCC. In the infiltrating BCC, FA2 staining was also present in the interstitial stroma between the tumor islands. FA2 was absent in areas with inflammatory cell infiltrates and elastoid degeneration. Epithelial and tumor cells were FA2 negative. The distribution of FA2 was clearly different from that of laminin and collagen type 4. Collagen type 4 and laminin were present as a continuous linear band corresponding to the BM surrounding the tumors. The close contact to the BM and the increased content of FA2 in the reactive stroma around BCC suggest that FA2 is involved in the matrix and/or BM changes taking place during tumor growth and invasion.

Different cellular receptors for human placental laminin and murine EHS laminin

Human placental laminin purified without the use of proteases promotes similar levels of cell attachment ? murine EHS laminin. The major HT1080 cell binding site of human placental laminin is thermally less labile than that of EHS laminin. Monoclonal antibody GOH3, which recognises the integrin α6 subunit, inhibits HT1080 and B16 cell attachment to EHS laminin but not to human placental laminin, indicating that the cells use different receptors to bind to these two types of laminin. Antibodies P1E6 and P1B5, which recognise the integrin α2 and α3 subunits respectively, do not affect HT1080 cell binding to either type of laminin.

Murine trabecular meshwork cells in tissue culture

Trabecular meshwork cells from an inbred strain of mice (A/J) were established in tissue culture. Within 1 hour of enucleation, tissue containing the cornea and the chamber angle was excised and placed in tissue culture. Two to five days later, three cell types grew from the explants. Two of these cell types, corneal endothelium and fibroblasts, grew together, with the fibroblasts preferentially spreading on top of the endothelial cells. The trabecular meshwork cells extended from the explant as a distinct morphological type. The corneal endothelium and its associated fibroblasts were then removed from the culture flask with a sterile cotton swab, leaving a monolayer of pure trabecular meshwork cells. These cells required 3-4 weeks to reach confluency and could be passaged five times. They were actively phagocytic in culture and exhibited immunoreactivity to antibodies against two extracellular matrix components, laminin and collagen type IV. Mouse trabecular meshwork cells also expressed receptors for acetylated low-density lipoprotein, a property shared by trabecular meshwork cells derived from other species. The availability of trabecular meshwork cells from an inbred strain of mice will facilitate future in vivo functional studies of these cells in a syngeneic system, as well as investigations of potential immunoregulatory properties of the trabecular meshwork.

Studies on human laminin and laminin-collagen complexes

Intact human laminin and laminin type IV collagen complexes were extracted from term placental membranes and their structures were examined by electroimmunoblot and by rotary shadowing electron microscopy. Rotary shadowing electron microscopy revealed a structure of human laminin which is essentially similar to the cruciform structure of the mouse tumor (EHS) laminin, but with some notable differences. The observed lengths of the short arms in the human laminin are different. One short arm has an average length of 34 nm and another short arm a length of 42 nm. In the mouse laminin all three short arms are of equal length. The average length of the long arm is 97 nm, which is longer than that of mouse tumor laminin (77 nm). The distal portion of the long arm has two clearly separated globular domains instead of the single one observed with tumor laminin. Human laminin was found in various states of aggregation including dimers, trimers and higher aggregates. Laminin molecules appeared to attach to a point in type IV collagen located 87 nm from C-terminus, or at 174 nm from C-terminus and one with less frequency, at 251 nm from C-terminus. A small number of molecules appeared to bind at the N-terminus of the collagen. These laminin-collagen interactions occurred via the distal globular domains of both the short and long arms of laminin. The data suggest that human laminin extracted from placenta is structurally different from that isolated from the mouse EHS tumor.

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.

Cell biological studies of primary and metastatic tumors of the rat prostate

The widespread occurrence and high mortality rate in older men associated with disseminated adenocarcinoma of the prostate provide a strong impetus for basic studies of the normal and neoplastic prostate gland. The present studies have been undertaken with the objective of gaining a fuller appreciation of histo- and cytologic interrelationships between metaplastic prostatic tumors and their host organs.The Dunning rat adenocarcinoma has provided a model for some years for the study of primary prostatic tumors, and recently a tumor subline has been identified which preferentially metastasizes to lung (Dr. David M. Lubaroff, U. IA). Cells of the metastatic subline colonize lung following intravenous injection, and produce widespread lesions within the lung parenchyma, and deep to the visceral pleura, in a matter of weeks. Correlative light microscopic (LM), transmission (TEM) and scanning (SEM) electron microscopic studies have been conducted of both the primary Dunning tumor, and the metastatic lesions within lung, using routine preparative techniques. Morphological examination has been conducted in concert with immunocytochemical localization of components of the extracellular matrix, within both lung and tumor, and with studies of the growth and attachment characteristics of tumor cells cultured in wells coated with such extracellular matrix components as fibronectin, laminin, and collagen types I and IV.

Distribution of basement membrane antigens in the uterine cervical adenocarcinomas: An immunohistochemical study

The distribution of laminin and collagen type IV in the basement membrane of 45 uterine cervical adenocarcinomas was studied using immunohistochemical techniques. Staining patterns of basement membranes were divided into three types, according to the intensity of staining: thick and discontinuous (type I), thin and discontinuous (type II), and fragmentary (type III). In well-differentiated adenocarcinomas, 17 of 24 cases (71%) showed type I and the remaining 7 showed type II. In contrast, in poorly differentiated adenocarcinomas 4 of 5 cases (80%) showed type III. Five of six cases (83%) of adenoma malignum, even though extremely well differentiated, showed type III. Staining patterns of the basement membranes correlated with histological grade. Five-year survival rate of patients with type I and II staining (74%) was better than that of patients with type III (55%).

The extracellular matrix in large-cell lymphomas: A histopathologic, immunohistochemical, and ultrastructural study

We studied the histopathologic, immunohistochemical, and ultrastructural features of the extracellular matrix (ECM) in 20 immunologically defined large-cell lymphomas, including immunoblastic sarcoma of B cells (three patients), peripheral T cell lymphoma (five patients), large non-cleaved follicular center cell (FCC) lymphoma (nine patients), and non-marking large-cell lymphoma (three patients). Immunohistochemical studies were performed with antibodies to laminin, fibronectin, and collagen types I, III, IV, and V. The immunologically defined subgroups demonstrated characteristic differences in ECM in light microscopic appearance, composition, and ultrastructural features. Immunoblastic sarcomas of B cells showed delicate intercellular bands that were apparent only at high power but were distinct in exhibiting focal staining for basement membrane elements (laminin and type IV collagen) in addition to type I collagen and fibronectin. Electron microscopically, no basal lamina were apparent, although the collagen fibers were embedded in a dense matrix not seen in the other lymphomas. All peripheral T cell lymphomas exhibited a packeting pattern of intercellular bands and were distinguished by the frequent presence of intense pericellular staining for type V collagen as well as by focal pericellular staining for types I and III collagen. The latter finding corresponded to extensive areas of direct contact between tumor cells and the ECM by electron microscopy. The large non-cleaved FCC lymphomas and the non-marking large-cell lymphomas demonstrated both diffuse and compartmentalizing arrangements of intercellular bands that frequently coexisted and stained predominantly for fibronectin and types I and III collagen. All groups demonstrated myofibroblasts and fibroblasts partially or completely separating the ECM from tumor cells, suggesting that most of the ECM is part of a reaction to these lymphomas. These studies show more variation in light microscopic appearance, composition, and ultrastructural relationships of the intercellular and pericellular ECM than was apparent in earlier studies of cleaved FCC lymphomas.

Isolation, Characterization and Immunological Determination of Basement Membrane-Associated Heparan Sulfate Proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.

Serum laminin and procollagen type III propeptide in patients with respiratory distress syndrome: Potentially useful markers of therapy success

Serum laminin and procollagen type III propeptide in patients with respiratory distress syndrome: Potentially useful markers of therapy success

Quantitative assessment of the diagnostic criteria of serum laminin and N-terminal type III procollagen peptide for portal hypertension in chronic liver diseases

Quantitative assessment of the diagnostic criteria of serum laminin and N-terminal type III procollagen peptide for portal hypertension in chronic liver diseases

Immunohistochemical studies of basal cell carcinomas transplanted into nude mice

Xenografting into nude mice forms a system for analysis of human tissues under experimental conditions. In this study, normal skin samples and basal cell carcinomas were investigated, prior to and after transplantation, using immunofluorescence methods with antibodies against keratins, laminin, and collagen type IV. Three groups of transplants were studied: intact tissue samples, human epithelium (either normal or neoplastic) recombined with normal human dermis and, human epithelium recombined with normal mouse dermis. Transplants recovered after 3 weeks showed the following characteristics. The xenograft system was satisfactory in terms of host survival and rate of successful tissue recovery except for recombinants between human epithelium and mouse dermis. Intact and recombined samples of normal skin retained their preexisting patterns of architecture, cytodifferentiation, and basement membrane staining. Solid nonfibrosing basal cell carcinomas showed altered architecture and differentiation of both the epithelium and the basement membrane zone after transplantation: the solid tumor pattern changed towards spreading of tumor cells, a more squamous differentiation pattern was apparent and was confirmed by reactivity with antibodies against large keratins. Discontinuities of the basement membrane zone were detected with antibodies against laminin and collagen type IV. These changes were seen in both intact and recombined tumor transplants.

Partial Reversion of the Metastatic Phenotype in a Wheat Germ Agglutinin-Resistant Mutant of the Murine Tumor Cell Line MDAY-D2 Selected With <italic>Bandeiraea simplicifolia</italic> Seed Lectin<xref ref-type="fn" rid="FN2">2</xref>

MDW4, a wheat germ agglutinin (WGA)-resistant (WGAr) mutant of the metastatic murine tumor cell line MDAY-D2, was previously shown to be nonmetastatic in the syngeneic DBA/2 host. A substantial portion of the asparagine (Asn)-linked carbohydrate in MDW4 terminated in N-acetylglucosamine (GlcNAc) and appeared to be a premature truncation product of the sialylated poly-N-acetyllactosamine-containing complex found in MDAY-D2 cells. This lesion in carbohydrate structure has been shown to contribute to the more adhesive behavior of MDW4 cells on laminin, fibronectin, and type IV collagen and to the increased sensitivity of MDW4 to natural killer (NK) cell lysis in vitro. For further characterization of the relationship between Asn-linked carbohydrate structures, cell adhesion, NK cell sensitivity, and metastasis, mutants of MDW4 were selected for resistance to the GlcNAc-binding lectin from Bandeiraea simplicifolia seeds (BSII). Three independently selected BSII-resistant (BSIIr) mutants of MDW4 chosen for further study had a lectin resistance phenotype intermediate between that of MDAY-D2 and that of MDW4. Plasma membrane glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with iodinated WGA, BSII, and leukoagglutinin also indicated an intermediate phenotype, with the presence of both GlcNAc-terminating structures and sialylated complex. Compared to MDW4, the BSIIr mutants of MDW4 showed a return to the more malignant phenotype of MDAY-D2 when injected iv. The double mutants were less sensitive to NK cell lysis in vitro and to the in vivo effects of the NK cell boosting agent polyinosinic-polycytidylic acid. The double mutants retained the ability to attach to fibronectin, laminin, and collagen type IV in vitro, a property that may have contributed to their low malignancy when the cells were injected sc. MDW4 cells have been shown to fuse at low frequency with host-derived bone marrow cells at the sc site of injection, thereby acquiring the wild-type lectin resistance and metastatic phenotypes. The same process appears to occur in mice given an injection of the double mutants. The results suggest that the WGA-binding oligosaccharides found in MDAY-D2 and the BSIIr mutants of MDW4 enhance the malignant phenotype of the cells in the experimental metastasis assay.

Pathogenesis of Desmoplasia. I. Immunofluorescence Identification and Localization of Some Structural Proteins of Line 1 and Line 10 Guinea Pig Tumors and of Healing Wounds 1,2<xref ref-type="fn" rid="FN2">2</xref>

The structural proteins of the scirrhous line 1 and the medullary line 10 bile duct carcinomas, both syngeneic in strain 2 Sewall-Wright inbred guinea pigs, were studied. Tumor structural proteins were compared with those of healing wounds. A provisional stromal matrix of cross-linked fibrin and fibronectin was initially deposited in both tumors and wounds and was subsequently replaced by granulation tissue containing collagen types I and III. The amounts of stromal fibrin-fibronectin and collagen were characteristic of each tumor: Line 1 contained significantly greater amounts than line 10. These differences were augmented when line 1 tumor rejection was prevented with cyclosporine, permitting time for stromal maturation. In tumors and wounds laminin and collagen type IV were found only in basement membranes. The findings suggest that 1) tumor desmoplasia is analogous to wound healing, 2) both processes involve replacement of an antecedent fibrin-fibronectin provisional matrix, 3) the extent of fibrin-fibronectin is characteristic of each tumor, and 4) tumor desmoplasia correlates with the amount of fibrin-fibronectin matrix deposited.

Collagens and non collagenous proteins in the human eye. I. Corneal stroma in vivo and keratocyte production in vitro.

Human corneal stroma in situ and keratocyte activity in vitro were studied by the indirect immunofluorescent technique using highly purified antibodies to the various types of collagens, fibronectin and laminin. Studying the localization of the various collagens in situ, it was observed that only type I collagen is present in the human corneal stroma. No positive immunofluorescence is obtained for type II or type III collagen. With type IV collagen, a bright immunofluorescence is observed at the level of Bowman's and Descemet's membranes. However, in vitro, the keratocyte cultures demonstrated the production of both type I and III collagens. No evidence for the production of type II or IV collagens was observed. Along with the collagens, human keratocytes in vitro also produced fibronectin. As for the non collagenous components of basement membrane (laminin), although a positive immunofluorescence was obtained the involvement of other cell-membrane glycoproteins in this reaction is not excluded. The possibility that a deregulation of type III collagen synthesis in vivo might be occurring also during certain pathologies and in corneal wound healing is postulated.

Enzyme-linked immunoassay (ELISA) for connective tissue components

Enzyme-linked immunoadsorbant assays have been developed for types I, II, III, and IV collagen and for laminin and fibronectin. These assays offer a specific, sensitive, and convenient method for the measurement of various connective tissue components either separately or simultaneously. Plastic microtiter wells are coated with purified antigen, then antibodies to the antigen are allowed to bind to the insolubilized antigen in each well. The amount of bound antibody is determined by incubation with a second antibody which is covalently linked to alkaline phosphatase or horseradish peroxidase. The amount of bound enzyme is assayed after adding an appropriate substrate. The level of protein in a sample is estimated from its ability to inhibit the binding of the first antibody to the antigen-coated well. Specificity of the assay depends on the purity of the adsorbed antigen and allows for highly specific tests. Under optimum conditions the sensitivity of the assay is determined by the K B of the antibody and allows 10–20 ng of a specific type of collagen or laminin and 1 ng fibronectin to be detected.