BritishJournal ofOphthalmology 1994; 78: 632-637

Localisation of ac(2,3) and cc(2,6) linked terminalsialic acid groups in human trabecular meshwork

S A Chapman, R E Bonshek, RW Stoddart, K R Mackenzie, D McLeod

AbstractSialic acid specific lectins were used to localiseisomers of sialyl glycosides in human trabecu-lar meshwork (TM) at the ultrastructural level.A lectin immunogold method demonstratedthat sialic groups were concentrated on theendothelial surface of Schlemm's canal (SC)and in the adjacent juxtacanalicular tissueJCT). One sialyl glycoside, a(2,6) linkedN-acetyl neuraminic acid, was present mainlyon the luminal aspect of the SC endotheliumand in the cytoplasm oftheJCT cells. Another,a(2,3) linked N-acetyl neuraminic acid, waslocalised predominantly to the extracellularfibriliar material ofthe JCT. The existence of atopographical segregation of these two sialylglycosides within the TM supports the viewthat highly charged anionic molecules may beof significance in regulating aqueous outflow.(Br_J Ophthalmol 1994; 78: 632-637)

Department ofPathological Sciences,University of ManchesterS A ChapmanR E BonshekR W Stoddart

Department ofOphthalmology,University of ManchesterR E BonshekK R MackenzieD McLeodCorrespondence to:Dr R E Bonshek, Departmentof Pathological Sciences,University of Manchester,Oxford Road, ManchesterM13 9PT.Accepted for publication22 February 1994

The trabecular meshwork (TM), Schlemm'scanal (SC), and collector channels are involved inthe regulation ofaqueous outflow and the controlof intraocular pressure (IOP). Primary openangle glaucoma (POAG) is considered to arisefrom a 'functional disturbance' of the outflowsystem, no pathognomonic morphologicalfeatures having been identified.' Resistance toaqueous outflow within the TM is thought, onthe basis of physiological and ultrastructuralstudies, to occur mainly at the level of thejuxtacanalicular tissue (JCT) (also known as theendothelial meshwork or cribriform layer),located beneath the inner wall of the SC andconsisting of several layers of endothelial cellssurrounded by extracellular matrix.' 2 Recently,interest has been revived in the role ofhyaluronicacid in the regulation of anterior chamber out-flow.,The cells lining the TM, including those of the

JCT, perform a variety of functions includingelaboration of extracellular matrix compon-ents,45 and may be important in maintenance ofthe integrity of the outflow pathway.6 The pas-sage of aqueous from the JCT into the canal ofSchlemm is generally thought to occur by anintracellular route through 'giant vacuoles'found in the inner wall endothelium.7-9 A'paracellular' route, with channels bound bynegatively charged membranes, has also beensuggested. '

Sialic acids are highly charged anions and arethe terminal constituent of many cell surfaceglycoconjugates, gangliosides, and extracellularglycoproteins, including fibronectin andlaminin. Previous studies have demonstrated thepresence within the TM of sialic acid, which hasthus been implicated in the maintenance of the

normal IOP and in the pathogenesis of POAG.At the ultrastructural level, investigations usingpolycationic ferritin (PCF) and colloidal iron(CI) concluded that sialyl residues are present onthe endothelial surfaces of the canal of Schlemmand the trabecular beams, and also within thecores of beams." Using the same techniques, acomparative study of normal TM and TM fromcases ofPOAG revealed an increase in the level ofsialic acids on the abluminal surface of the SCendothelium in glaucomatous eyes. 2

The term 'sialic acid' is generic for a group ofnatural derivatives of neuraminic acid, an acidamino sugar in pyranose form where the aminogroup is substituted by an acetyl or glycolylgroup; in humans N-acetyl neuraminic acid(Neu5NAc) is the most abundant. Sialic acidsare ca-glycosidically linked from C2 of the pyra-nose ring usually to C3 or C6 of the subterminalmonosaccharide group. The most common sialylgroupings in human tissue are Neu5NAc linkedc(2,3) to galactose (Gal) and Neu5NAc linkedct(2,6) to galactose (Gal) or N-acetyl galacto-samine (GalNAc). 13 The type of linkage isimportant in determining the properties of thegrouping - for example, ca(2,3) linkages arehydrolysed much faster than ut(2,6) bonds. " Thehigh electronegative charge of sialic acidsinfluences the conformation of glycoproteins andis thought to be important in the stabilising ofcell membranes'4 " and other functions such asresistance to proteolytic degradation."

It is now possible to differentiate betweensome of the sialic acids via their glycosidiclinkages using lectins with different specificbinding requirements. The lectin from the barkof elderberry, Sambucus nigra (SNA) binds withhighest affinity to the terminal glycoconjugatesequence Neu5NAccL(2,6)Gal/GalNAc.'6 Theleucoagglutinin from Maackia amurensis (MAA)binds specifically to Neu5NAca(2,3)GalP(1,4)(Glc)/GlcNAc, but not to Neu5NAcc(2,6)glycoconjugates. '7 A more general sialic acidspecificity is claimed for the lectin from theyellow slug, Limax flavus (LFA)." We haveapplied biotinylated SNA, MAA, and LFA tonormal human TM and have visualised lectinbinding at the ultrastructural level by animmunogold technique. We find a distinct,although not absolute, topographical specificityfor the two sialic acid isomers. By differentiatingbetween the at(2,3) and ut(2,6) linkages andproviding information on their ultrastructurallocalisation within the human TM, further

Abbreviations: Neu5NAc=N-acetyl neuraminic acid; Gal=galactosamine; Glc=glucosamine; 3'sialyllactose=Neu5Act(2,3)Gali3( 1 ,4)Glc; 6'sialvllactose= Neu5Acsx(2,6)Gal3( 1 ,4)Glc.

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insight has been gained into the topographicalrelations of these biochemical components of thehuman outflow pathway.

Materials and methods

Figure I Typical lowpower view ofthe trabecularmeshwork adjacent to theSchlemm's canal (SC).(Magnification 2900; scalebar=5 pm approx).JCT,juxtacanalicular tissue;CM, corneoscleralmeshwork; e, endothelialcell, inner wall ofcanal ofSchlemm; v, giant vacuole;tb, trabecular beam.

TISSUETM was taken from five human eyes at postmortem within a maximum of 11 hours fromdeath. The ages of the patients ranged from 32 to82 years and there was no history of glaucoma.The eyes were macroscopically and histologicallynormal. Four of the globes were initially takeninto 10% (v/v) buffered formalin and subse-quently part of the iridocorneal angle was

removed and cut into 1 mm thick pieces forfurther fixation in 2-5% (v/v) glutaraldehyde in0.1 mol/litre sodium cacodylate buffer contain-ing 3 mmol/litre calcium chloride for 2-4 hours.One globe was fixed immediately in 2 5% (v/v)glutaraldehyde. The tissue was then processedinto London resin white (LRW) for transmissionelectron microscopy (TEM).

REAGENTSBiotinylated LFA was obtained from EYLaboratories Inc (suppliers in the UK, BradsureBiologicals Ltd, Market Harborough, Leics);biotinylated SNA and MAA were obtained fromBoehringer Mannheim (Lewes, East Sussex,

Sc

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UK); bovine serum albumin (BSA, A7906) andNeu5Ac were obtained from Sigma Chemical Co(Poole, Dorset, UK); gold labelled anti-biotinantiserum, raised in goat, was supplied by Bio-cell Research Laboratories (Cardiff, UK);3'sialyllactose, and 6'sialyllactose were obtainedfrom Biocarb Chemicals (suppliers in the UK,Russell Fine Chemicals, Chester); Tween 20 wassupplied by BDH (Poole, Dorset); London resinwhite was obtained from Taab Laboratories(Reading, Berks, UK).

LECTIN IMMUNOGOLD LABELLINGA method was developed using biotinylatedlectin and gold labelled antibiotin. The anti-biotin gold technique was adapted from that ofSlot and Geuze.'9 Ultrathin sections (90 nmapprox) on nickel grids were incubated succes-sively as follows: 0 5 mol/litre Tris bufferedsaline, pH 7-6 (TBS) with 1% (w/v) BSA for5 minutes; TBS with 1% (w/v) gelatine for 10minutes followed by TBS with 0-2 mol/litreglycine for 3 minutes. The grids were thenwashed in TBS containing 1 mmol/litre CaCl2and 0-5% (v/v) Tween 20 before incubating withthe biotinylated lectins (concentration 10, 50, or100 [ig/ml) for 1 hour at room temperature.After washing as before, the grids were finallyincubated in antibiotin gold (10 nm or 15 nm)diluted 1:30 with TBS containing 1% (w/v) BSA.The grids were finally washed in distilled waterbefore counterstaining with 2% (w/v) uranylacetate and Reynold's lead citrate before exami-nation on a Philips 301 transmission electronmicroscope at 80 kV.

CONTROLSA negative control with 1% (w/v) BSA substi-tuted for the lectin was included with everyexperiment.The specificity of staining was confirmed for

each of the lectins with inhibitory sugars asfollows.

SNASNA was mixed with 4 mmol/litre 3'sialyllactose.A second aliquot of SNA was mixed with6 mmol/litre 6'sialyllactose. After 30 minutesgrids were then incubated separately with eachmixture and the normal labelling procedure wasthen followed. Labelling with SNA was reducedvery slightly by 3'sialyllactose and was reducedconsiderably by 6'sialyllactose. The principalspecificity is therefore for the sialyl group inct(2,6) linkage, with much lower affinity for thect(2,3) linkage. This is in agreement with thework of Shibuya et al. 16 Also, it has been reportedthat SNA has some affinity for the subterminalgroup GalNAc2' which explains the residuallabelling after inhibition with 6'sialyllactose.SNA was used at a concentration of 10 ,tg/ml tokeep low affinity binding to a minimum.

MAAAn aliquot of MAA was likewise mixedwith 3'sialyllactose and a second aliquot with

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SNASC

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Figure 2 Sambucus nigraagglutinin (SNA) bindingto the Schlemm's canal (SC)endothelium. (a)Immunogold label issomewhat heavier on theluminal aspect ofthe SCendothelial cells comparedwith the albuminal surface.It is also present within acytoplasmic vesicle(arrowhead). Very littlelabel is seen on the granularmaterial (G) oftheextracellular matrix.3ruxtacanalicular tissue cells(JC) are not labelled.(Magnification 38 750; scalebar=0-25 pm approx). (b)Immunogold label is visibleon the luminal andabluminal aspect ofthe canalendothelium and, to someextent, within the cytoplasmofthe endothelial cell shown.In this view, as opposed to(a), the cytoplasm ofajuxtacanalicular tissue cell(JC) is labelled.(Magnification 54 250; scalebar=0-25 pm approx).

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6'sialyllactose and the same prifollowed. MAA labelling was redu(zero with 3'sialyllactose and was u

6'sialyllactose confirming that MAling specifically the a(2,3) linkegroup. MAA was used at 50 andlabelling experiments.

LFALFA was mixed with Neu5NAc (31and after 30 minutes the usual 1cedure was followed. LFA labellingto zero by the inhibiting sugar cc

specificity of the lectin in this prowas used at a concentration of 10 g

ResultsThe preservation and resulting u

was variable because of the differentbefore fixation. This did not appear

labelling which was consistent inexamined. Figure shows at low matypical area ofJCT and adjacent strione of the cases studied. Labellisections with the three lectins was Ione case where a large filled vacuo

endothelium was identified.

SNALabelling was observed mainly on the endo-thelial cell membrane ofthe inner and outer wallsof SC, with a greater concentration on theluminal rather than the abluminal aspect of thecells (Fig 2a, 2b). Label was also present withincytoplasmic vesicles of the SC endothelium and,to a lesser extent, within the cytoplasm ofendothelial cells. Label was seen within thecytoplasm of a few JCT cells (Fig 2b). Veryoccasionally label was seen on fine fibrillarmaterial of the extracellular matrix (ECM)within the JCT. The filled vacuole which was

t serially sectioned, shown at low magnification inFig 3a, clearly showed SNA label predominantlyon the luminal surface of the endothelium, withoccasional label within the cytoplasm of endo-thelial cells and sparsely decorating the fibrillarmaterial of the ECM (Fig 3b). In the corneo-scleral meshwork, generally there was negligiblelabelling of the external surfaces of trabecularendothelial cells and within their cytoplasm, butvery occasional areas of moderately dense labelwere observed. The cores of trabecular beams

i showed sparse labelling.

,t,.o. MAA

Occasionally there was label on the cellSo- membrane of the SC endothelium and the

- extracellular granular material of the JCT, butlabelling was concentrated on the fine fibrillarmaterial beneath the endothelial cell layer of the

,. inner and outer walls of SC. There was consider-able variation in the amount of fibrillar materialobserved in the subendothelial matrix and label-led fibrils sometimes took the form of a narrowband adjacent to abluminal surface of the SCendothelial cell layer (Fig 4a) or larger tangles inthe JCT extending away from the endothelial celllayer (Fig 4b). Label also decorated basal lamina

ocedure was which was present focally beneath the SC endo-ced almost to thelium (Fig 4a). In the serial sections (Fig 3),Lnaffected by the vacuole which bound SNA predominantly onA was label- its luminal surface (Fig 3a) was observed to bindd sialic acid MAA mainly to the fibrils of the subendothelial100 [ig/ml in space (Fig 3b). Very occasional grains of label

were seen decorating the cytoplasm of the SCendothelial cell lining the vacuole. Trabecularbeams of the corneoscleral meshwork showedsparse labelling in the basement membrane of

0 mmol/litre) the trabecular endothelium.abelling pro-was reducednfirming the LFAcedure. LFA Moderately dense label was seen on the endo-;/ml. thelium of the inner and outer walls of SC (Fig

5a). It was concentrated in the glycocalyx of theluminal aspect of the endothelial cells, with lesslabel on the abluminal side. Label was also

ltrastructure observed in cytoplasmic vesicles and occasion-time periods ally in the cytoplasm of the SC endothelium. Theto affect the density of label on the SC endothelium wasall the cases greater than that observed with SNA. In theignification a JCT,label was seen on the plasmalemmal surfaceuctures from of cells of the JCT (Fig 5a) and on the fibrillaring of serial material beneath the SC endothelium (Fig 5b).possible with The granular material and elastic material werele on the SC not convincingly labelled. The vacuole which

clearly showed the differential localisation of

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Figure 3 Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinitibinding to the same filled vacuole on the Schlemm's canal (SC) endothelium. (a) Lcview ofthe vacuole and adjacent structures. Serial sectionsfrom areas adjacent to thdelineated by the box are shown at higher magnification in (b) and (c). (Magnificatiscale bar=2-5 tm approx). (b) SNA: label is heaviest on the luminal aspect ofthe iendothelium (arrowheads). There is only slight binding to the subendothelialfibrils.appears to be present within the endothelial cell cytoplasm at the top left hand comeareflect tangential cutting ofthe cell at this point. (Magnification 38 750; scale bar=4approx). (c)MAA: label is concentrated on the subendothelialfibrils ofthe extracellwith very occasional label on theSC endothelium (arrows and box). (Magnificatiorscale bar=0-25 [tm approx).

a(2,6) and a(2,3) isomers, showeddifferentiation with LFA. In the cornmeshwork, sparse label was obsetrabecular endothelial cells, in the bas;and on the collagen of the trabecular be5c).

DiscussionThe results show that, by applying biolectin probes followed by gold labelled a

antiserum, it is possible to localise residues ofdifferent isomeric sialyl glycosides in human TMat the ultrastructural level. ct(2,6) Sialic acid,demonstrated by SNA, was concentrated mainlyin the glycocalyx of the luminal aspect of endo-thelial cells of the CS; it was present to a muchlesser extent on the endothelium of trabecularcells of the comeoscleral mesh and within thecores of trabecular beams. Conversely, sialic acidin the a(2,3) linkage, as shown by MAA, wasobserved mainly in the fine fibrillar materialbeneath the endothelium of the CS and to a muchlesser extent in the basement membrane oftrabecular beams. LFA confirmed the findingswith SNA and MAA, bindirig to both the CSendothelium and the fine fibrils of the subendo-thelial extracellular matrix as well as to the

SNA endothelium of trabecular cells. The pattern ofLFA binding, with its broad specificity for sialylglycosides, is in broad agreement with studiesusing polycationic ferritin (PCF) and colloidaliron (CI) where it was found that, in normalhuman TM there is a greater concentration ofneuraminidase sensitive label on the luminalthan on the abluminal aspect of SC endothelium,with lesser amounts present within the extra-

V cellular matrix.'2A ~With respect to this and other similar studies it

should be noted that both PCF and CI areQ general markers for polyacids which include

polyuronides, sialyl glycoconjugates, poly-4* ~ sulphated and polyphosphorylated molecules.

Staining at pH 18-19 suggests sulphate, notsialic acid and sialyl glycosides would, in anycase, undergo an appreciable degree of hydroly-

V sis at this low pH. Binding ofPCF and CI abovepH 3-3 suggests non-sialyl carboxyl. Only reduc-tion of staining with neuraminidase pretreat-ment provides good evidence for sialic acid, butdoes not prove that the label has bound directlyto it. The label may have bound to a non-sialylacidic molecule which was attached to the sialic

: . acid, hence showing reduction in staining withneuraminidase treatment. Our reagents detectsialyl glycosides directly and do not cross reactwith, for example, hyaluronic acid which showsa broadly similar distribution in other studiesusing PCF and Cl."-23 Despite these chemicaland methodological considerations in interpret-

i (MAA) ing PCF and CI data, it is nevertheless of interestvpower that there appears to be a concentration of a

iat variety of highly anionic species in the distalion 4650; regions of the TM. A recent study employingSome label cationic dyes and an anti-hyaluronic acid mono-r. This may clonal antibody suggests that glycosamino-0a25 tmn glycans including hyaluronic acid may have anrm38750 'osmotic' function in controlling water flow at

the level of the chamber angle in addition to a'structural' function within the TM itself.3

no such The binding patterns we observed with SNAieoscleral and MAA indicate that there is molecular andrved on spatial heterogeneity in the expression of sialylal lamina glycosides within the human TM. It is likely thatams (Fig they are constituents of different sialoglycocon-

jugates with different roles in aqueous outflow.a(2,6) Linked sialic acid appears to be associatedpredominantly with the cell membrane and maythus be a component of membrane-bound glyco-

tinylated protein or glycolipid. It is known from nuclearintibiotin magnetic resonance spectroscopy to form a more

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Figure 4 Maackiaamurensis agglutinin(MAA) binding tosubendothelialfibrils ofthejuxtacanalicular tissue(7CT). (Magnification50 000; scale bar=0 5 p.mapprox). (a) Label can beseen onfibrillar tangles oftheJrCT as well asfocally on thebasal lamina of theSchlemm's canal (SC)endothelium (arrows). Veryoccasional label is present onthe luminal surface oftheendothelium (arrowheads).(b) Label is visible mainly onfibrils closelyjuxtaposed tothe basal lamina ofthe SCendothelium. No label isvisible on the luminalsurface.

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Figure 5 Limax flavus agglutinin (LFA) binding to trabecular meshwork. (a) Hetlabel is on the luminal aspect ofthe Schlemm's canal (SC) endothelium, but it can alsin the cytoplasm and on the abluminal surface. Label is also present on the plasmalensurface ofjuxtacanalicular cells. (Magnification 30875; scale bar=0 5 p.m approx).is visible on the SC endothelium and onfibrils ofthe3JCT. (Magnification 40625; sc

bar=0'5 p.m approx). (c)A trabecular beam ofthe corneoscleral mesh shows sparse 1on the trabecular cell surface and in the cytoplasm. There is some binding to the basallamina (BL) with very occasional label in the core. (Magnification 40 625; scale barapprox).

flexible structure than the a(2,3) form'4 and,because of the exposed terminal, position andhigh electronegative charge, it is likely to beinvolved in functions such as stabilisation ofmembranes'5 and maintenance of cellular attach-ment to basement membrane.24 A decrease insialyl residues at the cell surface has been shownto be associated with an increase in cell mem-brane permeability and endocytosis in myeloidsinusoidal endothelium25 and renal glomeruliY.2Further, a sialoprotein, identified on rat glome-rular epithelial membrane, shows a decreasedsialic acid content in the rat model of humanrenal 'minimal change disease'.27 Gluco-corticoids, which increase the expression ofsialyltransferase activity,28 29 are used thera-peutically to reverse minimal change disease inthe kidney and may affect aqueous outflowthrough a similar mechanism ('steroidglaucoma'). The a(2,3) linked sialic acid residueswere localised mainly in the fine fibrillar materialin the subendothelial region of the JCT, the so-called 'type I plaque',30 and were also noted to beassociated with the small amount of basallaminar material seen focally here. Other highlynegatively charged molecules such as heparansulphate, chondroitin sulphate, and dermatansulphate are also found in this location. It hasbeen suggested that these proteoglycans, whichare associated with a mixture of fine fibrils andbasal lamina-like material, may have a role inaqueous outflow resistance.3' The co-localisationof MAA binding to this fibrillar material in ourstudy implies an ECM based role of sialic acids inaqueous outflow.

Laminin, a multidomain sialoglycoprotein ofthe extracellular matrix, has been identified(together with type I, III, and IV collagen) in thesubendothelial layer ofthe inner wall of SC in theaged human outflow system.3233 The finefilamentous material described in these reportsappears to correlate closely with the MAA bind-ing fibrils observed in our study and laminin isknown to be a basal lamina constituent. How-ever, while the sialic acid of laminin from the

a LFA

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EHS murine tumour has been found to bepresent solely in the a(2,3) linkage,34 it is notpossible to draw a direct parallel with the humanTM as both species and tissue variations aboundin the structure and amino acid content oflaminin. As yet no data exist for the precisenature of the sialic acid of human laminin andwhile double labelling using MAA and anti-laminin may provide further information, bio-chemical analyses would be necessary to confirmunequivocally that the a(2,3) sialyl groups hereindemonstrated are located specifically on laminin.The greater density of gold label observed

with LFA compared with SNA suggests thatsialic groups in linkages other than a(2,3) anda(2,6) may also be present on the SC endo-thelium - for example, a(2,8) as seen in neuralcell adhesion molecules, for instance.35 Ultra-structural localisation of a whole range of specificsialic acid residues, including comparativestudies on the relative proportions and topo-graphy in normal, aged and glaucomatous TM,may offer further insights into the pathogenesisofPOAG.

This work was generously supported by a grant from the NorthWestern Regional Health Authority. We wish to thank AdamPrest, ophthalmic illustration, Manchester Royal Eye Hospital,for assistance in the preparation of photomicrographs.

1 Grierson I. In: McAllister JA, Wilson RP, eds. Glaucoma.London: Butterworths, 1986: 1-29.

2 Maepea 0, Bill A. Pressures in the juxtacanalicular tissue andSchlemm's canal inmonkeys. ExpEyeRes 1992; 54: 879-83.

3 Carreras FJ, L6pez Caballero JJ, Porcel D. A gel of glyco-saminoglycans lining the anterior and posterior chambers inman: histochemical evidence at the light and electronmicroscopy levels. Eye 1992; 6: 574-82.

4 Floyd BR, Cleveland PH, Worthen DM. Fibronectin inhuman trabecular drainage channels. Invest OphthalmolVis Sci 1985; 26: 797-804.

5 Yun AJ, Murphy CG, Polansky JR, Newsome DA, AlvaradoJA. Proteins secreted by human trabecular cells. InvestOphthalmol Vis Sci 1989; 30: 2012-22.

6 Bill A. Some aspects of anterior chamber drainage. Eye 1993;7: 14-9.

7 Inomata H, Bill A, Smelser GK. Aqueous humor pathwaysthrough the trabecular meshwork and into Schlemm's canalin the cynomolgus monkey ('Macaca irus'): an electronmicroscopic study. AmJ7 Ophthalmol 1972; 73: 760-89.

8 Tripathi RC. Ultrastructure of Schlemm's canal in patho-genesis of primary open-angle glaucoma. Exp Eye Res 1968;9:335-41.

9 Grierson I, Lee WR. Light microscopic quantitation of theendothelial vacuoles in Schlemm's canal. AmJI Ophthalmol1977;84:234-46.

10 Epstein D, Rohen JW. Morphology of the trabecular mesh-work and inner wall endothelium after cationized ferritinperfusion in the monkey eye. Invest Ophthalmol Vis Sci 1991;32: 160-71.

11 Tripathi BJ, Millard CB, Tripathi RC. Qualitative andquantitative analyses of sialic acid in the human trabecularmeshwork. Exp Eye Res 1990; 51: 601-6.

12 Tripathi RC, Tripathi BJ, Spaeth GL. Localization ot sialicacid moieties in the endothelial lining of Schlemm's canal innormal and glaucomatous eyes. Exp Eye Res 1987; 44: 293-306.

13 Corfield AP, Schauer R. In: Schauer R, ed. Sialic acids:chemistry, metabolism andfunction. Cell Biology Monograph.New York: Springer Verlag, 1982; 10: 195-261.

14 Schauer R. Chemistry, metabolism and biological functions ofsialic acids. Adv Carbohydr Chem Biochem 1982; 40: 131-234.

15 Quinton PM, Philpott CW. A role for anionic sites in epithelialarchitecture. Cell Biol 1973; 56: 787-96.

16 Shibuya N, Goldstein IJ, Brockaert WF, Nsimba-Lubaki M,Peeters B, Peumans WJ. The elderberry (Sambucus nigra L)bark lectin recognizes the Neu5Ac(a2-6)Gal/GalNAcsequence.JBiolChem 1987; 282: 1596-601.

17 Knibbs RN, Goldstein IJ, Ratcliffe RM, Shibuya N.Characterization of the carbohydrate binding specificity ofthe leukoagglutinating lectin from 'Maackia amurensis'.JBiol Chem 1991; 266: 83-8.

18 Miller RL, Collawyn JFJ, Fish WW. Purification and macro-molecular properties of a sialic acid-specific lectin from theslug 'Limax flavus'. Biol Chem 1982; 257: 7574-80.

19 Slot JW, Geuze HJ. In: Polak JM, Varndell IM, eds.Immunolabellingfor electron microscopy. Amsterdam: ElsevierScientific, 1984: 129-42.

20 Taatjes DJ, Roth J, Peumans W, Goldstein IJ. Elderberrybark lectin-gold techniques for the detection of Neu5Ac(a2,6) Gal/GalNAc sequences: applications and limitations.Histochemj 1988; 20: 478-90.

21 Segawa K, Localization of acid mucopolysaccharides in thehuman trabecular meshwork using Mowry's colloidal ironmethod. JapJ Clin Ophthalmol 1970; 24: 363-7.

22 Grierson I, Lee WR. Acid mucopolysaccharides in the outflowapparatus. ExpEyeRes 1975; 21: 417-31.

23 Armaly MF, Wang Y. Demonstration of acid mucopoly-saccharides in the trabecular meshwork of the rhesusmonkey. Invest Ophthalmol Vis Sci 1975; 14: 507-16.

24 Kanwar YS, Farquhar MG. Detachment of endothelium andepithelium from the glomerular basement membrane pro-duced by kidney perfusion with neuraminidase. Lab Invest1980; 42: 375-84.

25 De Bruyn PPH, Michelson S. Changes in the random distribu-tion of sialic acid at the surface of the myeloid sinusoidalendothelium resulting from the presence of diaphragmedfenestrae. CellBiol 1979; 82: 708-14.

26 Blau EB, Haas JE. Glomerular sialic acid and proteinuria inhuman renal disease. Lab Invest 1973; 28: 477-81.

27 Keriaschki D, Vernillo AT, Farquhar MG. Reduced sialyla-tion of podocalyxin - the major sialoprotein of the rat kidneyglomerulus - in aminonucleoside nephrosis. Am i Pathol1985; 118: 343-8.

28 Kolinska J, Baudysova M, Zakostelecka M, Kraml J,Kadlecova L. Regulation of sialylation of intestinal brush-border enzymes and of sialyltransferase activity in organcultures by dexamethasone. Biochem Int 1990; 22: 495-508.

29 Wang XC, Smith TJ, Lau JTY. Transcriptional regulation ofthe liver ,1-galactoside a2,6-sialyltransferase by gluco-corticoids. JBiol Chem 1990; 265: 17849-53.

30 Rohen WR, Futa R, Lutjen-Drecoll E. The fine structure ofthe cribriform meshwork in normal and glaucomatous eyesas seen in tangential sections. Invest Ophthalmol VisSci 1981;21: 574-85.

31 Tawara A, Varner HH, Hollyfield JG. Distribution andcharacterization of sulfated proteoglycans in the humantrabecular tissue. Invest Ophthalmol Vis Sci 1989; 30: 2215-31.

32 Marshall GE, Konstas AG, Lee WR. Immunogold localizationof type IV collagen and laminin in the aging human outflowsystem. ExpEye Res 1990; 51: 691-9.

33 Marshall GE, Konstas AGP, Lee WR. Immunogold ultra-structural localization of collagens in the aged humanoutflow system. Ophthalmology 1991; 98: 692-700.

34 Knibbs RN, Perini F, Goldstein I. Structure of the majorconcanavalin A reactive oligosaccharides of the extracellularmatrix component laminin. Biochemistry 1989; 28: 6379-92.

35 Troy FA. Polysialylation: from bacteria to brains. Glycobiology1992; 2:5-23.

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