Human corneal epithelial cell adhesion to laminins.

Purpose: To analyze alpha-integrin mediated adhesion of human corneal epithelial cells to placental and EHS laminin isoforms.

Methods: Western blot analysis was used to partially characterize commercially available preparations of laminin isolated from the mouse EHS sarcoma and from human placenta. Using the human corneal epithelial cell line HCE-T, adhesion to laminin isoforms and fibronectin was determined using a colorimetric adhesion assay.

The lack of extracellular laminin beta2 chain deposition correlates to the loss of conjunctival epithelial keratin K4 localization in culture.

Purpose: To examine the effects of external modulation of epithelial-mesenchymal interaction on conjunctival epithelial cell differentiation characteristics.

Methods: Keratin K4 and laminin beta2 chain protein localization was examined in an organotypic model which facilitates the comparison of differentiation characteristics of conjunctival epithelium interacting with conjunctival basement membrane or corneal basement membrane. In addition, keratin K4 and laminin beta2 chain localization was examined in primary cultures of conjunctival epithelial cells and fibroblasts.

The role of laminin-5 in TGF alpha/EGF-mediated corneal epithelial cell motility.

Transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) stimulate corneal epithelial cell wound closure. However, the role of these growth factors in regulating corneal epithelial cell motility on basement membrane proteins such as laminin has not been elucidated. In the present study we demonstrate that in an in vitro model of corneal wound healing, TGF alpha has no deleterious effects on the deposition of the laminin-5 isoform into the extracellular matrix structure underlying epithelial cells resurfacing bare collagenous stroma.

Localization of basement membrane-associated protein isoforms during development of the ocular surface of mouse eye.

The developmental localization patterns of collagen type IV alpha1-5 chains, laminin-1, laminin-5, and laminin alpha2 chain were analyzed in the embryonic mouse eye using isoform specific antibodies and immunofluorescence microscopy. Laminin-1 isoform and alpha1-2(IV) were ubiquitously expressed along the ocular surface basement membranes at a very early stage of eye development. Alpha3-5(IV) were first detected at later stages of development, and exhibited a variable distribution pattern along the ocular surface basement membrane.

Differential effects of transforming growth factors on localization of adhesion complex proteins following corneal epithelial cell wounding.

Purpose: The differential effects of transforming growth factor (TGF) alpha, beta 1 and beta 2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing.

Methods: Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGF alpha, TGF beta 1 or TGF beta 2. After 24 or 48 hours in culture, cryostat sections of explants were processed for immunofluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1.

Plasma membrane calcium ATPase in synaptic terminals of chick Edinger-Westphal neurons.

The plasma membrane calcium ATPase pump (PMCA) is one of two major mechanisms known to be involved in extruding calcium from cells. The monoclonal antibody 5F10 was used to examine the distribution of PMCA in chick Edinger-Westphal neurons, a population of cholinergic preganglionic neurons whose cells bodies reside in the Edinger-Westphal nucleus in the brainstem and whose axons form synaptic terminals on parasympathetic neurons in the ciliary ganglion. Definitive PMCA immunoreactivity was undetectable in Edinger-Westphal cell bodies in the brainstem.

Nuclear matrix proteins of bovine corneal and conjunctival epithelium.

Purpose: To present a preliminary biochemical and immunochemical analysis of nuclear matrix proteins isolated from ocular surface epithelium.

Methods: Nuclear matrix protein-enriched fractions were prepared from bovine corneal and conjunctival epithelial cells. The preparations were analyzed by 1D and 2D SDS-PAGE and Western immunoblotting.

Some P. aeruginosa pilus-binding proteins of human corneal epithelium are cytokeratins.

Purpose: The purpose of this study was to determine whether any of the previously identified human corneal epithelial pilus-binding proteins were cytokeratins.

Methods: Soluble human corneal epithelial proteins (hcep) were separated by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis under reducing conditions and transferred to nitrocellulose membrane for Western blot analysis. To characterize pilus-binding hcep (major proteins of < 21, 38, 45, 66 and 97 kDa and minor proteins, including a 55 kDa protein), blots were immunostained using three anti-keratin antibodies, including Pruss monoclonal antibody (MAb), specific for all classes of IFs, AE5 MAb, specific for a 64 kDa cytokeratin, and J7 polyclonal antibody (PAb), specific for a 55 kDa cytokeratin.

Ultrastructural, immunohistological and biochemical characterization of cultured mouse corneal epithelial cells.

We developed a method to culture mouse corneal epithelium. Cultured cells tested by 1-D-SDS-PAGE exhibited protein mobility patterns similar to freshly isolated epithelia. Western blots with antibodies broadly recognizing cytokeratins showed a similar pattern for both fresh and cultured cells, but only the fresh sample stained with J7, specific for a 55-kD 'corneal' cytokeratin.

Localization of a corneal basement membrane glycoconjugate in bovine eye.

Lectin histochemistry was used to analyze the ocular surface basement membrane in order to identify novel, tissue-specific glycoconjugates. Soybean agglutinin (SBA), a lectin marker for N-acetylgalactosamine residues, recognized a 130 kDa glycoconjugate in corneal but not conjunctival basement membrane. Following corneal epithelial cell wounding in vitro, the glycoconjugate was not expressed at the epithelial cell-stromal interface until 72-96 h in culture, much later than the expression of other basement membrane molecules.

Expression of keratins K12, K4 and K14 during development of ocular surface epithelium.

The 55 kDa keratin K12 and the 59 kDa keratin K4 were used as biochemical markers of differentiated corneal and conjunctival epithelium, respectively, to follow the temporal and spatial appearance of these cell types during embryonic development of the mouse eye. K12 was first detected in corneal epithelial cells of day 15 mouse embryos in a small subpopulation of superficial cells. At later developmental stages only suprabasal corneal epithelium expressed K12, however, in post-natal and adult cornea all cell layers were K12-positive.

Expression of insulin-like growth factors and their receptors in human meningiomas.

Meningiomas have previously been shown to highly express the gene for insulin-like growth factor-II (IGF-II), and receptors for IGF-I have been demonstrated by binding studies in membranes prepared for meningiomas. The co-expression of insulin and the insulin-like growth factors with their corresponding receptors in meningiomas has not been explored. Immunofluorescence microscopy was carried out on twelve meningotheliomatous meningiomas using antibodies directed against insulin, IGF-I, IGF-II, insulin receptor and IGF-I receptor.

The role of the basement membrane in differential expression of keratin proteins in epithelial cells.

Extracellular matrix is considered to play an important role in determining the phenotype of cells with which it interacts. Here we have investigated the possibility that extracellular matrix is involved in specifying the pattern of keratin expression in epithelial cells. For these studies, we have developed an explant system in which epithelial cells from one type of stratified epithelial tissue, namely conjunctiva, are maintained on an extracellular matrix substrate derived from a different tissue, namely cornea.

Adhesion complex formation after small keratectomy wounds in the cornea.

The adhesion complex of the corneal epithelium consists of the hemidesmosome and its associated structures, such as anchoring filaments, lamina densa of the basement membrane, and anchoring fibrils. It contributes to the adhesion of the corneal epithelium to Bowman's layer. To understand the adhesion complex better, an electron microscopic and immunofluorescence analysis was done of the reformation of the adhesion complex in small (1 mm) keratectomy wounds in the guinea pig cornea.

Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing.

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S.

Differential expression of insulin-like growth factor II in human meningiomas.

Two glioma tumor lines and brain tumor specimens from 10 patients were analyzed to determine genic expression of insulin-like growth factor II. Messenger ribonucleic acid encoding insulin-like growth factor II was found to be expressed in significant amounts in two of two meningiomas but not in any of the gliomas. Immunofluorescence studies on these tumors and normal brain confirmed the presence of insulin-like growth factor II in both meningiomas, but not in any of the gliomas or in normal brain.

A function for the integrin alpha 6 beta 4 in the hemidesmosome.

Many epithelial cells appear to use cell-substratum adhesion complexes known as hemidesmosomes as the main means of anchorage to the connective tissue. Initially recognized as distinctive electron-dense images, hemidesmosomes are still poorly understood at the biochemical level. The regulation and mode of their assembly, which is disrupted in certain blistering diseases and is critical to proper wound repair, also remains to be elucidated.

A novel hemidesmosomal plaque component: tissue distribution and incorporation into assembling hemidesmosomes in an in vitro model.

The hemidesmosome and its associated structures, such as anchoring fibrils, form a complex structure, the polypeptide composition of which has only recently begun to be elucidated. We describe the characterization of a monoclonal antibody, mAb6A5, directed against a 200-kDa polypeptide found in the cytoplasmic-most area of the hemidesmosomal plaque. This 200-kDa polypeptide is immunologically distinct from the 180- and 230-kDa hemidesmosomal plaque components recognized by bullous pemphigoid (BP) autoantibodies.

Analysis of wound healing in an in vitro model: early appearance of laminin and a 125 x 10(3) Mr polypeptide during adhesion complex formation.

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K = 10(3) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively.

Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells.

Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.

Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium.