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Udgivelsesdato: 1998-Mar We isolated and identified the major protein present in corneas with granular dystrophy (GCD). We compared Coomassie-blue-stained protein bands obtained on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the extracts of corneas with GCD, corneas with other disorders, and normal human corneal tissue. After SDS-PAGE and transfer to a polyvinylidene difluoride membrane, bands of interest were analyzed by amino acid sequencing and by Western blotting. Corneas with GCD were also examined immunohistochemically. On SDS-PAGE a 63-kd band just below albumin was present in extracts of all corneas. The albumin/63-kd ratio was normally approximately 3:1, suggesting that the protein is a dominant constituent of the cornea. This band was much more plentiful than normal in corneas with GCD. Amino-terminal sequence analysis of the protein revealed a Gly-Pro-Ala-Lys-Ser-Pro-Tyr-Gln-Leu-Val-Leu-Gln-His-Ser-Arg sequence indistinguishable from an amino-terminal protein sequence deduced from a cDNA clone designated beta ig-h3, and it as well as the abnormal accumulations in GCD cross-reacted with beta ig-h3 antiserum. The presence of excessive beta ig-h3 in human corneas with GCD together with reported mutations in the beta ig-h3 gene in GCD suggests that the mutated gene product is a fundamental constituent of the characteristic corneal accumulations in GCD.

Udgivelsesdato: 1997-Dec PURPOSE: To isolate the protein that collects in increased amounts beneath the corneal epithelium in familial subepithelial corneal amyloidosis (FSCA), also known as gelatinous droplike corneal dystrophy, and to identify it by N-terminal amino acid sequencing. METHODS: Peptides resulting from pepsin digestion of a unique protein isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from frozen tissue from two corneas with FSCA were purified by high-pressure liquid chromatography followed by protein sequence analysis. The protein was identified by amino acid sequencing, Western blotting, and immunohistochemistry. RESULTS: A protein was identified in two corneas with FSCA that was not present in normal corneas or in corneas with other disorders. The amino acid sequences of two peptides derived from this protein were identical to portions of lactoferrin. The unique protein reacted with rabbit antihuman lactoferrin after Western blotting. The presence of lactoferrin in the amyloid within affected corneas was confirmed using the immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and lactoferrin antiserum. CONCLUSIONS: Corneal tissue with FSCA contains lactoferrin, and this is the first form of amyloidosis found to be associated with this protein. Because lactoferrin is a product of lacrimal glands, the corneal lactoferrin may be derived from the tears. Because the gene for lactoferrin is on chromosome 3 (3q21-q23), this locus is a potential site for the FSCA gene.

Autosomal recessive macular corneal dystrophy (MCD) is a heterogeneous disorder leading to visual impairment. Sixteen American and Icelandic families (11 type I and 5 type II) were analyzed for linkage, by use of 208 polymorphic microsatellite markers. A significant maximum LOD score Zmax of 7.82 at a maximum recombination fraction (thetamax) of .06 was found with the 16q22 locus D16S518 for MCD type I. In addition, a peak LOD score of 2.50 at a recombination fraction of .00 was obtained for the MCD type II families, by use of the identical marker. These findings raise the possibility that MCD type II may be due to the same genetic locus that is involved in MCD type I.

The expression of the human T-cell lymphotropic virus type 1 (HTLV-1) tax gene in transgenic mice has been shown to result in the development of neurofibromas. Further characterization of these transgenic mice has revealed other significant pathologic similarities between this transgenic mouse model and human neurofibromatosis (NF). Pheochromocytomas of the adrenal medulla and hamartomas of the iris are well-recognized manifestations of human NF. Adrenal medullary tumors have been found in 68% of transgenic animals that were studied. They appear, however, not to be pheochromocytomas, but rather composed of undifferentiated spindle cells. Proliferation of fibroblastlike cells in the iris also occurs in one-half of the transgenic animals surviving more than 6 months. Melanocytes, however, have not been found in the transgenic iris lesion, although they are characteristically found in the Lisch nodules of human NF. The similarities between human neurofibromatosis and this transgenic mouse model (in which the overexpression of a single gene results in neoplasia) are discussed. This transgenic system may provide further insights into molecular mechanisms involved in the pathogenesis of neurofibromatosis.

Udgivelsesdato: 1991-Feb-15 Amino acid (aa) sequence data from Staphylococcus areas V8 protease-digested bovine corneal 54-kDa protein (BCP54) fragments were utilized to derive mixed oligodeoxyribonucleotide (oligo) primers complementary to the reverse translation products of these sequences. These degenerate oligo primers were used to prime the amplification of BCP54 sequence from bovine corneal epithelial cell cDNA. The cDNA probe generated by this mixed oligo-primed amplification of cDNA was cloned and dideoxy-sequenced. A search of the GenBank database (version 63.0) revealed extensive sequence similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). Nucleotide (nt) and aa sequence alignment of the BCP54 translation product reveals it is 78% and 84% homologous with RATALD at the nt and aa levels, respectively. Conservation of aa sequence elements common to the aldehyde dehydrogenase family thought to be of structural/functional significance is further substantiated by this analysis. Included in the discussion is the likelihood that gene sharing (genes encoding metabolic enzymes and other stable proteins) may extend to the cornea.

Udgivelsesdato: 1990-Aug The most abundant soluble protein of bovine cornea, BCP 54 (Bovine Corneal Protein, molecular weight 54 kD) was isolated and digested under both limited and complete digestion conditions with Staphylococcus aureus V8 protease. The fragments resulting from limited digestion were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, visualized by Coomassie Blue staining, cut out, and submitted to N-terminal protein sequence analysis. Complete digestion fragments were separated on a Vydac C18 reverse-phase HPLC, collected, and concentrated prior to sequencing. Using this method, we obtained amino acid sequence data from three internal V8 protease derived fragments of BCP 54 and a number of HPLC fragments. Comparison of these amino acid sequences, corresponding to 30% of the BCP 54 molecule, to those sequences contained within release 22 of the National Biomedical Research Foundation Protein Identification Resource revealed no extended sequence similarity of known proteins to BCP54.

Studies of corneal explants in the hamster cheek pouch chamber have demonstrated that blood vessels invade the cornea only if the tissue is first infiltrated by leukocytes. In view of this observation, a comparative study of the events that precede and accompany corneal vascularization was undertaken in various experimental models. A variety of established methods were used to induce corneal vascularization, including exposure of the cornea to noxious agents, intracorneal injection of antigens into sensitized animals, as well as maintaining animals on diets deficient in vitamin A or riboflavin. In all models studied, the corneal vascularization was a manifestation of the reparative phase of the inflammatory response. A conspicuous leukocytic infiltrate of the cornea preceded and accompanied the corneal vascularization in all of the models. Although the lesions varied in several respects in the different models, all models displayed three phases with regard to vascularization: an early prevascular phase of leukocytic infiltration, a second phase where blood vessels persisted in the cornea in the absence of leukocytes. The latent period that preceded vascularization was directly related to the time of the initial leukocytic infiltration. The models in which a delay occurred in the leukocytic invasion displayed a subsequent delay in the vascular ingrowth. Conversely, in experiments where there was a rapid and extensive leukocytic invasion, there was also an early and enhanced corneal vasoproliferative response. In the various modesl investigated, the sites of the leukocytic infiltration and subsequent vascular ingrowth into the cornea paralleled each other. The data further support the hypotheses that leukocytes are a prerequisite to corneal vascularization and that leukocytes produce one or more factors which stimulate directional vascular growth.

Melanin pigmentation of the corneal epithelium was induced in pigmented guinea pigs by the topical application of colchicine to their eyes or by corneal cauterization with silver nitrate. With colchicine the pigmentation was preceded by the development of an abnormal corneal epithelium in which numerous cells became arrested in cell division. The corneal melanosis resulted largely from the migration of melanocytes into the corneal epithelium from the normally pigmented contiguous conjunctiva and to a lesser extent from the presence of melanin granules within corneal epithelial cells. In both models a leukocytic and vascular invasion of the cornea proceded and accompanied the migration of melanocytes into the corneal epithelium. Electron microscopy disclosed cells with the same morphology as conjunctival melanocytes between the epithelial cells of the cornea. Mature melanin granules were also present within some squamous epithelial cells as individual granules or as clusters. The ultrastructural findings are viewed in relation to how melanin granules are transferred from melanocytes to epithelial cells. Evidence is presented which suggests that malanin granule transfer may follow the fusion of the membranes of the melanocytes and epithelial cells.

Investigations on several experimental models in the past have supported the hypotheses that corneal vascularization is a manifestation of the inflammatory response and that leukocytes perform an essential role in stimulating corneal vascular ingrowth. To evaluate the possible role of leukocytes further in this phenomenon, the effect of leukocyte elimination on corneal vascularization induced by silver nitrate cauterization was investigated. Weanling Fischer albino rats received doses of total body x-irradiation ranging from 1100 to 2100 rads to deplete circulating leukocytes, and corneal silver nitrate cauterization was performed 4 days later. In this model, animals that received 1500 rads or more total body x-irradiation became severely leukopenic within 4 days. As a rule, neither leukocytes nor blood vessels invaded the cauterized corneas, whereas both a leukocytic and vascular invasion occurred at lower doses of irradiation that did not totally eliminate circulating leukocytes. Corneal vascularization ensued if the corneal cauterization was performed immediately after total body x-irradiation with 1500 rads before the leukopenic effect of x-irradiation occurred. Control studies in which the cornea was cauterized 4 days after only the head received 1500 rads x-irradiation ruled out the possibility of irradiation-induced limbal endothelial damage as the explanation for the vascular suppression observed by x-ray treatment. In nonirradiated rats, silver nitrate cauterization of the cornea consistently induced corneal vascularization by 2 to 3 days. In further experiments, methylprednisolone acetate was administered subconjunctivally after corneal cauterization. This corticosteroid inhibited the infiltration of leukocytes and the subsequent vascular invasion into the corneal stroma, if administered immediately after silver nitrate cauterization. However, when the same glucocorticoid was administered 1 day after cauterization, both a leukocytic infiltration and vascular ingrowth occurred but to a less severe degree than in non-glucocorticoid-treated cauterized corneas. These investigations together demonstrated that a vascular ingrowth of the cornea did not follow corneal cauterization with silver nitrate in the absence of leukocytes, and gives further support to the hypothesis that leukocytes serve a crucial function in corneal vascularization.

The glycosaminoglycans synthesized by 5 corneal explants with macular corneal dystrophy were analyzed by sequential degradation using heparitinase (and/or butyl nitrite), chondroitin ABC lyase, chondroitin AC lyase, and keratan sulfate endo-beta-galactosidase. The tissue was first pulse-labeled by incubation in phosphate-buffered saline (pH 7.3) containing 100 muCi/ml 3H-glucosamine and approximately 1000 muCi/ml 35S-sulfate for 1 hour and chased with Eagle's minimum essential medium containing 10% fetal calf serum. Notable differences were detected between the products of these corneal explants with macular corneal dystrophy and controls that were prepared and analyzed in the same manner. As assayed by its susceptibility to keratan sulfate endo-beta-galactosidase degradation, corneal tissue with macular corneal dystrophy incorporated less 35S-sulfate, and usually less 3H-glucosamine, into keratan sulfate than normal corneas. The corneal explants with macular corneal dystrophy also synthesized prominent fractions which eluted from DEAE-Sephacel columns in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 7.2) with 0.15 M and/or 0.25 M lithium chloride after pronase digestion. Since normal corneas produced small quantities of material with a similar elution profile after preparation and analysis under identical conditions it is possible that the fractions are normal synthetic products of the cornea. The significance of these findings in the pathogenesis of macular corneal dystrophy remains to be determined.

Purpose To evaluate the TGFBI gene and the encoded transforming growth factor beta-induced protein (TGFBIp) in a 47-year-old African-American patient with an unusual atypical asymmetric lattice corneal dystrophy (LCD). Methods The eyes of the proband and his brother were examined by slit-lamp biomicroscopy and their clinical records were reviewed. All 17 exons of TGFBI were evaluated in genomic DNA extracted from blood or buccal epithelial cell samples from the proband and his family members. The corneal tissue of the proband was examined histopathologically, and TGFBIp was analyzed in half of an excised corneal button. Results The proband (who had an unusual atypical asymmetric LCD) and his brother (who had mild bilateral deep stromal opacities) were found to have homozygous Val624Met mutations in TFGBI. The proband’s daughter who was heterozygous for the Val624Met mutation had no reported ophthalmic abnormalities. The corneal tissue from the proband contained TGFBIp with the Val624Met mutation. Patients with LCD have different clinical phenotypes based on their genotype. Molecular genetic analyses are becoming increasingly important in making precise diagnoses and prognostic predictions about inherited corneal disorders. Conclusions A novel Val624Met homozygous mutation in TGFBI was associated with atypical LCD in two family members. Symptomatic corneal disease was absent at the age of 24 years in the offspring of the proband who was heterozygous for this mutation. This is an apparent example of a TGFBI mutation that becomes evident when it is homozygous. The finding of Val624Met mutated TGFBIp in a ~65 kDa protein band in a reduced sodium dodecyl sulfate (SDS) gel suggests that the accumulated protein was intact TGFBIp and not a fragment of TGFBIp.