Immunolocalisation of P2Y receptors in the rat eye

Nucleotides present an important role in ocular physiology which has been demonstrated by recent works that indicate their involvement in many ocular processes. P2Y are important among P2 receptors since they can control tear production, corneal wound healing, aqueous humour dynamics and retinal physiology. Commercial antibodies have allowed us to investigate the distribution of P2Y receptors in the cornea, anterior and posterior chamber of the eye and retina. The P2Y1 receptor was present mainly in cornea, ciliary processes, and trabecular meshwork. The P2Y2 receptors were present in cornea, ciliary processes and retinal pigmented epithelium. P2Y4 was present in cornea, ciliary processes, photoreceptors, outer plexiform layer and ganglion cell layer. The P2Y6 presented almost an identical distribution as the P2Y4 receptor. The P2Y11 was also detectable in the retinal pigmented epithelium. The detailed distribution of the receptors clearly supports the recent findings indicating the relevant role of nucleotides in the ocular function

Analysis of copy number variation at DMBT1 and age-related macular degeneration

BACKGROUND: DMBT1 is a gene that shows extensive copy number variation (CNV) that alters the number of bacteria-binding domains in the protein and has been shown to activate the complement pathway. It lies next to the ARMS2/HTRA1 genes in a region of chromosome 10q26, where single nucleotide variants have been strongly associated with age-related macular degeneration (AMD), the commonest cause of blindness in Western populations. Complement activation is thought to be a key factor in the pathogenesis of this condition. We sought to investigate whether DMBT1 CNV plays any role in the susceptibility to AMD. METHODS: We analysed long-range linkage disequilibrium of DMBT1 CNV1 and CNV2 with flanking single nucleotide polymorphisms (SNPs) using our previously published CNV and HapMap Phase 3 SNP data in the CEPH Europeans from Utah (CEU). We then typed a large cohort of 860 AMD patients and 419 examined age-matched controls for copy number at DMBT1 CNV1 and CNV2 and combined these data with copy numbers from a further 480 unexamined controls. RESULTS: We found weak linkage disequilibrium between DMBT1 CNV1 and CNV2 with the SNPs rs1474526 and rs714816 in the HTRA1/ARMS2 region. By directly analysing copy number variation, we found no evidence of association of CNV1 or CNV2 with AMD. CONCLUSIONS: We have shown that copy number variation at DMBT1 does not affect risk of developing age-related macular degeneration and can therefore be ruled out from future studies investigating the association of structural variation at 10q26 with AMD

Quantification of MUC2 and MUC5AC Transcripts in Human Conjunctiva

PURPOSE. Transcripts of mucins 1, 4, and 5AC have been identified in human conjunctival tissue. Of these, only MUC5AC has been localized to goblet cells. MUC2 is a goblet cell mucin originally identified in the intestinal mucosa. The presence of MUC2 transcripts and levels of MUC2 and MUC5AC transcripts in normal human conjunctiva, as determined by quantitative polymerase chain reaction (PCR), is reported. METHODS. RNA from conjunctivae of six donors (3 men, 3 women, 44 to 69 years, all white) was isolated and subjected to competitive reverse transcription-PCR. Internal standards, which are dsDNA molecules with ends complementary to a given primer pair but containing nonhomologous central sequences, were prepared for each gene assayed. Titration of a constant amount of cDNA against serial dilutions of the internal standard allowed quantification of the template cDNA. MUC2 and MUC5AC levels were compared to levels of the "housekeeping" gene, ␤ 2 -microglobulin (␤ 2 M). The identity of PCR products was confirmed by sequencing. RESULTS. In the six individual samples tested, ␤ 2 M mRNA is expressed, on average, at approximately 10 Ϫ20 moles per sample (1 g RNA) or approximately 63.5 ϫ 10 4 molecules. The mRNA encoding MUC5AC, a relatively abundant ocular mucin, exists at levels 10-fold lower than ␤ 2 M. In contrast to previous reports of MUC2 mRNA being absent at the ocular surface, these results show that MUC2 transcripts are present and expressed at levels 5900-fold lower than for MUC5AC. Apparently, MUC2 transcripts exist on the order of only approximately 100 to 1000 molecules/g of RNA in the analyzed samples. CONCLUSIONS. MUC2 transcripts are present in human conjunctival tissue and their abundance is much lower than that of MUC5AC. This is the first application of competitive PCR to the quantitative analysis of mucin expression in human ocular tissue. The sensitivity of this method allows the detection of MUC2 transcripts that were not detected by Northern blot analysis or in situ hybridization in previous studies. It also makes possible the comparison of relative levels of expression for ocular mucins. (Invest Ophthalmol Vis Sci. 2000;41:703-708) T he preocular tear film is a trilayered structure consisting of an outermost layer of Meibomian lipids, a middle aqueous layer, and an inner, hydrated mucus layer. The mucus layer, a fluid-filled meshwork of glycoproteins, functions to lubricate and protect the ocular surface. It anchors the aqueous tear film to the underlying conjunctival and corneal surfaces and protects this epithelium from sheer force damage, drying, and bacterial invasion. The tear film is the first protective layer encountered by particulates, pathogens, and noxious agents. In other tissues, 1-4 mucins function as antioxidants and inhibit bacterial adherence; similar roles are likely for tear film mucins. Recently, the thickness of the mucus layer has been reexamined. The depth of this layer was revised to between 30 and 40 m, 7 Resolution of these differences will require further study. Dilly 6 proposes that the mucus layer is stable and anchored to the ocular surface epithelium and that the aqueous layer, containing dissolved mucins, provides a cleavage plane for lid movement. The more viscous mucus layer dissipates these movements to protect the underlying epithelium. Although the structure of the tear film is becoming more clearly understood, the mucin composition and extent to which these molecules contribute to the functions of the tear film is less clear. Mucins comprise a heterogeneous family of glycoproteins expressed by most specialized epithelial tissues of mucosal surfaces. To date, nine distinct mucin genes have been reported (MUC1 to 4, MUC5AC, MUC5B, MUC6 to 8). Historically, expression of these genes was thought to be tissuespecific. However, it appears that many of the mucin genes are expressed in a wide variety of tissues with overlapping patterns of expression. 8 -10 Of these, three have been detected on the ocular surface: MUC1, MUC4 (ASGP-2), and MUC5AC. 11-14 MUC1 and ASGP-2 are membrane-associated mucins produced by nongoblet conjunctival epithelial cells