摘要:

The surface of 8 different corneal quadrants was assessed by scanning electron microscopy. Cells were identified by their electron reflex (light, medium, dark) and their areas, longest and shortest dimensions measured using a manual digitizer tablet/computer-interfaced system. Analyses of over 8200 cells show that while light cells are predominantly small (uncorrected mean area of 39.7μm2), there can be light cells ten times larger than this. Medium reflex cells show a very wide range of area (uncorrected values ranging from 16 to 1257μm2) while dark reflex cells show an even wider range (24 to 2218μm2). Analysis of the longest to shortest dimensions (LS ratio) reveals light cells to be more elongate than medium cells while dark cells tend to be rounder.

ISSN:0271-3683    

DOI:10.3109/02713689008999618

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

To investigate the effects of extracellular matrix components on cellular function, we cultured several types of ocular cells on substrates composed of extracellular matrix materials that were layered on culture dishes either as dried films or as gels. We measured cellular proliferation on these substrates and on a series of gels composed of varying proportions of rat tail tendon type I collagen and Matrigel™, a commercially available extract of a basement membrane-producing murine tumor. In addition, we studied the biosynthesis of collagens and of proteoglycans by these cultured cells using [3H]-L-proline and [35S]-sulfate. The proliferative abilities of the various types of ocular cells on the dried film substrates, on uncoated plastic culture vessels, and on pure type I collagen gel, were similar. However, proliferation of ocular cells cultured on gels composed of≥90% Matrigel was markedly reduced. There was little or no inhibition of growth of two types of non-ocular cells: rat C6 astrocytoma cells, and human dermal fibroblasts. Histologic studies showed that the ocular cells tested often formed long strands and capillary-like tubes, and tended to“burrow”beneath the surface of substrates containing high percentages of Matrigel. Fibroblasts infrequently formed tubes, and exhibited the burrowing property also on gels containing primarily type I collagen, while C6 cells showed neither of these behaviors on any of the matrices tested. The elution pattern of newly synthesized [3H]-labeled and [35S]-labeled macromolecules produced by all of the cultured cell types, and detected by Sepharose CL-4B chromatography in the medium and in the cell layer plus matrix fractions did not vary following culture on the different substrates. Approximately twofold more of the newly synthesized collagens and proteoglycans were deposited in the cell layer plus matrix, and proportionately less appeared in the medium, when cells were cultured on type I collagen gels and on Matrigel than on the dried film substrates. These experiments demonstrate the influence of the extracellular matrix on several aspects of cell behavior, and provide further evidence that modification of the composition of the extracellular matrix may be an important determinant of normal or pathological cell function.

ISSN:0271-3683    

DOI:10.3109/02713689008999619

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The migration of retinal pigment epithelial (RPE) cells from their normal anatomic position to a new position in the vitreous cavity is a critical feature of proliferative vitreous retinopathy. To determine if insulin-like growth factor I (IGF I), which is present in the vitreous fluid of diabetics, stimulates RPE cells, we examined the effects of IGF I on the proliferation, chemotaxis, and release of plasminoqen activator by these cells.At the concentrations of IGF I tested, significant proliferation of RPE cells is seen. Significant chemotaxis of the RPE cells also is seen at all the concentrations of IGF I tested. The mean number of migrating cells per high-powered field in control studies was 43±13 (x±SEM), and for IGF I at 2.5 ng and 50 ng/ml the mean numbers of migrating cells were 96±17 and 483±62, respectively (P<0.001 for each comparison). The IGF I response was noted to be dose-dependent. The chemotactic response noted at 50 ng/ml of IGF I was greater than the positive chemotactic control of 10% fetal calf serum. Addition ofαIR-3, an IGF I receptor antibody, eliminated the IGF I chemotactic response.The effect of IGF I on the secretion of plasminogen activators was assessed using an immunological assay for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). Media conditioned by RPE cells have measureable levels of PAI and t-PA antigen. IGF I supplementation resulted in an increase of t-PA secretion and PAI secretion over basal levels. These studies support a role for IGF I in modulating RPE cell function.

ISSN:0271-3683    

DOI:10.3109/02713689008999620

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Six neutral glycosphingolipids were isolated from porcine corneas using silicic acid column chromatography and preparative thin-layer chromatography. Five of these glycolipids were partially identified by gas-liquid chromatography. Two were glucosylceramides, two were lactosylceramides and one was tetrahexosylceramide containing galactose, glucose and N-acetylgalactosamine in the molar ratio of 2:1:1. Glucosylceramides were found to be the predominating component, with lactosyl- and tetrahexosylceramides being the minor constituents. Sphingosine was the major long-chain base in all fractions. The fatty acids of the corneal neutral glycosphingolipids were variable in chain length. This represents the first investigation of neutral glycosphingolipids in corneas of any species.

ISSN:0271-3683    

DOI:10.3109/02713689008999621

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Previous attempts to prepare monoclonal antibodies (MAbs) against S-antigen, a photoreceptor cell protein involved in the visual process and a potent autoantigen for the induction of experimental autoimmune uveitis (EAU), have yielded MAbs which define only carboxyl terminal epitopes. In this study we devised alternate strategies to prepare five MAbs directed to other regions of the molecule. MAbC10C10 and MAbH11-A2 were prepared against synthetic peptides known to be uveitopathogenic and they were selected for more detailed studies. MAbC10C10 was generated against synthetic peptide BSA281-302 which contains a predictive consensus sequence for defined T cell epitopes (GIALD) as well as a consensus sequence for GTP-binding proteins. One human adenosine deaminase synthetic peptide containing an extensive amino acid sequence homology to BSA281-302 was a potent inhibitor of MAbC10C10 binding in a competitive inhibition radioimmunoassay. MAbH11-A2 was generated against peptide BSA303-332 which also contains a uveitopathogenic site. The binding site of MAbH11-A2 was determined to be within amino acid positions 305 to 314 (NLASSTIIKE) in S-antigen. This binding site corresponded closely to the binding site of an affinity-purified rat polyclonal antibody raised to human S-antigen. MAb5C6.47 was isolated from a mouse hyperimmunized with bovine S-antigen and was specific for a highly conserved sequence near the amino terminus, amino acid residues 42 to 48 (DGVVLVD). Both MAbC10C10 and MAb5C.47 were useful in screening gtll cDNA libraries expressing S-antigen polypeptides as fusion proteins. Our results demonstrate the feasibility of producing site-specific MAbs potentially useful in the study of T cell-mediated immune mechanisms in EAU as well as in the phototransduction of vision.

ISSN:0271-3683    

DOI:10.3109/02713689008999622

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Interstitial retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein which is a highly pathogenic autoantigen for the induction of experimental autoimmune uveitis (EAU). In this study we produced a series of monoclonal antibodies (MAbs) which define different epitopes in the native molecule. These MAbs were further subdivided into three distinct groups based on a radioimmunoassay, and by ELISA assay using native IRBP and synthetic peptides corresponding to its entire amino acid sequence. Group I MAbs (MAbD7-B1 and MAbC6-B4) bound to native IRBP but not to any synthetic peptides, suggesting that their antigenic epitopes are strictly conformation dependent. Group II MAbs (MAbC7-D3 and MAbG8-H4) bound weakly to multiple peptides which shared amino acid sequence similarity located within each of four homology domains indicating that these epitopes are also conformation dependent. In group III (MAbH3-B5, MAbH7-A5, and MAbB6-D12) MAb binding was localized to a specific peptide. The MAbH3-B5 binding site was further refined to amino acid positions 361 to 367 in the native molecule. MAbH3-B5 was also useful in localizing IRBP in the mouse retina by immuno-histochemical techniques. The application of these MAbs in the study of EAU and interphotoreceptor transport mechanisms is discussed.

ISSN:0271-3683    

DOI:10.3109/02713689008999623

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The synthetic profile of glycosaminoglycans (GAGs) of human trabecular meshwork in perfusion organ culture was studied in a series of 34 human eyes. The anterior segments of these eyes were cultured for periods of two to 28 days and received medium containing3H-glucosamine and35S-sulfate during the final 48 hours of culture. The meshwork was then dissected and the GAGs isolated and subjected to sequential enzyme digestion. Active labelling of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate was found in all time periods. Eyes cultured seven and 14 days had similar incorporation profiles to“fresh”eyes (cultured 48 hours to allow for labelling). Eyes cultured 21 days showed an increase in dermatan sulfate labelling and a slight decrease in keratan sulfate labelling when compared with“fresh”eyes. Light microscopic autoradiography confirmed the trabecular meshwork incorporation of the radiolabelled precursors at all time periods. Thus, the trabecular meshwork remains metabolically active and GAG synthetic profiles remain reasonably similar to fresh eyes for up to three weeks in a perfusion organ culture system. This system may serve as a model for future studies of human trabecular meshwork GAGs.

ISSN:0271-3683    

DOI:10.3109/02713689008999624

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The interaction of adrenergic and peptide receptors linked to adenylate cyclase and the inhibition by bioactive peptides of stimulated cyclic AMP production has been investigated in intact, excised rabbit ciliary processes. Cyclic AMP production stimulated by isoproterenol, vasoactive intestinal peptide, or forskolin was inhibited by the biologically active peptides neuropeptide Y, somatostatin, and the synthetic somatostatin analogue SMS 201-995. IC508 determined from dose-response curves of inhibition are consistent with the known abilities of these ligands to modulate cyclic AMP and physiological responses in other tissues. Inhibition by neuropeptide Y or SMS 201-995 was unaffected by the specific alpha2-adrenergic antagonist yohimbine, which shows that peptide inhibition is not occurring via peptide binding to the inhibitory alpha2-adrenergic receptor. These results suggest that endogenous peptides may participate in modulation of cyclic AMP production and subsequent physiological events influenced by cyclic AMP levels in rabbit ciliary processes by inhibiting stimulated cyclic AMP synthesis.

ISSN:0271-3683    

DOI:10.3109/02713689008999625

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

We have investigated the transcription of IGF-I and IGF-II genes in the adult rat eye. Each eye was dissected into cornea, lens, sclera+choroid+pigment epithelium, and neural retina. Total cellular RNA was isolated from each of these tissues. Northern blot analysis using rat coding region cDNA probes revealed the presence of IGF-I and IGF-II specific transcripts in the retina and the sclera but not in the lens and cornea. Retina demonstrated the highest level of IGF-I and IGF-II expression. Our results suggest autocrine and paracrine effects of the IGFs and their possible involvement in normal eye physiology as well as in the pathogenesis of certain eye diseases.

ISSN:0271-3683    

DOI:10.3109/02713689008999626

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The binding of [3H]inositol 1,4,5-trisphosphate ([3H]IP3) to bovine iris sphincter microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites for [3H]IP3 (Kd=3.52 nM, Bmax=218 fmol/mg protein). The concentration of IP3 receptors in the iris sphincter is much higher than that of the corneal epithelium (99 fmol/mg protein), but considerably lower than that of the rat brain cortex (2250 fmol/mg protein). Kinetic studies on bovine iris sphincter microsomes showed: (a) an extremely rapid time-course of [3H]IP3 binding with a half-maximal binding achieved in 55 sec and reached equilibrium by 10 min; (b) that the [3H]IP3 binding was readily reversible with a t1/2 value of 36 sec, and (c) that the specificity of IP3 receptor sites can be demonstrated by their lack of affinity for 1,3,4-IP3, IP6 and cyclic IP1, and a much weaker affinity for IP1, IP2 and IP4. The results presented provide the first data on the affinity of [3H]IP3 to smooth muscle microsomal membranes. These data support the hypothesis that the [3H]IP3 binding reported here represents a putative physiologically important IP3 receptor which can be quantified in iris sphincter membranes.

ISSN:0271-3683    

DOI:10.3109/02713689008999627

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor