Showing papers by "Anthony P. Corfield published in 1997"

Soluble mucins in human aqueous tears.

Abstract: Nine mucin genes have been identified in non-ocular tissues. The many mucin gene products share a common structure with highly glycosylated variable number tandem repeat (VNTR) regions separated by naked nonVNTR regions [ 1,2]. How mucin structure relates to their function in the tear film awaits definitive characterisation of the mucins. Also to be elucidated is the relationship between mucins within the preocular mucus gel layer and mucins in aqueous tears over the gel. It is not yet clear whether these phases exist as separate entities. If they do then they might have shared and specific functions including lubrication, hydration of the gel and general and specific mucosal defence [ 1,3]. Gel mucins may require prior biochemical change or degradation for solubilisation to occur, or they may be secreted as a distinct mucin subgroup which makes its way through the gel to the aqueous phase. We aimed to assess the presence of soluble mucins in buman aqueous tears overlaying the preocular gel and to identify a source of these mucins. Unstimulated tears were carefully collected by touching a micro-capillary tube against the lower aqueous tear meniscus. These tears were from 4 normal volunteers aged 16, 31, 12 and 48 years. They were stored in guanidine hydrochloride with protease inhibitors [4] until a volume of 200 pl had been collected. Conjunctival mucins were extracted into guanidme hydrochloride with inhibitors from tissue obtained from the Bristol Eye Bank. Following caesium chloride density gradient centrifugation, native and reduced-alkylated tears and tissue extracts were gel filtrated on Sepharose CL2B then the excluded volume (V,) electrophoresed in 1% agarose gels [5]. Vacuum blots of the gels onto polyvinylidine difluoride membranes were probed with HRP-linked wheatgerm agglutinin (WGA) or antibodies antiMUC2 or anti-MUC5AC against non-VNTR regions of the mucin peptide cores [a]. Antibody binding was detected with HRP-linked second-layer antibody. Serial sections of formalin-fixed wax-embedded bulbar or eyelid conjunctiva were hybridised in situ with "SadATP-labelled 48mer antisense oligonucleotides complementary to messenger RNA for MUC 1-8 genes (VNTR regions). These probes have been applied successfully to several other mucosae [7]. Known positive control tissues and competition controls with the addition of unlabelled probe (50 x excess) were run at the same time. Slides were dipped in photographic emulsion auto-exposed, developed, counter-stained and examined with a microscope to localise MUC gene message. Some sections, and conjunctival impression cytology specimens on nitrocellulose membranes, were probed with the antibodies anti-MUC2 or anti-MUC5AC. Second-layer-antibody only served as a negative control. Tear mucins cross-reacted with aM1 only in the range 1.3-1.4 g/ml (Figure I), a more restricted range than found for tissue mucins. Sepbarose CLZB V, mucins of buoyant density 1.3-1.35 g/ml in native tears were found to migrate with subunit mucins from conjunctival tissue extract (Figure 2). This pattern is not repeated in the range 1.35-1.4 g/ml.