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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.

Varicella-zoster virus (VZV) expresses four proteins that influence viral transcriptional events and that also are homologous to herpes simplex virus type 1 (HSV-1) immediate-early proteins. However, their transcription and the mechanisms by which it is regulated are not yet resolved. To identify the promoter regions, a precise knowledge of the initiation and termination of the encoded RNAs is first required. In this report, we summarize the complete and precise mapping of the RNA transcripts of two of these genes--those from open reading frames 4 and 63. In addition, several elements of their promoter regulatory regions have been identified and predicted. Structural and functional studies of the regulatory sequences suggest that these two VZV genes may be regulated in a fashion different from that of their HSV-1 counterparts.

Three overlapping genomic clones to chick lumican were isolated and then characterized using restriction enzyme analyses, Southern blot analyses with cDNA probes, and by DNA sequencing. The results showed chick lumican gene to consist of 3 exons with a 2.9-kb first intron and a 4.2-kb second intron. Transcription initiation sites, identified by S1 nuclease experiments using genomic fragments containing exon 1 and by primer extension analysis of RNA, indicated the first exon to be 303 b. Two TATA sequences were 31 and 49 bases upstream of the first exon. The first exon contained all 5' untranslated sequence. The second exon was 896 b and contains 20 b of untranslated sequence, and codes for the start methionine to the end of the 10th leucine rich repeat. The third exon is 880 b and codes for the remainder of the core protein, and 724 b of untranslated 3' sequence. A 1-kb genomic fragment containing a portion of exon 1 and upstream sequence in a luciferase reporter sector showed specific promotor activity in the forward, but not the reverse direction when transfected into corneal fibroblasts. These results show the chick lumican gene to consist of three exons, and that regulatory elements are present within 1 kb upstream of the first exon.

Corneal proteoglycans have chondroitin/dermatan and keratan sulfate (KS) chains and belong to the leucine-rich proteoglycan gene family. Corneal KS is N-linked to Asn of an NX(S/T) site through a complex oligosaccharide linkage region. Only some sites receive KS, whereas others remain in a high mannose form. To determine whether the attachment of KS was biased toward specific sites, we isolated trypsin-digested KS-containing fragments of chick corneal proteoglycans and sequenced the peptides. Results showed that all of the peptides sequenced aligned to the deduced amino acid sequence of either chick lumican or chick keratocan at the first, third, and fourth potential N-linked sites. Sites 1 and 4 in lumican and keratocan are in a homologous location. By analogy with the structure of ribonuclease inhibitor (a Leu-rich repeat containing protein), the KS chains would extend outward on the outer face of a horseshoe-like structure. The amino acid sequences surrounding the potential N-linked sites were also compared. Sites receiving KS tend to have a higher occurrence of aromatic residues, in particular Phe, located within 3 amino acids of NX(S/T). These conserved Phe residues may have a role in the conversion of high mannose N-linked oligosaccharides to polylactosamine and/or keratan sulfate.

The corneal stroma consists of a highly structured extracellularmatrix that is maintained by specialized fibroblasts called kerato-cytes. The transparency of the cornea is dependent on the presenceof highly sulfated proteoglycans (PG) that can bind collagen andcan regulate collagen fibril diameter (Rada et al. 1993). The cor-neal proteoglycans that have been identified contain numerousLeu-rich repeats and all belong to the same gene family, the Leu-rich proteoglycans (LRP). These include a chondroitin/dermatansulfate proteoglycan (CS/DSPG), decorin, and three keratan sul-fate proteoglycans (KSPG), lumican, keratocan, and osteoglycin(Funderburgh et al. 1991, 1993; Blochberger et al. 1992; Corpuz etal. 1996). The corneal KSPGs are glycoproteins exclusively duringearly corneal development before corneal transparency (Cornuet etal. 1994) and in macular corneal dystrophy type I, an inheriteddisease that is characterized by corneal opacity and deposits(Klintworth et al. 1977, 1983; Nakazawa et al. 1984). These pre-vious studies show a direct correlation between corneal transpar-ency and the presence of keratan sulfate. The core protein andpoly-lactosamine side chains alone appear to be insufficient extra-cellular matrix components for sustaining a transparent structure inthe cornea.The primary structure of keratocan, based on the deducedamino acid sequence from bovine and chick cDNA sequences,shows N- and C-terminal globular domains containing four andtwo Cys residues forming two and one disulfide bond, respec-tively, and 11 highly conserved Leu-rich repeats located mainlybetween the two globular domains (Corpuz et al. 1996; Dunlevy etal. 1998). The two-dimensional model of chick keratocan, basedon the X-ray crystallography structure of ribonuclease inhibitor, amolecule comprised entirely of Leu-rich repeats, (Kobe et al. 1993,1995), shows the Leu-rich repeats coiled in a spiral with theseregions in close proximity to each other, forming a horseshoestructure (Dunlevy et al. 1998). This model also shows two out ofthree KS chains extended outward from the outer surface of thehorseshoe with the inner surface more likely to be involved incollagen binding.On the basis of mRNA levels, keratocan is most abundant incornea and sclera, but is also found at substantially lower levels inligament, artery, cartilage, skeletal muscle, and skin (Corpuz et al.1996), and keratocan is more specifically expressed in cornea thanis lumican. Consequentially, we are interested in gaining furtherinsights into its tissue-specific regulation of expression, its role indevelopment and maintenance of corneal transparency. In the pre-sent study, we report the cDNA sequence of mouse keratocan andlocalization of the gene to distal Chromosome (Chr) 10 by segre-gation analysis of restriction fragment length variants (RFLVs) inrecombinant inbred (RI) strains of mice.Total RNA was isolated from mouse whole eyes by use of aguanidinium thiocyanate-phenol-chloroform procedure (Chomc-zynski and Sacchi 1987). An mRNA purification kit (PharmaciaBiotech) was used to further purify the RNA, and ∼10 mgofmRNA was used to prepare a cDNA library (Stratagene CustomLibrary Department) in the Uni-Zap XR vector system. The librarywas screened with a high-specific-activity

The lacrimal gland secretes and metabolizes retinoids and responds to retinoic acid in culture. Like other retinoid responsive organs it is expected to express the nuclear retinoid receptors. The goal of this study was to identify the retinoic acid receptors (RAR) in the lacrimal glands of rats, rabbits, and humans. Total RNA was prepared from whole lacrimal glands and rat lacrimal gland acinar cells grown in culture. RNA was subjected to Northern blot analysis and probed for the RAR alpha, RAR beta, and RAR gamma mRNAs. Nuclear extracts of rat and rabbit lacrimal glands were incubated with 3H-all-trans retinoic acid and analyzed by gel filtration chromatography. Western blots of the nuclear extracts were probed using monoclonal antibodies to RAR alpha and RAR beta. Rat lacrimal gland expresses RAR alpha mRNA with two transcripts (3.8 and 3.0 kb), a single RAR beta mRNA transcript (3.3 kb), and a single RAR gamma mRNA transcript (3.3 kb). Cultured rat lacrimal acinar cells also expressed the mRNA for all three RAR subtypes. Rabbit lacrimal glands express mRNAs for RAR alpha (3.7 and 2.9 kb) and RAR beta (3.2 kb) but RAR gamma mRNA is not detectable. Human lacrimal glands also express mRNA for RAR alpha (3.5 and 2.3 kb), RAR beta (3.4 kb) and RAR gamma (3.0 kb). Lacrimal gland nuclear extracts contain proteins in the 50 kDa range that specifically bind retinoic acid with Kd = 1.25 nM in rat lacrimal gland and 0.3 nM in rabbit. The monoclonal antibodies identified RAR alpha and RAR beta in both rat and rabbit lacrimal glands. The results of this study support a role for retinoids in maintaining the structure and function of the lacrimal gland. The presence of RARs suggests potential interactions of these receptors with other members of their superfamily, including androgen and thyroid receptors, which also may be involved in lacrimal function.

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin 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 presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.

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.

The effect of age on the compositional analysis of human Descemet’s membrane was examined. Membranes were obtained from human corneas ranging from 0.5 to 95 years and purified by sodium dodecyl sulfate (SDS) extraction. Substantial age-related changes were obtained in the amino acid compositions of the SDS-insoluble basement membranes. These results suggested that compositional changes occur during the aging process, which appeared to reflect an alteration in the subunit make-up of the basement membrane.

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.

Two glycoproteins, GP-1 and GP-2, have been isolated from an extracellular membrane synthesized in cell culture by an embryonal carcinoma-derived cell line. The amino acid and carbohydrate compositions have been determined. Both proteins are rich in half-cystine residues and contain approximately 12–15% carbohydrate. Antibodies have been obtained against one of the glycoproteins, GP-2, in rabbits. The antibody reacts with basement membranes from adult mouse and human kidney glomeruli and tubules, and all basement membranes tested from mouse embryonic tissues. The molecular properties of GP-2 are superficially similar to LETS protein; however, immunological and other criteria show that they are distinct proteins. The presence of LETS protein and GP-2 in basement membranes suggests that there are subtle interactions which are important in adhesion of epithelial cells to basement membranes.

Alkaline β-elimination and sodium borohydride reduction were used to study the O-glycosidic linkage of N-acetylgalactosamine to seryl and threonyl residues in the high molecular weight crude glycoprotein fractions isolated from human ocular mucus. Pure mucins, BSM and OSM, were used as models. Data are presented for the existence of such O-glycosidic linkages. It was estimated from sodium borohydride reaction that at least 22% of the total peptide bonded hydroxyamino acid residues are linked O-glycosidically. These findings, as a continuation of our previous work on the isolation and chemical characterization of human ocular mucus, provided additional evidence of the mucin nature of the glycoprotein isolates.

A system for studying the biosynthesis of tear glycoproteins by human lacrimal gland is described. Sufficient quantities of the newly-synthesized glycoproteins were obtained to permit some analyses of their physical and chemical properties. Separation of glycoproteins by gel filtration on Sephadex G-200 showed three major heterogeneous glycoprotein fractions with molecular weights of 580 000, 100 000 and 39 000 daltons. The sulfate, carbohydrate, and amino acid composition of each fraction was determined. The protein moieties were found to be acidic in nature. The smaller molecular weight fractions were moderately richer in carbohydrate [26% (w/w) than the higher molecular weight fractions (8% w/w)]. The low carbohydrate contents were comparable to those of plasma-type proteins. DEAE ion exchange chromatography of the major fraction also showed an elution pattern characteristic of plasma-type proteins. Using immunodiffusion techniques, at least five serum-type proteins, including IgA, IgG, albumin, transferrin and cerulo-plasmin were identified from the different fractions of the cultured lacrimal gland secretions. It appeared that human lacrimal gland secreted similar plasma proteins to those found in the tears. The significance of these identified tear proteins to the physiology of tears is discussed.