Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

Immunolocalisation of thrombospondin 1 in human, bovine and rabbit cornea

Light- and electron-microscopic immunohistochemical techniques were used to investigate the distribution of the matricellular protein thrombospondin 1 in normal human, bovine and rabbit cornea. Light-microscopic immunoreactivity for thrombospondin 1 was observed in the epithelial basement membrane, posterior Descemet's membrane and endothelium of human and bovine cornea. The bulk of the stroma, the stromal cells (keratocytes) and the anterior part of Descemet's membrane in human and bovine cornea were devoid of detectable thrombospondin 1 and the protein could not be demonstrated in any of the layers of the rabbit cornea. Electron-microscopic immunogold studies of human and bovine cornea revealed that thrombospondin 1 labelling of corneal endothelial (and basal epithelial) cells included focal deposits at cell membranes. It is postulated that thrombospondin 1 regulates interactions between cells and their basement membrane, and perhaps cell-to-cell interactions, in the normal human and bovine corneal endothelium and basal epithelium.     

Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment

OBJECTIVE: The study was designed to investigate the corneal changes at various times after epikeratophakia performed on rabbit cornea. METHODS: The process of epithelial repair, or interlayer healing, the changes of endothelial cells and Langerhans cells (LC) in corneal epithelium were observed at different intervals after surgery by using histochemistry technique. RESULTS: The epithelial repair of the graft was completed by 4-12 postoperative days. The repopulation of keratocytes was seen firstly at peripheral and superior part of the lenticule at 7-14 postoperative days and completed to normal by postoperative 2 months. No changes were observed in the endothelial cells. The proliferation of LC was observed in the limbal epithelium at day 3, reached the peak by day 14 and recovered to normal at month 2 postoperatively. CONCLUSION: Epikeratophakia is available and safe, The proliferation of LC might result from wound healing not from immune rejection.     

Unique peptide maps of the three largest proteins specified by the flavivirus Kunjin

Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [35S]methionine-labeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA.     

Conservation of the Notch signalling pathway in mammalian neurogenesis

PURPOSE: To determine whether there is an association between epidermal growth factor (EGF)-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase) and stimulation of wound closure in rabbit corneal epithelial cells. METHODS: Immortalized rabbit corneal epithelial cells were cultured in 24-well plates until they became confluent. Circular wounds were created in confluent cultures by cell denudation and then incubated in the absence and presence of EGF for varying intervals. Wound closure was monitored by staining the cells with Giemsa and quantifying the wound area with SigmaS can computer program. Cell proliferation during wound repair was estimated by measuring the incorporation of [3H]thymidine into nuclear DNA. Changes in PI 3-kinase activity were assessed by measuring the production of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] in 32P-labeled cells as well as by immunoprecipitating and assaying PI 3-kinase activity with phosphatidylinositol 4,5-bisphosphate and [gamma-32P]ATP as substrates. The enzyme product, PIP3, was analyzed by a combination of thin-layer and high-pressure liquid chromatography. RESULTS: Addition of 10 ng/ml EGF to the wounded corneal epithelial cells stimulated wound closure in a time-dependent manner, and the wound closed completely within 48 hours. The effect of EGF was dose dependent, and maximal wound closure occurred at 10 ng/ml EGF. As the epithelial cells were undergoing EGF-stimulated wound closure, there was a time-dependent increase in PI 3-kinase activity. The enzyme activity increased maximally at 24 hours and then decreased gradually as the incubation was continued to 48 hours. When the cells were treated with wortmannin, a PI 3-kinase inhibitor, the EGF-stimulated PIP3 formation as well as the wound closure were inhibited significantly. Treatment of the cells with genistein or tyrphostin B42 also decreased both EGF-stimulated PIP3 formation and wound closure in a dose-dependent manner. Concomitant with stimulation of wound repair, the growth factor increased [3H]thymidine incorporation into nuclear DNA, and this effect was inhibited by pretreatment of the cell with wortmannin. CONCLUSIONS: The data suggest a close correlation between EGF-stimulated wound closure and activation of PI 3-kinase in corneal epithelial cells. It can be concluded that PI 3-kinase might be an important component in signal transduction cascade initiated by EGF-receptor interaction, which leads to mitosis and cell proliferation during wound closure in corneal epithelial cells.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.     

The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).     

Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo

PURPOSE: To determine the potential of somatic gene transfer as a technique for modulating corneal wound healing after superficial keratectomy. METHODS: The transduction of human and rabbit keratocytes with beta-galactosidase and herpes simplex virus thymidine kinase genes was performed. In vitro, human and rabbit keratocytes were transduced with retroviral vectors bearing beta-galactosidase or HStk (herpes simplex virus thymidine kinase) genes. In vivo, rabbit keratocytes were transduced by topical application of vector supernatant after a superficial keratectomy. In vitro and in vivo, expression of the beta-galactosidase gene was examined with histochemical staining. In vitro, ganciclovir cytotoxicity in HStk gene-transduced keratocytes and bystander effect in co-cultures of HStk(+) and HStk(-) keratocytes were measured by determining the degree of confluency of cells in 6-well plates after 10 days of incubation. Corneal haze in rabbits was measured after transduction with Hstk and subsequent treatment with topical ganciclovir. RESULTS: In vitro, both human and rabbit keratocytes were transduced successfully with both beta-galactosidase and HStk genes. Transduction efficiency was greater with human (22%) than with rabbit (16%) cells, and both HStk-transduced cell lines showed dose-dependent ganciclovir cytotoxicity and a significant bystander effect. In vivo, expression of beta-galactosidase within vimentin-positive corneal stromal cells confirmed transduction of keratocytes in the rabbit after superficial stromal keratectomy with an efficiency of 25% to 40%. Postoperative application of topical ganciclovir reduced corneal stromal haze in rabbits. CONCLUSIONS: The ability to genetically transduce stromal keratocytes provides a new strategy for understanding the important cellular and molecular events that influence corneal wound healing, thus offering a potential approach to decrease or prevent corneal haze and scarring after superficial keratectomy.     

Surfactant replacement in adults and children with ARDS--an effective therapy?

[3H]-Thymidine radioautography showed that 0.005% p-aminobensoic acid (PABA) solution applied three times per day on penetrating wounds in central part of the adult rat cornea selectively stimulates proliferative activity of corneal stroma keratoblasts. In control rats, a curve showing changes in index of labeled nuclei in corneal stroma had two peaks, on the second and sixth day after injury (about 5 and 3%, respectively), whereas in animals receiving PABA treatment it had a single peak on the second day (about 12%). On days 7-14, indices of labeled nuclei in corneal stroma of both experimental and control rats become similarly low. No difference between experimental and control animals was revealed in indices of labeled nuclei in corneal epithelium: in both groups, corresponding curves had two peaks (about 9%) on the first and fifths days, and proliferation still continued two weeks after injury.     

Vectorial competence of Glossina tachinoides Westwood and Glossina palpalis gambiensis Vanderplank infected by Trypanosoma brucei brucei EATRO 1125]

In this work the relationship between the proliferation of bovine corneal epithelial cells and PGE2 has been studied. Our data indicate that PGE2 plays an important role in the growth of corneal epithelial cells. Actually, epithelial cells cultured on a keratocyte feeder-layer and exposed to indomethacin, a cyclooxygenase inhibitor, have shown a decrease in growth rate at drug concentrations which otherwise did not induce a reduction in the viability of the keratocytes as well as in epithelial cells in separate cultures. This effect has been reversed by an exogenous PGE2 addition to the culture media. Moreover, significant increases have been found in the growth of epithelial cells cultured in the presence of keratocytes, with basal medium and with conditioning medium after adding exogenous PGE2 at concentrations equal to or lower than 10(-6) M. Significant decreases in the dimensions of the corneal epithelial cells have been found only when PGE2 has been added to basal and to conditioning medium, suggesting that the autacoid maintains cell dimension and morphology. The appearance of keratins with high molecular weight (54 and 57 kDa) coupled with the tendency to stratification of the cells cultivated with media supplemented with PGE2, indicates that the autacoid could favour cell differentiation. The action of PGE2 on the corneal epithelial cells does not seem to be influenced by the presence of the fibroblasts and their products, since PGE2 has induced increases in cell growth and morphological variations, independent of cultural conditions and therefore also only in the presence of basal medium.     

Alternative paths of zinc transepithelial transport in the small intestine

PURPOSE: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.     

Primary neural precursors and intermitotic nuclear migration in the ventricular zone of adult canaries

New neurons continue to be born in the ventricular zone (VZ) of the lateral ventricles in the brain of adult birds. On the basis of serial section reconstruction and electron microscopy, we determined that the VZ of the adult canary brain is composed of three main cell types (A, B, and E). Type A cells were never found in contact with the ventricle and had microtubule-rich processes typical of young migrating neurons. Type B cells were organized as a pseudostratified epithelium, all contacted the ventricle, and most had a characteristic single cilium. Type E cells, also in contact with ventricle, were ultrastructurally similar to the mammalian multiciliated ependymal cells. After six injections of [3H]-thymidine (1 every 12 hr), Types A and B cells were found labeled. Type E cells were never [3H]-thymidine labeled. One to two hours after a single injection of [3H]-thymidine, all labeled cells corresponded to Type B cells. At survivals of 5, 24, and 74 hr after [3H]-thymidine injection, the proportion of labeled Type B cells decreased and that of Type A cells increased, indicating that Type B cells were the primary precursors. Most [3H]-labeled nuclei at 1-2 hr after [3H]-thymidine injection were separated from the ventricular cavity, but most of the mitotic cells were adjacent to the ventricle. This observation and measurements of the distance between labeled nuclei and the ventricular surface at 1, 5, 7, and 11 hr after [3H]-thymidine injection indicate that Type B cell nuclei move toward the ventricle to divide. This work reveals the architecture of the VZ in an adult vertebrate brain, identifies the primary precursor of new neurons, and describes nuclear translocation of these precursors during the cell cycle.     

Histone composition of nucleosomes isolated from cultured Chinese hamster cells

Nuclei isolated from cultured Chinese hamster cells were treated with micrococcal nuclease and lysed, and the resulting chromatin subunit classes (nucleosomes) were purified by sedimentation and resedimentation through isokinetic sucrose gradients. Nucleosomes isolated from [3H]thymidine-labeled cells were analyzed for DNA size using both polyacrylamide gel and electron microscopic techniques. Nucleosomes isolated from [14C]lysine-labeled cells were analyzed for protein content using a sodium dodecyl sulfate-polyacrylamide gel system. The results from monitoring the [14c]lysine in each protein indicate that, in the nucleosome classes (monomer through tetramer), the molar ratios of histones H2A, H2B, H3, and H4 are equivalent. Furthermore, in each population of the nucleosome classes monomer through tetramer, it was possible to demonstrate that this histone unit (H2A + H2B + H3 + H4) is present, on the average, in the amount of two for monomers, four for dimers, six for trimers, and eight for tetramers. This is direct experimental confirmation of the prediction of R.D. Kornberg [(1974) Science 184, 868] concerning the substructure of chromatin.     

Detection of neurone-specific enolase in long-term cultures of human corneal endothelium

BACKGROUND: Human corneal endothelial cells cultivated in monolayer culture for protracted periods undergo morphological dedifferentiation, whereby they assume a more fibroblast-like appearance. These cultures may also become overgrown with contaminating stromal fibroblasts and/or with keratocytes, when non-selective media are employed, thus rendering identification of actual endothelial cells difficult on a strictly morphological basis. METHODS: The endothelium of the human cornea stains for neurone-specific enolase (NSE) in situ, and we therefore wished to study the expression of this marker in primary and long-term monolayer cultures of these cells. Ten such cultures were established, six being stained for NSE at the primary and first-passage stage, the other four for 6, 8, 10 and 12 months. The NSE-staining pattern manifested in co-cultures of corneal endothelium and fibroblasts or keratocytes (first to fifth passage cultures) was also investigated, and co-cultures established from each of the latter two cell types served as controls. RESULTS: In monolayers of corneal endothelium which had retained their cobblestone-like morphology, NSE could be demonstrated even after more than 20 passages, which amounted to 1 year in culture. Dedifferentiated or degenerating endothelial cells stained poorly and inhomogeneously. Control cultures of fibroblasts or keratocytes were consistently NSE-negative, and when each of these cell types was co-cultured separately with corneal endothelium, only the latter expressed the marker protein. CONCLUSION: Since antibodies against NSE are commercially available, practical use may be made of this marker protein for confirming corneal endothelial status in long-term cultures.     

Interaction of invasive bacteria with host signaling pathways

PURPOSE: Programmed cell death (apoptosis) is the controlled death of cells that occurs with minimal collateral damage to surrounding cells or tissue during development, homeostasis, and wound healing. The authors hypothesize the keratocyte apoptosis is an initiating factor in the wound-healing response after refractive surgical procedures. To evaluate the effects of different corneal manipulations, keratocyte apoptosis was examined qualitatively and quantitatively after traditional epithelial scrape-photorefractive keratectomy (PRK), transepithelial PRK, removal of a cap of superficial cornea using a microkeratome, production of a flap of superficial cornea with a microkeratome, and laser-assisted in situ keratomileusis (LASIK) compared with unwounded controls in rabbit corneas. METHODS: Refractive surgical procedures or their components were performed in rabbit eyes. Keratocyte apoptosis was monitored using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay to detect DNA fragmentation. Cellular morphologic changes were evaluated by electron microscope examination. RESULTS: Keratocyte apoptosis was noted with each refractive procedure or corneal manipulation and was variable from eye to eye with each procedure. Transepithelial PRK was associated with the lowest levels of central corneal apoptosis, even if the stromal surface was scraped after the procedure. Keratocyte apoptosis is confined to the superficial stroma extending to a depth of approximately 50 microns to 75 microns after epithelial scrape-PRK and transepithelial PRK. Apoptosis was noted in the deeper central corneal keratocytes located anteriorly and posteriorly to the lamellar cut in LASIK. CONCLUSIONS: There are qualitative and quantitative differences in keratocyte apoptosis between LASIK, epithelial scrape-PRK, and transepithelial PRK. Epithelial injury is an important factor modulating keratocyte apoptosis. The level and distribution of keratocyte apoptosis, along with subsequent repopulation by activated stromal keratocytes, are likely to be important determinants of corneal wound healing associated with variability and regression after PRK and LASIK. Transepithelial PRK induces low levels of keratocyte apoptosis, and, therefore, this approach may be useful for treating higher levels of myopia and for retreatment after regression.