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Although there is currently no FDA approved antiviral treatment for adenovirus (Ad) infections, the broad spectrum antiviral cidofovir (CDV) has demonstrated potent inhibitory activity against many Ad serotypes in vitro and in an in vivo ocular replication model. The clinical potential of CDV prompted the assessment for the emergence of CDV resistance in Ad5. Serial passage of Ad5 in increasing concentrations of CDV resulted in derivation of four different Ad5 variants with increased resistance to CDV. CDV resistance was demonstrated by ability to replicate viral DNA in infected cells at CDV concentrations that inhibit the parental virus, by ability to form plaques in CDV concentrations of >20 microg/ml and by increased progeny release following infection and growth in media containing CDV. Using marker rescue, the loci for CDV resistance in variant R1 was shown to be mediated by one residue change L741S, one of two mutations within the R1 encoded DNA polymerase. The CDV-resistant variants R4, R5 and R6 also contained mutations in their respective DNA polymerase sequences, but these were different from R1; variant R4 contained two changes (F740I and V180I), whereas both R5 and R6 variants contained the non-conserved mutation A359E. R6 contained additional alterations L554F and V817L. The location of the R1 change is close to a region of the DNA polymerase which is conserved with other polymerases that is predicted to involve nucleotide binding.

The cornea contains, as a major element, a transparent stroma produced and maintained by keratocytes (fibroblasts). Through molecular biology studies using cultured human corneal fibroblasts, a cDNA that was shown to be novel was isolated and sequenced. This novel gene product, named SH3-domain binding protein 4 (SH3BP4), contains a 5.6-kb message that is present in normal human corneal fibroblasts and all tissues examined, with higher levels in pancreas, placenta, heart, and kidney. SH3BP4 was localized by FISH analysis to human chromosome 2q37.1-q37.2 near the telomere. The deduced sequence for SH3BP4 was found to contain a 963-amino-acid open reading frame that has homology to a 479-amino-acid protein in GenBank called EH-binding protein. Although the entire sequence of EH-binding protein aligns with SH3BP4, the alignment is not complete or contiguous. Therefore, SH3BP4 has an additional 73 amino acids at the N-terminus and an additional 411 amino acids near the C-terminus that are not present in EH-binding protein. Consensus sequence domains identified in SH3BP4 include a SH3 domain, three N-P-F motifs, a P-X-X-P motif noted for binding to SH3 domains, a bipartite nuclear targeting signal, and a tyrosine phosphorylation site. SH3BP4 homologies and consensus sequence sites indicate that it may be involved in a newly identified cascade of proteins involved in endocytosis, intracellular sorting, and the cell cycle.

A 1.9-kb cDNA clone to chick lumican (keratan sulfate proteoglycan) was isolated by screening an expressing vector library made from chick corneal RNA with antiserum to chick corneal lumican. The cDNA clone contained an open reading frame coding for a 343-amino acid protein, Mr = 38,640. Structural features of the deduced sequence include: a 18-amino acid signal peptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 62% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I, where X represents any amino acid. Lumican contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The sequential arrangement of these repeats and their spacing suggest that this region arose by duplication. The deduced sequence shows five potential N-linked glycosylation sites, four of which are in the leucine-rich region. These sites are also potential keratan sulfate attachment sites. The cDNA clone to lumican hybridizes to a 2.0-kb mRNA found in tissues other than cornea, predominantly muscle and intestine. Radiolabeling and immunoprecipitation studies show that lumican core protein is also synthesized by these tissues. The primary structure of lumican is similar to fibromodulin, decorin, and biglycan, which indicates it belongs to the small interstitial proteoglycan gene family. The expression of lumican in tissues other than cornea indicates a broader role for lumican besides contributing to corneal transparency.

A series of biochemical analyses were carried out with keratoconus and normal corneas to determine the amount of stromal collagen, degree of posttranslational modification of collagen and the solubility of collagen. Our results revealed there was no obvious alteration in the degree of posttranslational modification of collagen in keratoconus corneas. However, the amount of collagen decreases and solubility of collagen increases in keratoconus corneas. It was also found that keratoconus corneas in organ culture produce substantially more collagenase and gelatinase activities than normal corneas. Our results suggest that keratoconus may represent a collagenolytic disease.

In this paper, a new, more versatile single-column methodology is presented for the analysis of basement membrane and other collagen-like proteins. The methodology is designed for use on the Beckman 121-M microcolumn amino acid analyzer. Excellent analyses of imino acids and amino acids including tryptophan, as well as the amino sugars glucosamine and galactosamine, can be obtained at the same time if the protein is hydrolyzed with 3 n methanesulfonic acid. This methodology is suitable for hydrolyzates from reduced and alkylated basement membrane collagen as well as performic acid oxidized basement membrane collagen and similar collagen-like materials.

Crude human ocular mucus was extracted with 0.154 M-NaCl to separate soluble protein components from mucus. Small amounts of lipoglycoprotein of high molecular weight, as well as twelve plasma proteins, were detected in the soluble extract by gel filtration and immunodiffusion studies. After the NaCl extraction, the remaining mucus residue was further extracted with 6 M-urea-0.2 M-Tris-phosphoric acid buffer. From this portion of soluble extract, a relatively larger amount of lipoglycoprotein of high molecular weight, as well as a lower molecular weight fraction containing eight detectable plasma proteins, were both isolated by gel filtration. The glycoprotein moieties of the lipoglycoproteins of high molecular weight had similar chemical composition. Both contained approximately 40-43% protein and 57-60% carbohydrate, giving a carbohydrate--protein ratio of 1.30 to 1.48. Fucose, galactose, N-acetylhexosamine and N-acetylneuraminic acid comprised about 423 516 residues per 1000 amino acid residues, while serine and threonine constituted about 285 299. All analyses indicated mucin-like character in the lipoglycoproteins of high molecular weight. Plasma proteins constituted approximately three-fifths of the macromolecular components in ocular mucus. These proteins also appeared to be in complexes with lipids, but to a much lesser extent than the high molecular weight fractions. The relevance of present findings to the structure and composition of precorneal tear film is discussed.

Changes in expression of proto-oncogenes have been suggested as a key element in controlling the proliferative and differentiated status of a cell. We studied the expression of several of these genes in proliferating normal and metaplastic RPE cells. Using cDNAs to the c-myc, N-ras and n-myc genes we performed Northern blot analysis on the cytoplasmic RNA in normal and metaplastic RPE cells. We found that proliferating normal and metaplastic RPE cells exhibit similar patterns of expression of the c-myc gene. In contrast, when compared to normal cells, proliferating metaplastic RPE cells exhibited early and abnormally high levels of a 4 kb N-ras RNA. These changes in N-ras expression precede the vitreous-induced phenotypic change from an epithelial to a fibroblastic morphology. Understanding what role, if any, the N-ras gene plays in this metaplasia may lead to a better understanding of proliferative disorders such as proliferative vitreoretinopathy.