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Biomedical subjects

I K Gipson

Publications and source records attributed to I K Gipson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cellular origin of mucins of the ocular surface tear film.

In summary, we have demonstrated that the ocular surface epithelia express at least three mucin genes. We suggest that the gel-forming mucin MUC5AC is a major mucin forming the mucus gel of the tear film. We further suggest that MUC1 facilitates the spread of the MUC5-containing mucus on the ocular surface and, along with the mucus gel, prevents cell and debris adhesion to the ocular surface. The function of MUC4 at the ocular surface remains to be elucidated.

Animals↗
PubMed1998

Actin 'purse string' filaments are anchored by E-cadherin-mediated adherens junctions at the leading edge of the epithelial wound, providing coordinated cell movement.

At the leading edge of healing embryonic epithelium, cables of actin filaments appear to extend from cell to cell, forming a ring around the wound circumference. It has been hypothesized that this actin filament cable functions as a contractile 'purse string' to facilitate wound closure. We have observed this cable in large, circular healing epithelial wounds in corneas of adult mice. To elucidate the role of the actin filament cable, we characterized the molecular components associated with the cell-cell junction where the actin filament cable inserts and with the actin filament cable itself, and we studied the effect of disruption of the cable using an E-cadherin function-blocking antibody, ECCD-1. Localization of E-cadherin and the direct association of catenins with actin filament cable at the cell-cell interface of the actin cable confirmed that the cell-cell junction associated with the actin filament cable is an adherens junction. The E-cadherin function-blocking antibody caused disruption of the actin filament cable and induction of prominent lamellipodial extensions on cells at the leading edge, leading to a ragged uneven epithelial wound margin. These data demonstrate that cell-to-cell associated E-cadherin molecules link the actin filament cable, forming a functional adherens junction, and that the actin filament cable plays a role in coordinating cell movement.

Actins↗
PubMed1998

Alteration of mucin in human conjunctival epithelia in dry eye.

PURPOSE: The objective of this study was to determine whether alteration in mucins could be detected in patients with dry eye symptoms by using the monoclonal antibody H185, which recognizes carbohydrate epitopes on mucin molecules. METHODS: Immunohistochemistry and immunoelectron microscopy was used to examine binding of H185 antibody to conjunctival cells obtained by nitrocellulose filter paper stripping (impression cytology). Two study populations were examined. Study I included 22 patients with dry eye symptoms and 13 normal volunteers. Study II included 16 aqueous-deficient dry eye patients and 14 age-matched control subjects. RESULTS: Results of the studies demonstrated significant differences in binding patterns of H185 to conjunctival cells in normal eyes compared with those of patients with dry eye symptoms. In normal eyes, the antibody bound to apical cells in a mosaic pattern, with cells exhibiting either light, medium, or intense binding. A predominant pattern in patients with dry eye symptoms was loss of the mosaic pattern with replacement by a "starry sky" pattern in which there was a lack of apical cell binding (hence, dark sky) but increased binding to goblet cells (hence, stars in the sky). The starry sky pattern correlated with rose bengal staining. CONCLUSIONS: From these studies it is concluded that there is an alteration either in mucin distribution or mucin glycosylation on the surfaces of apical conjunctival cells in dry eye and that glycosylation of goblet cell mucins changes with the disease.

Adult↗
PubMed1997

Cloning of rat Muc5AC mucin gene: comparison of its structure and tissue distribution to that of human and mouse homologues.

The human mucin gene MUC5AC codes for a large mucin which has tandem repeat units and cysteine rich regions characteristic of several members of this class of glycoproteins. Human epithelia expressing the mucin include that of stomach, bronchus/trachea, endocervix and conjunctiva. We report here a 3.8 kb partial sequence of a rat homologue for the human MUC5AC gene and compare its tandem repeat sequence and cysteine rich domains to those of the human and mouse gene. Rat and mouse have the same number of amino acids (16) in their Muc5AC tandem repeat units and share 69% sequence similarity, whereas human MUC5AC has only 8 amino acids in its tandem repeat. In rat, the tandem repeat domain is flanked at its 3' end by a non-repeat region coding for 1142 amino acids. Four cysteine rich subdomains were identified in this region; one of these has 64% similarity to a corresponding region in human MUC5AC and 80% similarity to a mouse MUC5AC cysteine rich region. Southern blot analysis revealed cross hybridization of a probe for the rat cysteine rich region, to human, mouse, rabbit, and porcine genomic DNA; the rat tandem repeat probe hybridized with mouse and rabbit only. Unlike humans, rat expressed MUC5AC message detectable by Northern blot and in situ hybridization only in stomach epithelium and conjunctival goblet cells.

Amino Acid Sequence↗
PubMed1997

Visualization of conjunctival goblet cell actin cytoskeleton and mucin content in tissue whole mounts.

The purpose of the work was to visualize goblet cells, their cytoskeletons, and their content in tissue whole mounts of conjunctiva using confocal microscopy. Conjunctival tissue from rat, mouse, rabbit, and human were excised, fixed in 4% paraformaldehyde, and incubated either with rhodamine-labeled phalloidin to label filamentous actin or in combination with fluoresceinated lectins to label carbohydrates on goblet cell mucins. Tissues were evaluated by confocal microscopy. Phalloidin labeling of excised tissue facilitated visualization of goblet cells within the stratified epithelium of the conjunctiva. Phalloidin labeled the perimeter of the goblet cells, so that either in optical section or three-dimensional constructs of optical sections, the goblet cells appeared as plump empty clusters of cells in rat and mouse and as individual plump empty cells in rabbit and human tissues. Double labeling of excised tissue with lectins demonstrated that the plump cells visualized by labeling with phalloidin bound the label and were indeed goblet cells. At very high resolution one could see individual mucin packets within the goblet cells. Variation in intensity of lectin labeling of these mucin packets both between cells and within cells was evident. Streams of labeled mucin emanating from apical surfaces of goblet cells could be visualized using the fluoresceinated lectins. The labeling of excised conjunctiva with fluorochrome-conjugated phalloidin allows visualization by confocal microscopy of goblet cells within the stratified epithelium. Double labeling with fluorochrome-conjugated lectins allows visualization of their content. These methods may be useful in studies of goblet cell differentiation, mucin content and secretion.

Actins↗
PubMed1997

Mucin genes expressed by human female reproductive tract epithelia.

Recent characterizations of mucins at the molecular level indicate that at least eight mucin genes are expressed by epithelia of mucosal surfaces. The purpose of this study was to determine whether these cloned mucins, designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7, are expressed by epithelia of the female reproductive tract. Northern blot analysis, in situ hybridization, and immunohistochemistry were performed using RNA and tissue from surgically removed human reproductive tract specimens including endocervix, ectocervix, vagina, endometrium, and fallopian tube. Complementary DNA to the tandem repeat regions of MUCs 1, 2, 3, 5AC, 5B, and 6; oligonucleotides to the tandem repeat regions of MUCs 4, 6, and 7; and antibodies that recognize unique mucin tandem repeats were used. The data demonstrate that the endocervical epithelium expresses five mucin genes: MUCs 1, 4, 5AC, 5B, and 6. The ectocervical and vaginal epithelia express MUCs 1 and 4, although vaginal expression of MUC4 appears patchy. Endometrial epithelium expresses MUC1 and low amounts of MUC6. MUC6 immunoreactivity was detected only is scattered endometrial glands located in the basalis region in specimens from pre- and postmenopausal women. The only mucin detected in the fallopian tube was MUC1. These data indicate that the endocervical epithelium expresses multiple mucin genes and that the stratified epithelia of the ectocervix and vagina also produce mucins that may function in reproductive processes and protection of the reproductive tract tissues.

Base Sequence↗
PubMed1996

Expression of secretory mucin genes by human conjunctival epithelia.

PURPOSE: To determine whether human conjunctival epithelium expresses any of the human mucin genes designated MUC2 through MUC7. METHOD: Northern blot analysis was performed using total RNA isolated from surgically removed conjunctival tissues. Complementary DNA or oligonucleotides to the tandem repeat region of each mucin gene were labeled and hybridized to conjunctival RNA. In situ hybridization also was performed to determine the distribution of mucin mRNA. RESULTS: Only MUC4 and MUC5 probes hybridized to conjunctival RNA by Northern blot analysis. Both probes bound in a polydispersed pattern, which is characteristic of mucin genes. Using in situ hybridization, MUC4 mRNA was detected in the cells of the stratified conjunctival epithelium, whereas MUC5 mRNA expression was limited to goblet cells MUC4 or MUC5 probes did not hybridize to sections of corneal epithelium. CONCLUSIONS: The mucins MUC4 and MUC5 are expressed by the human conjunctiva. These mucins may play an important role in forming the tear-film layer at the air and ocular surface epithelium interface.

Adult↗
PubMed1995

Stratified squamous epithelia produce mucin-like glycoproteins.

The stratified squamous epithelia of the ocular surface, larynx, and vagina are mucus-coated epithelia, apices of which are subject to abrasive pressure from epithelia-epithelia interactions from eyelid, vocal cords, or vaginal folds, respectively. Mucus coats on these epithelia have generally been considered to be derived from the specialized mucin-producing cells embedded either in the epithelia or in adjacent tissues. Here we report the isolation, partial characterization, and cellular localization of a mucin-like glycoprotein produced by these stratified epithelia. In all three epithelia, the mucin-like molecule is present on cytoplasmic vesicles in subapical cells. As cells differentiate to their apical-most position adjacent to their mucus coat, the mucin-like molecule moves to the cell membrane where it is particularly prominent on microplicae folds. Lectin affinity chromatography was used to isolate the molecule from rat vaginal and corneal epithelium. Isolated material was approximately 60% carbohydrate and 40% protein. The major monosaccharide was N-acetylgalactosamine with lesser amounts of N-acetylglucosamine, galactose, mannose, xylose and fucose. Amino acid analysis demonstrated the predominant amino acids to be glycine, serine, threonine and proline. These data plus PAS and Alcian blue binding to the isolate indicate a mucin-like glycoprotein.

Animals↗
PubMed1995

Extracellular matrix and growth factors in corneal wound healing.

Healing of corneal wounds is a complex process involving epithelial, keratocyte, and endothelial interactions that are affected by their associations with wound bed matrix and by cytokine availability and activation. The spectrum of possible cellular-matrix-growth factor interactions is indeed great and growing. Several of the significant contributions made during the past year include development of an organotypic organ culture model of the cornea that allows in vitro assembly of the epithelial extracellular matrix-anchoring complex, demonstration of epithelial synthesis of Bowman's layer collagens, demonstration of transforming growth factor-beta 2's inhibition of stromal cell collagenase synthesis, and demonstration of the paracrine pathway of keratinocyte growth factor action in the cornea.

Animals↗
PubMed1995

Human corneal and conjunctival epithelia produce a mucin-like glycoprotein for the apical surface.

PURPOSE: The authors have determined that the corneal and conjunctival epithelia of the rat produce a mucin-like glycoprotein at the apical surface of the epithelium. The purpose of this study was to determine if human ocular surface epithelium produces similar glycoproteins. METHODS: Because our initial attempts at production of monoclonal antibodies yielded blood type A-specific antibodies, corneal epithelia from blood type O donor eyes were used for the production of monoclonal antibodies. Screening of hybridoma products was accomplished by immunofluorescence microscopy of cryostat sections of blood type O donor eyes. The monoclonal antibody produced was used for immunofluorescence microscopy and immunoelectron microscopy to determine tissue and cellular distribution, respectively. Immunoblot analysis of SDS-PAGE-separated proteins from corneal epithelial tissue and from cultured human corneal epithelium was used to determine molecular weight range and epitope binding after periodate oxidation, N-glycanase, and O-glycanase treatment. RESULTS: A monoclonal antibody, designated H185, that binds to apical cell layers of human corneal, conjunctival, laryngeal, and vaginal epithelium was produced. The antibody binds to apical membranes of apical cells, particularly at the tips of microplicae. In subapical cells, the antibody binds to small cytoplasmic vesicles. Cultured human corneal epithelium produces H185 antigen. By immunoblot analysis, H185 binds a high molecular weight protein, > 205 kD, from corneal epithelium and cultured corneal epithelium. The protein band visualized by immunoblot analysis cannot be stained by Coomassie or silver stain on SDS-PAGE, but it does stain with Alcian blue followed by silver stain, indicating that the protein is highly glycosylated. H185 binding to blotted proteins is destroyed by sodium periodate treatment and O-glycanase incubation, suggesting that the epitope of H185 is an O-linked carbohydrate. CONCLUSION: Human corneal and conjunctival epithelia produce a molecule, similar in size, cellular localization, and distribution to the mucin-like glycoprotein of rat ocular surface epithelium. These data suggest that the entire ocular surface epithelium produces mucins for the tear film.

Animals↗
PubMed1995

Differential gene expression in healing rat corneal epithelium.

PURPOSE: The authors used and validated a recently developed method, mRNA differential display, to detect and clone genes that are differentially expressed in healing compared to stationary corneal epithelium. METHODS: RNAs from unwounded and 18-hour postwound corneal epithelia were isolated and subjected to mRNA differential display analysis. The generated cDNAs were used as probes in Northern blot analysis and in situ hybridization to confirm their differential expression and to clone longer or full-length cDNAs from a healing corneal epithelial cDNA library. RESULTS: Changes in the pattern of gene expression in healing epithelium, compared with that in stationary cells, were noted. To date, 15 combinations of 5'- and 3'- primers were used with approximately 1500 mRNA species screened. Differential expression of nine mRNA species were observed. These included four known proteins. They are nonmuscle tropomyosin TM-1, cytokeratin K14, small GTP binding protein rab 11, and amyloid beta-A4 precursor-like protein-2. One is a sequence with homology to type II cytokeratin, and four represent genes with sequences that are unreported. The differential expression of five of these genes was confirmed by Northern blot analysis, in situ hybridization, or both. CONCLUSION: mRNA differential display provides a unique and powerful experimental system to study differential gene expression in wound healing and cell migration. Using this system, differential expression of nine genes was observed. Detection of genes differentially expressed in healing epithelium may prompt studies that will define the specific role of each of the proteins in wound healing.

Amino Acid Sequence↗
PubMed1995

Human corneal and conjunctival epithelia express MUC1 mucin.

PURPOSE: To determine if human corneal and conjunctival epithelial synthesize MUC1 mucin, a membrane-spanning mucin present in a variety of simple epithelia. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to examine the expression of MUC1 mRNA by epithelial cells, using total cellular RNA prepared from cultured corneal epithelial cells and conjunctival epithelial cells stripped from the ocular surface with nitrocellulose filter paper. Northern blot analysis was performed to examine the transcription of MUC1 gene in cultured corneal epithelial cells. In situ hybridization histochemistry was performed to determine distribution of MUC1 mRNA in ocular surface epithelium. Human milk fat globule antibody (HMFG-1) and monoclonal antibody 139H2, which are specific for MUC1 core protein, were used in immunoblot analysis, immunohistochemistry, or both, to determine the presence and distribution of MUC1. RESULTS: MUC1 mRNA was detected in cultured corneal and ex vivo conjunctival epithelial cells by RT-PCR. Northern blot analysis showed production of two different sizes of transcripts in the cultured corneal epithelium. Expression of MUC1 mRNA was observed in all layers of corneal epithelium. By immunoblot analysis, HMFG-1 binding (> 200 kd) was detected in human cultured corneal epithelium, and its binding was enhanced by neuraminidase pretreatment. Immunohistochemically, HMFG-1 binding was observed along the apical membranes of corneal epithelium after neuraminidase pretreatment. In the conjunctiva, HMFG-1 and 139H2 binding were detected in the basal region and sporadically on the apical surface, but not in the goblet cells. CONCLUSIONS: The stratified epithelia of the cornea and conjunctiva synthesize MUC1 mucin. This transmembrane mucin may have a role in tear film spread and may prevent adhesion of foreign debris, cells, or pathogens to the ocular surface.

Base Sequence↗
PubMed1994

Detection of blood group differences in human corneal epithelium using a monoclonal antibody and lectins.

OBJECTIVE: To determine if blood group variation in corneal epithelial glycoconjugates can be detected with the use of a monoclonal antibody and sugar-specific lectins. METHODS: Monoclonal antibodies were developed to isolated corneal epithelial cells using conventional hybridoma techniques. Hybridoma screening and studies of antibody binding to cryostat sections of human corneas of blood groups A, B, and O were performed with immunofluorescence microscopy. Correlation of lectin binding to antibody and blood group binding was done immunohistochemically with lectins conjugated to fluorochromes. RESULTS: Monoclonal antibody designated 1B3.29.36 binds to corneal epithelium of humans of blood group A only. The epitope of the antibody appears to be, in part, N-acetylgalactosamine. The lectins Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia lectin-I isolectin-B4 (GSL-IB4), and Ulex europeus agglutinin-I (UEA-I) bind to epithelium of subjects with blood groups A, B, and O, respectively, and soybean agglutinin binds to all three groups. CONCLUSION: Variations in corneal epithelium of subjects with different blood groups can be detected with the use of a monoclonal antibody and the lectins DBA, GSL-IB4, and UEA-I. Previous reports of lectin binding to ocular surface epithelium should be reevaluated since they did not take into account blood-group-specific binding.

ABO Blood-Group System↗
PubMed1993

Redistribution of the hemidesmosome components alpha 6 beta 4 integrin and bullous pemphigoid antigens during epithelial wound healing.

As basal cells of stratified squamous epithelia become migratory in response to wounding, they lose their cell-substrate adhesion junctions, the hemidesmosomes. We report here studies to determine the fate of the hemidesmosome components, alpha 6 beta 4 integrin and the bullous pemphigoid antigens (BPAGs), as recognized by bullous pemphigoid autoantisera (BPA), in migrating epithelium. In addition, we report studies to determine whether relative synthesis and amount of alpha 6 beta 4 is altered during migration. Mouse corneas with 1.5- to 2-mm-diameter central epithelial debridements were allowed to heal in vitro or in vivo for 1-18 h. In order to do preembedding immunoelectron microscopic localization of alpha 6 beta 4, sheets of stationary and migrating corneal epithelium were removed from their basal laminae after organ culture. BPA and antibodies to alpha 6 and beta 4 were used for immunofluorescence microscopy on frozen sections of intact corneas healing in vivo 1-18 h. Both alpha 6 and beta 4 were found to redistribute from their clustered location within hemidesmosomes to a more even distribution within the substrate-associated membrane of basal cells of the tip of the leading edge of migrating epithelium. Behind the tip of the leading edge, basal cells bound the integrin antibodies around their entire membrane. BPAGs moved from their location along the basal cell membrane of stationary epithelium to a diffuse location within the cytoplasm of migrating cells at the leading edge of migration. Quantitative immunoprecipitation and immunoblotting of alpha 6 beta 4 as well as beta 1 integrin from stationary and migrating epithelium were done to determine whether the synthesis or total amount of the integrins were altered during migration. The relative syntheses of alpha 6 beta 4 and beta 1 per milligram of protein or per cell do not appear to differ between stationary and migrating epithelium and the total amount of the beta 4 and beta 1 does not change despite increased rates of protein synthesis in migrating epithelium. Taken together, these studies suggest that as hemidesmosomes disassemble, their clustered integrin component distributes more evenly in the basal cell membrane, the components recognized by BPA and associated with intermediate filaments are released from the membrane, and these events occur in the absence of any measurable change in the synthesis or total amount of the alpha 6 beta 4 component.

Animals↗
PubMed1993

Synthetic epikeratoplasty in rhesus monkeys with human type IV collagen.

Human type IV collagen discs were found to support proliferation and adhesion of rabbit corneal epithelial cells in tissue culture. To assess the biocompatibility of this synthetic collagen for epikeratoplasty, seven eyes of seven rhesus monkeys underwent epikeratoplasty with lenticules made of human type IV collagen. Eye rubbing by the animals expulsed two of the lenticules and caused failure of two to epithelialize completely. The remaining three lenticules epithelialized, remained clear, and caused no adverse effects on the eye. Two of these lenticules developed focal areas of subepithelial thinning 3 months postoperatively and the third lenticule has remained stable for 30 months. The presence of epithelial attachment components at the epithelial-lenticule interface was demonstrated by immunolocalization. Histopathologic and ultrastructural examination revealed focal areas of epithelial invasion and degradation of the lenticule. Neutral proteases were detected in the thinning region of one specimen. Human type IV collagen supports epithelialization in vivo and may have potential as a biomaterial for epikeratoplasty, but the stability of the material must be improved.

Animals↗
PubMed1993

Eyelid opening induces expression of a glycocalyx glycoprotein of rat ocular surface epithelium.

PURPOSE: We previously characterized a monoclonal antibody against a glycoprotein of rat ocular surface glycocalyx (ROSG). This monoclonal antibody recognizes a carbohydrate epitope on a glycoprotein expressed in apical cells of the differentiated ocular surface epithelium. Our goal was to determine the developmental appearance of the ROSG glycoprotein. METHODS: We localized the antigen immunohistochemically and immunocytochemically in newborn (day 1) rats and in rats 2-5, 7, and 11-15 days after birth. RESULTS: Before eyelid opening (days 12-14), the ROSG antigen was localized in the palpebral conjunctiva near the lid margin. The binding extended to layers of the subapical flattened cells. However, the antigen was not found in the corneal epithelium while the eyelid remained closed. In contrast, at the first day of eyelid opening (days 12-15), the antigen was present contiguously in several cell layers from the eyelid margin along the entire ocular surface, including the corneal epithelium. Thus, the binding pattern seen upon eyelid opening was similar to that of adult rats. The phenomenon of appearance of the glycoprotein upon eyelid opening was further demonstrated in rats with asynchronous eyelid opening. Artificial, premature eyelid opening at days 8-11 also induced the expression of the antigen along the entire ocular surface epithelium, similar to the binding in naturally opened eyes. CONCLUSION: Eyelid opening may induce glycosylation or expression of a glycocalyx component that we hypothesize to be involved in mucin spread.

Animals↗