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Exploring the Molecular Adhesion of Ocular Mucins M. Berry,*,† T. J. McMaster,‡ A. P. Corfield,† and M. J. Miles‡ Mucin Research Group, Bristol Eye Hospital, University of Bristol, Bristol, BS1 2LX, U.K.; and H.H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol, BS8 1TL, U.K. Received December 12, 2000; Revised Manuscript Received January 15, 2001
Mucins have been ascribed both pro- and anti-adhesive functions. To clarify how both functions can be embodied in the same molecule we studied the interaction of human ocular mucins with mica and with mucins deposited on mica. Adhesion energy and forces of interaction were evaluated as a function of speed of approach, dwell time at maximum extension, and presence of divalent cations in the imaging buffer. Mucins were tethered to an AFM gold-coated tip. Repeated cycles of approach and retract to mica revealed a large number of adhesions in each cycle. Adhesion energy (0.2-48 aJ) and detachment forces (0.1-4 nN) increased with the addition of Ni(II) ions, and with lengthening dwell time. Speed of approach made little difference to the interactions. Most detachments occurred less than 40 nm from the surface. Interdetachment distances reflected the major periodicities of the mica basal plane. Short distances of interaction, magnitude of detachment forces, and imaging of mucins on SAM all suggest deformable compact mucin aggregates on the AFM tip. Inter-detachment distances suggest a large degree of interpenetration between neighboring molecules. Tip-tethered mucins did not adhere to mucins deposited on mica. This phenomenon is analogous with the nonadherence of the mucin gels on lids and on cornea during blinking. Introduction The behavior of polymeric molecules at solid interfaces has long been a topic of theoretical and practical interest.1,2 Adhesion at surfaces manifests itself in various ways. For two polymer surfaces brought into contact, there is often a degree of reptation and interpenetration of polymer chains,3 with “tie” molecules bridging both surfaces which then exhibit pullout or chain scission when the surfaces are separated. This ingress may also occur when one of the surfaces is porous or membranous and allows molecular diffusion.4 A third type of behavior is the interaction of polymers with solid impenetrable surfaces, with molecular adhesion being the dominant mechanism.5,6 On the basis of atomic force microscopy (AFM),7 new physical techniques have emerged which permit measurement of force-extension behavior at the single molecule level.8-10 Examples of this molecular force spectroscopy approach include reversible unfolding of IgG domains,11 sequence-dependent stretching characteristics of DNA,12 and polysaccharide chair-boat transitions.13 Submolecular structures taking part in adhesion were identified in the fibrinogen molecule from the distribution of distances between successive binding sites.14 Mucins are involved in both adhesive and anti-adhesive processes.15 Ocular mucus has been shown to inhibit the adherence of microorganisms to the cornea.16 Forty percent of normal ocular surfaces are culture negative for bacteria.17 However, a commensal flora has been described for the eye * Corresponding author. Division of Ophthalmology, Bristol Eye Hospital, Bristol BS1 2LX, UK. Telephone: + 44 117 928 48 53. Fax: +44 117 925 14 21. E-mail:
[email protected]. † Mucin Research Group, Bristol Eye Hospital, University of Bristol. ‡ H.H. Wills Physics Laboratory, University of Bristol,
that changes with contact lens wear.18 Salivary mucins are generalists in binding and aggregating a wide spectrum of bacteria.19-21 Specific adhesion to mucins may be mediated through carbohydrate moieties, e.g., sulfomucins for Helicobacter pylori22 or sialic acids that act as receptors for Pseudomonas aeruginosa in the eye.23,24 Cysteine-rich regions at the N and C termini of MUC7 and MUC5B have also been shown to participate in bacterial adhesion.25 Here we present a study of the molecular adhesion behavior of mucin glycoprotein molecules covalently attached to an AFM cantilever, as a function of environment. We have chosen to use the AFM in force spectroscopy mode to elucidate the nature and distribution of structures that mediate ocular mucin nonspecific (low-affinity) adherence interactions. Materials and Methods Mucins. Human ocular mucins were extracted and purified from cadaver conjunctivae using classical mucin techniques.26 Briefly, mucins were extracted in 4 M guanidine hydrochloride (GuHCl) with protease inhibitors, isolated after two successive cesium chloride density gradients, and fractionated by gel filtration on Sepharose CL2B in 0.5 M GuHCl. Mucin samples were free of DNA contamination (H 33258 Fluorescent Assay for DNA quantification, Amersham Pharmacia Biotech, Little Chalfont, U.K.). Mucin aliquots were extensively dialyzed against ultrapure water and kept at -20 °C until used. The largest mature mucins, isopycnic density 1.35-1.5 g/mL and excluded on Sepharose CL2B, were used in all experiments. Atomic Force Microscopy. AFM tip-cantilevers (DIVeeco, Santa Barbara, CA) were evaporatively coated with
10.1021/bm000145y CCC: $20.00 © 2001 American Chemical Society Published on Web 03/29/2001
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Mucin Adherence Table 1. Detachments during Approach-Retract Cycles of Mucin-Coated Tip to Micaa detachments approach speed (nm/s) 87.2 87.2 87.2 87.2 87.2 87.2 20.5 38.7
NiCl2 -b +c + + + +
dwell time (s) 0 5 20 0
no.
mean/ cycle
max dist (nm)
cycles
49 144 229 229 151 75 164 132
8 14 25 12 14 25 14 13
9.77 38.15 18.06 68.51 31.02 35.48 35.1 35.5
6 10 9 19 11 3 12 10
a Detachments were indicated by the sudden decrease in the force acting on the cantilever. Changes smaller than 0.1 nN or occurring at less than 5 nm from the surface were neglected for instrumental reasons. b Without Ni(II) present. c With Ni(II) present.
Table 2. Adhesion Energy as a Function of Speed, Dwell Time, and Ionic Environmenta approach speed (nm/s) 87.2
20.5 38.7 348.8
adhesion energy (aJ) NiCl2 + + + + + +
dwell time (s)
mean
SE
nb
0
3.76 10.79 24.41 43.28 21.88 119.55 10.73 12.08 9.32
0.228 0.862 2.651 2.49 5.64 48.28 0.59 1.78 0.63
6 20 9 19 11 3 12 10 10
5 20 0 0 0
a Adhesion energy was calculated as the area between the approach and retract curves, after corrections for instrumental sensitivity. b No. of curves analyzed.
thin layers of first chromium and then gold, 1 and 10 nm, respectively, using a quartz crystal thickness monitor. A selfassembled monolayer (SAM) of 11,11′-dithiobis(succinimidylundecanoate) (DSU) was prepared on the gold surface following the approach of Wagner et al.27 The succinimide group of the SAM binds specifically to primary amines, and whole mucins were attached via the N-terminus or exposed primary amine groups of the backbone polypeptide. The cantilever was dipped into a mucin solution for 10 min and then washed exhaustively with ultrapure water, and excess water was removed by spotting the edge of the cantilever substrate on filter paper. The mucin-functionalized tips were then used in a Dimension AFM (DI-Veeco) which was operated in a force spectroscopy mode. A total of 215 approach and retract curves were obtained over a range of approach distances and approach speeds between 20.5 and 348.8 nm s-1 (Tables 1 and 2), equal in the approach and retract parts of each individual cycle. The software also permitted time delays (dwell times) on the surface between the approach and retract portions of the force curve cycle. Dwell times of 0, 5, and 20 s were used in this study (Table 2). For approaches to a plane surface, freshly cleaved mica (Agar Scientific, Stansted, U.K.) was used. All force curves were obtained in a liquid environment. To investigate the effect of cations on molecular adhesion, NiCl2 solution (10 mM) was used, made up in 10 mM HEPES buffer pH 7.4. The cantilevers used ranged in force constants from 0.06 to 0.38 N m-1 (manufacturer’s
figures), with a precision of 10-20%. The largest force constant cantilevers were used exclusively for the measurement of mucin-substrate forces; softer cantilevers were used for the liquid imaging of mucin structures. Tapping mode cantilevers (nominal spring constant 30 N m-1) were used for imaging structures in air. For mucin-mucin adhesion, a mucin gel was formed by repeatedly depositing 10 µL aliquots of mucins onto the mica surface. In an analogous manner to mucin-tip preparation, mucins were deposited on a SAM and imaged using a Multimode AFM (DI-Veeco). These images were compared with the structures produced by repeated spraying of mucin aliquots onto mica, which were imaged in air. Analysis of the force curves was primarily carried out using spreadsheet macro programs (Windows 2000, Microsoft). This allowed zeroing of the force curves at the contact point of the cantilever on the surface and data offsets to compensate for any drift between the approach and retract null lines. The sensitivity of the photodiode (V/nm) was calculated from the slope of the hard repulsive portion of the approach curve. Calculation of the energy of adhesion was performed by a stepwise summation of the differences between the retract and approach force-distance curves. This calculation was carried out between the contact point on the surface and the last force interaction, which resulted in the cantilever returning to its null position. Fourier analysis (Mathematica, Wolfram Associates) was used to determine whether any periodicities existed in the inter-detachment spacings. Results No adhesions were observed between the SAM-coated tip and mica, irrespective of the presence or absence of NiCl2 in the buffer or the speed of approach. However, with mucin-coated tips, richly diverse force pulloff behavior was observed with two main types of pull-off event: short-length jumps in the force-extension curve denoted as “detachment events” and longer duration “stretching events” (Figure 1). Detachments occurred at distances much smaller than the known lengths of mucin molecules from previous AFM imaging experiments.26 The total number of detachments per cycle increased in the presence of NiCl2, (Table 1) and was maximal at 5 s dwell time. Parts of the force-displacement curve indicative of molecular stretching occurred more readily in the presence of the divalent cation than in its absence (Figure 1, arrows). When the two surfaces were in contact and moving together there were “steps” in both approach and retract portions of the force curve cycle. These disappeared after the first peak in the retract curve, and may well be related to stepwise lateral motion or slippage of the cantilever across the surface. They were not visible in the approach curve when not in contact (Figure 1 D). Adhesion energy increased in the presence of the divalent cations and with lengthening dwell time but was not affected by the approach speed (Table 2). Forces acting on the cantilever at detachments (Figure 2), including force at the last detachment, were smaller in the absence of NiCl2 than in its presence for each dwell time (Figure 2, parts A-C),
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Figure 1. Examples of force-distance retract curves for mucin-mica interactions. Key: (A) 0 s dwell, 38.7 nm s-1, no Ni(II); (B) as in A, but with 10 mM NiCl2 in the HEPES buffer; (C) as in A, but with 5 s dwell time; (D) as in C, in the presence of Ni(II). The arrows indicate typical single molecule stretchlike behavior. The detachment events are manifest as short sudden changes in the cantilever force. Part D also shows the approach portion of the force curve, slightly offset for clarity, illustrating the noise level of the data.
were not affected by speed (Figure 2D), but did increase with dwell time. There was no correlation between force magnitude and distance from the surface. Figure 2A presents data for two series of approach-retract cycles (circles and open diamonds) in the presence of NiCl2, performed at a few hours from each other. A cutoff of 5 nm from the contact point was applied in selecting inter-detachment analysis. In the absence of divalent cations, no major periodicity could be detected in the distribution of distances from the surface at which detachments occurred (Fourier analysis). Most inter-detachment distances were smaller than 4 nm (Figure 3). Neglecting inter-detachment distances smaller than 0.1 nm, to take account of instrument sensitivity, the distributions showed a single peak at less than 0.5 nm in the absence of divalent cations, (Figure 3A), and two peaks less than 1 nm in their presence (Figure 3B), irrespective of dwell time. Increasing the dwell time from 0 to 20 s, the proportion of the 1 nm peak in part B increases from 20 to 40%. Changes in the speed of approach do not seem to affect inter-detachment distances (Figure 3C). Figure 4A, part i, shows the featureless SAM surface before adsorption of mucins. Exposing SAM-coated mica to a mucin solution resulted in the formation of mucin aggregates (Figure 4A, parts ii-iv) with volumes between 105 and 106 nm3. Phase images clearly demonstrated that these aggregates are formed by mucin molecules. Spraying mucins on mica, followed by drying (Figure 4B), resulted in the formation of extended nets covering the entire imaged surface. Occasional single strands could also be seen, emerging from thicker molecular entanglements. Repeated cycles of approach and retraction, in different experimental conditions, did not alter the pattern of interac-
tion between a mucin-coated tip and mica. Presence of mucins on the mica, however, had a profound effect on the force curves. No adhesive interactions were recorded between tip mucins and a sessile mucin gel (Figure 5). Discussion AFM was used to investigate the interface behavior of normal ocular mucins and evaluate the strength of adhesion between tethered mucin molecules and mica, and tethered molecules and a mucin-coated surface. Since no adhesions were observed between tip and mica, it is safe to assume that all adhesions occurred between tip-tethered mucins and the surface. A necessary condition for the correct interpretation of our results is that mucins remain tethered to the tip. Lack of mucin-mucin adhesion and constancy of behavior after cycles of approach and retraction (Figure 2A, circles and open diamonds, obtained several hours apart) indicate that mucins remained attached to the tip throughout, without aging. The choice of Ni(II) as a cation was suggested by its concentration-dependent effect on mucin mobility on mica,26 and by studies showing the congruence between Ni atomic dimensions and the cavity in muscovite mica basal plane28 and its low enthalpy of hydration.29 Adhesion energy between mucins and mica is increased on addition of NiCl2, mainly as a result of an increase in forces at detachment. This change in interaction strength might follow from changes in charge distribution at the molecular surface, consistent with Ni(II) ions residing in cavities on the mica surface.29 Bonds between surface and mucin molecules were not established simultaneously, as
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Figure 3. Distribution of inter-attachment distances Intervals between successive attachments were calculated from force-displacements curves obtained (A) in the absence of Ni(II), or (B) with the divalent cation in the HEPES buffer, and (C) for approach-retract cycles carried out at different speeds in buffer containing NiCl2. Detachments at less than 5 nm from the surface also show peaks around 0.5 and 1.0 nm, respectively, in the absence and presence of Ni(II), but the relative frequency of these intervals is below 20%.
Figure 2. Force-displacement distributions Distributions were calculated for approach-retraction cycles with 0 (A), 5 (B), or 10 (C) s dwell. Varying the speed of approach (D) by 1 order of magnitude had no appreciable effect on either force magnitude or displacement for 0 s dwell on the surface. Most interactions took place at less than 40 nm from the surface. In the absence of Ni(II), closed symbols A-C, adhesion forces and displacements were smaller than in the presence of the cation. There was no correlation between force magnitude and distance from the surface. The data in A for adhesions in the presence of NiCl2 were obtained in two separate runs several hours apart. The absence of a statistically significant difference between the means of these two sets indicates that the structure of the mucins on the tip was unchanged by successive force cycles.
reflected in the increase in the number of interactions and the peak in adhesion energy at dwell of 5 s (Table 2). Some of these attachments seem to disappear or reform with a longer interaction time, supporting the idea of limited diffusion of mucin molecules attached to the tip. Ligandantigen bonds, such as between selectin on endothelial cells and adhesion molecules on T cells have been shown to strengthen during the first second after formation,30 possibly because of formation of multiple nonspecific bonds between the two partners in the interaction. Our results concur to suggest that nonspecific interactions may be slow to form and contribute to the increase in adhesion energy with time. That the force behavior does not vary with retract speed is not altogether surprising given the small range of rates
investigated. The effect of loading rate on binding forces was only apparent over several decades of loading rate.31,32 Forces acting on the cantilever at detachment are of nanonewton magnitude. These force magnitudes, given that mucins remain attached to the tip, support the idea that a number of mucin molecules bridge the gap between tip and mica, and act in parallel.14 Mucins are glycosylated along the whole length of the molecule, though the density of glycosylation varies; regions with dense oligosaccharide insertions alternate with more poorly glycosylated ones, where the height of the molecule suggests a naked peptide core.26 This detail of molecular structure suggests that potential adhesive sites, be they oligosaccharides or amino acids, are present throughout the length of the polymer. The short distances from the surface of less than 100 nm at which detachments occurred, compared with observed molecular lengths of micrometers,26 indicate that mucin molecules were far from fully extended during retraction cycles. Small displacements, large detachment forces and observation of mucin morphology on mica and SAM (Figure 4) suggest that mucin molecules formed a condensed structure on the tip, with only limited lengths of molecules protruding and interacting with the surface. The z-dimension of the mucin aggregates ranges from 1 to 10 nm, compared to the 1 nm thickness of single molecules on mica.26 The likelihood that each molecule is covalently attached at more than one point would constrain the molecule
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Figure 4. Images of mucin surface structures. (A) Surface structures produced on a DSU-SAM mimicking the preparation of the mucintips used in the force spectroscopy experiments. Image i was obtained in 10 mM HEPES buffer. Images ii-iv were obtained in 10 mM HEPES buffer by depositing 5 µL of 1.4-1.5 g/mL mucin onto the SAM, followed by rigorous washing with ultrapure water (to ensure that noncovalently bonded material was removed). (B) Surface structures produced when mucins in aqueous solution were sprayed onto mica and allowed to dry. Images i and ii are from the “neat” mucin solution (20 µL of 1.4-1.5 g/mL, 20 µL of 10 mM HEPES buffer). Images iii and iv are diluted 10 times, and v and vi 100 times.
in a condensed state. Organization of the mucins into a gellike phase tethered to the tip would also account for the
Berry et al.
Figure 5. Interactions between the mucin-coated tip and mucincoated mica. No mucin-mucin adhesive interactions were observed at dwells of either 10 (A), 5 (B), or 0 (C) s. Changes in the slope of the approach trace at nominal separations around 10 nm are indicative of electrostatic repulsive forces between the two mucin surfaces. The small kinks in the approach curves when the two surfaces are in physical contact may be attributed to the deformation of one or other of the mucin surfaces. The small steps on the retract cycle are much smaller than 0.1 nN.
rather long optimal dwell period, seconds, rather than milliseconds, as evaluated for fibrinogen.14 A lower cutoff of 0.1 nm was imposed on the analysis of distances between successive detachments by considerations of instrumental sensitivity. Interestingly, however, smaller intervals were observed experimentally. Attachment of mucins to the mica might be mediated by hydrophobic interactions between the peptide core and mica, and through divalent cations bridging between negative charges on both mica and mucin, as in the case of DNA.29 Ocular mucins
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carry a net negative charge. As the total charge on mucin subunits increases, the number of sialic acids (N-acetylneuraminic acids) decreases, while sulfation increases.33 Ni(II) ions can be tightly bound to mica in the hexagonal cavity between oxygen atoms in the mica basal plane.28 The center to center distance between adjacent cavities is either 0.52 or 0.9 nm,28 which correspond to the two peaks in inter-adhesion distances observed experimentally (Figure 2). In the absence of Ni(II) ions, the inter-attachment distances still reflect the dimensions of the mica lattice, indicating that a mechanism other than electrostatic attraction is operating; such a mechanism might entail sparsely glycosylated regions of the peptide core. From the ratio between the measured volume of molecular assemblies on the tip and that of a mucin molecule, of the order of 1 µm long and a diameter of 1 nm, it appears that each compact structure encompassed a few molecules only. The geometry of the tip (pyramidal macroscopically, segment of sphere at the scale of a molecular assembly) and the 200500 nm distances between neighboring mucin aggregates (Figure 4B) suggest that successive detachments occurred within one aggregate. We cannot infer whether these successive detachment points are in neighboring positions along the molecule, or indeed whether they belong to the same mucin molecule. The small distances between attachment points suggests, however, a large degree of self-crossing or interpenetration between mucin molecules, consistent with images of mucins deposited on mica 26 and Figure 4, parts A and B. In contrast, the anti-adhesive nature of mucins has been shown by the lack of interactions between two mucin gels (on the tip and on the mica substrate), which might be a result of electrostatic repulsion between the short and negatively charged oligosaccharides of the ocular mucins.33,34 In the eye, the mucin gel on the upper lid glides over the gel covering cornea and conjunctiva. Lack of adhesion between mucins might account for the persistence of foreign material (trapped in a mucin gel) in the same position through a number of blinks. Adhesionless gliding of gels over each other may act in tandem with shear thinning of preocular fluid during blink to protect underlying epithelia from being dislodged and to prevent sensory nerve activation. Conclusions Ocular mucins diffuse on the mica surface, and there appears to be an ongoing process of formation of adhesions. Short distances between consecutive detachments indicate the lack of a specialized structure for molecular adhesion, with both oligosaccharides and peptide core implicated in this process. The compact structures observed in AFM images, and the relatively small maximal distances of the force curve detachments, both support a high degree of mucin-mucin interpenetration. Interestingly, mucin-mucin interactions, as seen with force spectroscopy, are minimal, which may go some way to explain physiological observations of tear film stability. Acknowledgment. This work was supported by a BBSRC Bioimaging Initiative grant. We thank Dr Andrew
Round for useful discussions of the results and Andrew Humphris for help with data analysis. References and Notes (1) Haupt, B. J.; Ennis, J.; Sevick, E. M. Langmuir 1999, 15, 38863892. (2) Brown, H. R. Science 1994, 263, 1411-1413. (3) Krupenkin, T. N.; Taylor, P. L. Macromolecules 1995, 28, 58195826. (4) Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Biophys. J. 1999, 77, 542-552. (5) Chen, X.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Edwards, J. C. Langmuir 1997, 13, 4106-4111. (6) Glantz, P.; Arnebrant, T.; Nylander, T.; Baier, R. Scand. Odont. Acta 1999, 57, 238-241. (7) Binnig, G.; Quate, C. F.; Gerber, C. H. Phys. ReV. Lett. 1986, 56, 930-933. (8) Merkel, R.; Nassoy, P.; Leung, A.; Ritchie, K.; Evans, E. Nature 1999, 397, 50-53. (9) Radmacher, M. Phys. World 1999, 12, 33-37. (10) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science (Washington, DC) 1997, 276, 1109-1112. (11) Carrion-Vazquez, M.; Marszalek, P. E.; Oberhauser, A. F.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11288-11292. (12) Rief, M.; Clausen-Schauman, H.; Gaub, H. E. Nature Struct. Biol. 1999, 6. (13) Marszalek, P. E.; Pang, Y.-P.; Li, H.; El Yazal, J.; Oberhauser, A. F.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 78947898. (14) Hemmerle, J.; Altmann, S. M.; Maaloum, M.; Horber, J. K.; Heinrich, L.; Voegel, J. C.; Schaaf, P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6705-6710. (15) Van Klinken, B. J.-W.; Dekker: J.; Buller, H. A.; Einerhand, A. W. C. Am. J. Physiol. (Gastrointest. LiVer Physiol.) 1995, 32, G613G627. (16) Fleiszig, S. M. J.; Zaidi, T. S.; Pier, G. B. In Lacrimal Gland, Tear film, and dry eye Syndromes; Sullivan, D. A., Ed.; Plenum Press: New York, 1994; pp 359-362. (17) Willcox, M. D. P.; Stapleton, F. Med. Microbiol. ReV. 1996, 7, 123131. (18) Willcox, M. D. P.; Hume, E. B. H. Aus. N-Z J. Ophthalmol. 1999, 27, 231-233. (19) Groenink, J.; Ligtenberg, A. J. M.; Van Winkelhoff, A.-J.; Veerman, E. C. I.; Niew Amerongen, A. V. XIV International Symposium on Glycoconjugates; Chapman & Hall: Zurich, Switzerland, 1997. (20) Nieuw Amerongen, A. V.; Bolscher, J. G. M.; Veerman, E. C. I. Glycobiology 1995, 5, 733-740. (21) Liu, B.; Rayment, S.; Oppenheim, F. G.; Troxler, R. F. Arch. Biochem. Biophys. 1999, 364, 286-293. (22) Bravo, J. C.; Correa, P. J. Clin. Pathol. 1999, 52, 137-140. (23) Hazlett, L. D.; Moon, M.; Berk, R. S. Infect. Immun. 1986, 51, 687689. (24) Hazlett, L. D.; Masinick, S.; Barrett, R.; Rosol, K. Infect. Immun. 1993, 61, 5164-5173. (25) Liu, B.; Rayment, S. A.; Gyurko, C.; Oppenheim, F. G.; Offner, G. D.; Troxler, R. F. Biochem. J. 2000, 345, 557-564. (26) McMaster, T. J.; Berry, M.; Corfield, A. P.; Miles, M. J. Biophys. J. 1999, 77, 533-541. (27) Wagner, P.; Hegner, M.; Kernen, P.; Zaugg, F.; Semenza, G. Biophys. J. 1996, 70, 2052-2066. (28) Nishida, S.; Biggs, S.; Scales, P. J.; Healy, T. W.; Tsunematsu, K.; Tateyama, T. Langmuir 1994, 10, 4554-4559. (29) Hansma, H. G.; Laney, D. E. Biophys. J. 1996, 70, 1933-1939. (30) Pierres, A.; Benoliel, A. M.; Bongrand, P.; van der Merwe, P. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15114-15118. (31) Evans, E.; Ritchie, K.; Biophys, J. 1997, 72, 1541-1555. (32) Gergely, C.; Voegel, J.-C.; Schaaf, P.; Senger, B.; Maaloum, M.; Ho¨rber, J. K. H.; Hemmerle, J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10802-10807. (33) Ellingham, R. B.; Berry, M.; Stevenson, D.; Corfield, A. P. Glycobiology 1999, 9, 1181-1189. (34) Berry, M.; Ellingham, R. B.; Corfield, A. P. InVest. Ophthalmol. Vis. Sci. 2000, 41, 398-403.
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