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1.  Experimental epikeratophakia using tissue lathed at room temperature. 
This report presents for the first time the results of carrying out epikeratophakia with tissue lathed at room temperature. Using an experimental model of epikeratophakia in the rabbit, we evaluated tissue handling techniques for the preparation of donor lenticules. Details of the technique are described and the in-vivo and histopathological findings reported.
PMCID: PMC1041453  PMID: 3293653
2.  Measurement of intraocular pressure after epikeratophakia 
AIMS—To assess the accuracy of three commonly used tonometers in eyes after epikeratophakia.
METHODS—Five eye bank eyes with sutured epikeratophakia buttons were connected to a manometer and a pressure transducer. Intraocular pressure was adjusted in 5 mm Hg increments from 0 to 50 mm Hg. The intraocular pressure was measured at each increment using a Goldmann tonometer, a pneumatonometer, and a Tono-pen.
RESULTS—The difference between the manometer (actual pressure) and the Goldmann tonometer ranged from −19 to + 9 mm Hg (mean (SD) overestimation 2.6 (5.8) mm Hg). The pneumatonometer error ranged from −27.5 to + 5.5 mm Hg (mean (SD) overestimation 4.7 (6.1) mm Hg), and for the Tono-pen the range was −18 to + 11 mm Hg (mean (SD) overestimation 0.05 (7.9) mm Hg). The correlation coefficients for the three tonometers were 0.94, 0.92, and 0.87 for the Goldmann tonometer, pneumatonometer, and Tono-pen respectively.
CONCLUSION—The Goldmann tonometer had the best correlation with the manometer in eye bank eyes with epikeratophakia (correlation coefficient 0.94), but none of the tonometers was accurate over the entire range of pressures tested. Detection of glaucoma in eyes with epikeratophakia cannot rely on tonometry alone, but requires examination of the optic nerve and visual field.

PMCID: PMC1722219  PMID: 9274407
3.  Improved preservation of human corneal basement membrane following freezing of donor tissue for epikeratophakia. 
Current methods for the production of lenticules for epikeratophakia involve rapid freezing, cryolathing, and slow warming of the donor cornea. We have found that this procedure causes structural damage to the epithelial basement membrane in the donor cornea which may subsequently contribute to poor postoperative re-epithelialisation of the implant, leading to graft failure. Endeavouring to overcome these problems, the effects of cryoprotection of donor cornea were investigated, using dimethyl sulphoxide, in conjunction with different cooling and warming rates as part of the protocol for cryolathing. The structural integrity of the epithelial basement membrane zone (BMZ) was then assessed by electron microscopy and by immunofluorescence microscopy using antibodies to types IV and VII collagen, components of the basal lamina and anchoring fibrils respectively, and an antibody to a component of the anchoring filaments. No differences in the pattern of immunostaining for these components were detected, indicating that the composition of the BMZ was unaltered by the different treatment regimens applied. However, electron microscopy showed that preservation of basement membrane ultrastructure was markedly improved when cornea was warmed rapidly rather than slowly, both in cryoprotected and non-cryoprotected tissue. Epithelial cell retention and preservation of stromal architecture appeared superior in cryoprotected samples, while keratocyte structure was heterogeneous throughout the experimental groups. Further work is in progress to assess the efficacy of these protocols in the preservation of keratocyte viability in association with improved basement membrane structure in donor tissue for epikeratophakia.
PMCID: PMC504974  PMID: 7848985
4.  Epikeratophakia for aphakia, keratoconus, and myopia. 
A series of 67 cases of epikeratophakia is presented with an average time from surgery of 12.2 months. For aphakia there was a delay in the recovery of vision, but by nine months 83% of 57 patients achieved an acuity equal to, or within 1 line of, the preoperative value. 57% were corrected to within 3 dioptres of emmetropia, but in the latter part of the series 75% were within this range. Astigmatism and reduced contrast sensitivity, especially in the presence of glare, were important complications. For keratoconus, 86% of seven patients with over two months of follow-up achieved a spectacle corrected acuity of 6/9 or better. One patient had surgery for myopia and obtained the desired refractive correction.
PMCID: PMC1041992  PMID: 2310729
5.  Expression and potential role of major inflammatory cytokines in experimental keratomycosis 
Molecular Vision  2009;15:1303-1311.
The aim of this study was to investigate the expression and regulation of the four major inflammatory cytokines in fungal keratitis (FK) with the goal of further understanding its pathogenesis in order to develop more effective therapeutic approaches.
Aspergillus fumigatus and Candida albicans were the corneal pathogens selected for this study to establish murine FK using epikeratophakia with the aid of corneal epithelium erasion. One, three, five, and seven days post-infection, the corneal lesions and inflammatory responses were observed by slit-lamp and histopathology, and the expressions of the four inflammatory cytokines, macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant (KC), interleukin-1β (IL-1β), and interleukin-6 (IL-6), in the infected corneas were determined using reverse transcription polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). For the intervention experiment with neutralizing antibodies, the experimental mice were then injected subconjunctivally with 5 μl (2 ng/μl) MIP-2 or IL-1β polyclonal antibody 1 h before and 24 h after surgery. Reestablishment of the FK murine model was performed following injection. Effects of MIP-2 or IL-1β polyclonal antibody on the corneal diseases were observed by slit-lamp microscopy, histopathology, and ELISA.
Expression of MIP-2, KC, IL-1β, and IL-6 was upregulated significantly in the infected group one, three, five, and seven days after surgery. Following treatment with an MIP-2 polyclonal antibody, the corneal clinical scores and inflammatory responses decreased, the MIP-2 protein levels were downregulated significantly (p<0.01), and the KC protein levels decreased slightly (p>0.05). Upon administration of IL-1β polyclonal antibodies, the decrease in clinical scores, inflammatory responses, and protein levels of MIP-2 and KC was apparent at 1 and 3 days after infection (p<0.01).
A persistent, high level expression of MIP-2 and IL-1β is an important and even major factor in the corneal pathogenesis of FK. Specific polyclonal neutralizing antibodies may be administered to inhibit the major chemokines and cytokines responsible for corneal damage thus effectively relieving the injury caused by FK.
PMCID: PMC2707360  PMID: 19590756
6.  Rehabilitation of children with cataracts. 
Over a period of 10 years, 160 children with cataracts underwent operation at the University of Tennessee Medical Center, Memphis. The surgical, optical, and psychosocial rehabilitation of these patients was analyzed and studied. The optical rehabilitation included patients with glasses, intraocular lens implants, epikeratophakia, and contact lenses. Seventy three of these patients were chosen at random and reevaluated as to visual outcome, and 46 were subjected to a psychosocial test to evaluate their quality of life and their rehabilitation. Eighteen of these were also given a psychosocial test to evaluate the quality of life enjoyed by these children at an older age following treatment for the cataract. Surgical, optical, and psychosocial rehabilitation of such children is also discussed. This is the first report of the psychological evaluation of such children. The further needs of these children as they approach adulthood are discussed in detail.
PMCID: PMC1298408  PMID: 10360302
7.  Refractive Surgery: New Options for Visual Correction 
Canadian Family Physician  1986;32:1505-1510.
Refractive surgery is now at the forefront of ophthalmological development and has proved to be a safe and effective method of correcting refractive errors. The incisions performed during radial keratotomy flatten the cornea to reduce or eliminate myopia. In keratomileusis, resecting and reshaping a portion of the cornea can correct either myopia or hyperopia when the cornea is sutured back into position. In epikeratophakia, donor corneas become “living contact lenses”. They have the potential for correcting high degrees of myopia and hyperopia. Although there are controversial aspects to refractive surgery, most informed people consider that the benefits outweigh the risks for suitable candidates.
PMCID: PMC2327443  PMID: 21267102
ophthalmology; eye; refractive surgery
8.  Biomechanical properties of Achilles tendon repair augmented with a bioadhesive-coated scaffold 
The Achilles tendon is the most frequently ruptured tendon. Both acute and chronic (neglected) tendon ruptures can dramatically affect a patient’s quality of life, and require a prolonged period of recovery before return to pre-injury activity levels. This paper describes the use of an adhesive-coated biologic scaffold to augment primary suture repair of transected Achilles tendons. The adhesive portion consisted of a synthetic mimic of mussel adhesive proteins that can adhere to various surfaces in a wet environment, including biologic tissues. When combined with biologic scaffolds such as bovine pericardium or porcine dermal tissues, these adhesive constructs demonstrated lap shear adhesive strengths significantly greater than that of fibrin glue, while reaching up to 60% of the strength of a cyanoacrylate-based adhesive. These adhesive constructs were wrapped around transected cadaveric porcine Achilles tendons repaired with a combination of parallel and three-loop suture patterns. Tensile mechanical testing of the augmented repairs exhibited significantly higher stiffness (22–34%), failure load (24–44%), and energy to failure (27–63%) when compared to control tendons with suture repair alone. Potential clinical implications of this novel adhesive biomaterial are discussed.
PMCID: PMC3046464  PMID: 21266745
9.  Understanding Marine Mussel Adhesion 
In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.
PMCID: PMC2100433  PMID: 17990038
adhesion; biomimetics;  marine mussel (Mytilus edulis);  recombinant protein
10.  Roles of discontinuities in bio-inspired adhesive pads 
Morphological intricacies of the biological attachment pads generate considerable interest owing to their remarkable ability to control adhesion to various surfaces. Motivated by the adhesive microstructures of insects, we examine the behaviour of adhesion and crack propagation in patterned adhesive films. These films are made of silicone elastomers that were patterned with lateral, longitudinal or crosswise incisions from which a thin silanized glass plate was removed in a displacement-controlled peel experiment. The behaviours of crack propagation on these patterned adhesive films are controlled by simple incision patterns, their depths and spacing. With the crosswise incisions, significant enhancement (×10–20) of fracture energy has been achieved. These findings point towards an important mechanism by which of biological organisms might enhance adhesion, and provide a simple design principle for manipulating the interfacial fracture in a variety of artificial attachment devices.
PMCID: PMC1578261  PMID: 16849164
patterned adhesive film; interfacial fracture toughness; bio-mimetic adhesion
11.  The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading 
Molecular Biology of the Cell  2011;22(22):4380-4389.
GTPase regulator associated with focal adhesion kinase-1 (GRAF1) interacted with endocytic and adhesion proteins, and GRAF1 endocytic activity was up-regulated in spreading cells and concentrated at the leading edge of migrating cells. Depletion of GRAF1 resulted in profound defects in cell spreading. GRAF1 remodeled membrane microdomains at adhesions, aiding membrane turnover during cell morphological changes.
The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.
PMCID: PMC3216663  PMID: 21965292
12.  Investigation of Bioinspired Gecko Fibers to Improve Adhesion of HeartLander Surgical Robot 
In this paper, a way for improving adhesion of a mobile robot (HeartLander) on biological tissue is presented, that integrates bioinspired gecko adhesive fibers on the robot surface. HeartLander is a medical robot proposed to perform clinical procedures on a beating heart, overcoming limitations of current cardiac procedures. Biologically inspired gecko fibers have been proposed for adhesion on surfaces. The aim of this work is to assess the advantages of integrating these structures for enhancing the grip between the artificial device and the myocardial tissue. Experimental in vitro tests have been carried out assessing the performance of the HeartLander attached to muscular tissue. The effect of the adhesive fibers on improving the adhesion behavior on a slippery surface has been investigated, obtaining a friction increase of 57.3 %.
PMCID: PMC3563248  PMID: 23366040
13.  Cadherin adhesion in the intestinal crypt regulates morphogenesis, mitogenesis, motogenesis, and metaplasia formation. 
Molecular Pathology  1999;52(4):166-168.
The topographical organisation of the epithelium lining mucous membranes has been an intense point of research. One of the fundamental biological issues underpinning this and associated issues relates to the role and regulation of epithelial adhesion molecules. Adhesion between individual cells allows an intact layer to be formed, which is selectively permeable. In addition, the orchestrated regulation of multiple adhesion molecules allows the gradual transition from basal secretory cells to apical absorptive cells in the crypt-villus gradient. Moreover, it is becoming clear that no one class of adhesion molecule can sufficiently govern crypt architecture; however, the main cell-cell adhesion molecules are the cadherins and the related desmosomal cadherins. These latter molecules interact with the catenins, which bind directly or indirectly with cytoskeletal molecules such as Rho and Rac. In addition, other complex glycoproteins, such as the carcinoembryonic antigens, might contribute to adhesion, although their mechanisms of function are distinctly different. Integrins on the basal aspect of the cells also signal important morphoregulatory signals as a result of their binding to the extracellular maxtrix. The disruption of these physiological processes also provides a necessary and, in some cases, sufficient molecular mechanism for cancer invasion and metastasis, such as occurs in E-cadherin mutation positive familial gastric cancer.
PMCID: PMC395694  PMID: 10694934
14.  Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions 
The Journal of Cell Biology  1993;123(4):993-1005.
The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.
PMCID: PMC2200138  PMID: 8227154
15.  PtdIns(3,4)P2 instigates focal adhesions to generate podosomes 
Cell Adhesion & Migration  2009;3(2):195-197.
Cell-to-extracellular matrix (ECM) adhesion plays important roles in various biological events, such as proliferation, differentiation and migration. Distinct from other types of adhesion structures (focal complexes, focal adhesions and so on), podosomes and invadopodia are thought to have additional functions beyond attachment, possibly including invasion into the ECM. For podosomes and invadopodia to invade into the ECM, molecules involved in adhesion, actin polymerization and ECM degradation must be recruited to sites of action. Our recent study demonstrated that podosomes form near newly formed focal adhesions via the minimally expressed phosphoinositide PtdIns(3,4) P2-mediated recruitment of the Tks5-Grb2 scaffold, followed by the accumulation of N-WASP. Although this study demonstrated details of molecular interplay during the transformation of focal adhesion, its regulation in the in vivo invasion process remains to be clarified. Here, we discuss the molecular bases of the transformation of focal adhesions to podosomes/invadopodia based on current understanding.
PMCID: PMC2679885  PMID: 19262173
podosome; invadopodium; focal adhesion; Tks5; PtdIns(3,4)P2; N-WASP
16.  Loss of the Desmosomal Component Perp Impairs Wound Healing In Vivo 
Epithelial wound closure is a complex biological process that relies on the concerted action of activated keratinocytes and dermal fibroblasts to resurface and close the exposed wound. Modulation of cell-cell adhesion junctions is thought to facilitate cellular proliferation and migration of keratinocytes across the wound. In particular, desmosomes, adhesion complexes critical for maintaining epithelial integrity, are downregulated at the wound edge. It is unclear, however, how compromised desmosomal adhesion would affect wound reepithelialization, given the need for a delicate balance between downmodulating adhesive strength to permit changes in cellular morphology and maintaining adhesion to allow coordinated migration of keratinocyte sheets. Here, we explore the contribution of desmosomal adhesion to wound healing using mice deficient for the desmosomal component Perp. We find that Perp conditional knockout mice display delayed wound healing relative to controls. Furthermore, we determine that while loss of Perp compromises cell-cell adhesion, it does not impair keratinocyte proliferation and actually enhances keratinocyte migration in in vitro assays. Thus, Perp's role in promoting cell adhesion is essential for wound closure. Together, these studies suggest a role for desmosomal adhesion in efficient wound healing.
PMCID: PMC2902749  PMID: 20628490
17.  Induction of Cell Polarization and Migration by a Gradient of Nanoscale Variations in Adhesive Ligand Spacing 
Nano letters  2008;8(7):10.1021/nl801483w.
Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and signalling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand, can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 μm when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization, and initiate cell migration and signalling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.
PMCID: PMC3811077  PMID: 18558788
18.  Molecular markers of cell adhesion in ameloblastomas. An update 
Ameloblastoma is the most common odontogenic tumor of epithelial origin, and though it is of a benign nature, it frequently infiltrates the bone, has a high rate of recurrence and could potentially become malignant. Cellular adhesion potentially plays an important role in the manifestation of these characteristics and in the tumor biology of ameloblastomas. Losses of cell-cell and extracellular matrix adhesion and cohesion are among the first events that occur in the invasion and growth of tumors of epithelial origin. The present review includes a description of the molecules that are involved in cell adhesion as reported for various types of ameloblastomas and discusses the possible roles of these molecules in the biological behaviors of this odontogenic tumor. Knowledge of the complex mechanisms in which these molecules play a role is critical for the research and discovery of future therapeutic targets.
Key words:Ameloblastoma, cellular adhesion, molecular markers, cell-cell adhesion, extracellular matrix-cell adhesion.
PMCID: PMC3909437  PMID: 23986011
19.  Antithrombin significantly influences platelet adhesion onto immobilized fibrinogen in an in-vitro system simulating low flow 
Thrombosis Journal  2006;4:19.
Adhesion of platelets onto immobilized fibrinogen is of importance in initiation and development of thrombosis. According to a recent increase in evidence of a multiple biological property of antithrombin, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in-vitro flow system.
Platelets in anticoagulated whole blood (29 healthy blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 13 s-1 to 1500 s-1). Platelet adhesion onto fibrinogen-coated slips was assessed using a fluorescence laser-scan microscope and compared to the plasma antithrombin activity. Additionally the effect of supraphysiological AT supplementation on platelets adhesion rate was evaluated.
Within a first minute of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin were observed at 13 s-1 and 50 s-1 (r = -0.48 and r = -0.7, p < 0.05, respectively). Significant differences in platelet adhesion related to low (92 ± 3.3%) and high (117 ± 4.1%) antithrombin activity (1786 ± 516 U vs. 823 ± 331 U, p < 0.05) at low flow rate (13 s-1, within first minute) have been found. An in-vitro supplementation of whole blood with antithrombin increased the antithrombin activity up to 280% and platelet adhesion rate reached about 65% related to the adhesion rate in a non-supplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4 p = 0.008, respectively).
It appears that antithrombin in a low flow system suppresses platelet adhesion onto immobilized fibrinogen independently from its antithrombin activity. A supraphysiological substitution of blood with antithrombin significantly reduces platelet adhesion rate. This inhibitory effect might be of clinical relevance.
PMCID: PMC1618384  PMID: 17040572
20.  Stimulation of integrin-mediated adhesion of T lymphocytes and monocytes: two mechanisms with divergent biological consequences 
The Journal of Experimental Medicine  1994;179(4):1307-1316.
We show that the adhesion of T lymphoid cells to immobilized fibronectin can be increased by two distinct mechanisms. The first is by increasing the affinity of the fibronectin receptor/ligand interaction using the anti-beta 1 integrin monoclonal antibody 8A2. The second is by treating the cells with phorbol 12-myristate 13-acetate (PMA), which alters events that occur after receptor occupancy (e.g., cell spreading) without affecting receptor affinity. The effects of these two mechanisms on adhesion in the presence of physiological concentrations of soluble fibronectin suggest that they have different biological consequences. Under these conditions, the net effect of increasing the affinity of the fibronectin receptors is to decrease cell adhesion, whereas the increase in adhesion induced by PMA is unaffected. This suggests that the high affinity receptors are not primarily available for cell adhesion under these circumstances, and that they have an alternative function. We further show that high affinity binding of soluble fibronectin can be induced by either differentiation of the monocytic cell line THP-1 or by cross-linking the T cell receptor complexes on the T lymphoid cell line HUT-78. The differentiated monocytic cells express two populations of fibronectin receptors: a minority in a high affinity state, and the majority in a low affinity state. Thus they will both continue to adhere in the presence of physiological concentrations of soluble fibronectin and bind significant amounts of soluble fibronectin at the cell surface.
PMCID: PMC2191445  PMID: 7511685
21.  Cell adhesion and response to synthetic nanopatterned environments by steering receptor clustering and spatial location 
HFSP Journal  2008;2(5):276-285.
During adhesion and spreading, cells form micrometer-sized structures comprising transmembrane and intracellular protein clusters, giving rise to the formation of what is known as focal adhesions. Over the past two decades these structures have been extensively studied to elucidate their organization, assembly, and molecular composition, as well as to determine their functional role. Synthetic materials decorated with biological molecules, such as adhesive peptides, are widely used to induce specific cellular responses dependent on cell adhesion. Here, we focus on how surface patterning of such bioactive materials and organization at the nanoscale level has proven to be a useful strategy for mimicking both physical and chemical cues present in the extracellular space controlling cell adhesion and fate. This strategy for designing synthetic cellular environments makes use of the observation that most cell signaling events are initiated through recruitment and clustering of transmembrane receptors by extracellular-presented signaling molecules. These systems allow for studying protein clustering in cells and characterizing the signaling response induced by, e.g., integrin activation. We review the findings about the regulation of cell adhesion and focal adhesion assembly by micro- and nanopatterns and discuss the possible use of substrate stiffness and patterning in mimicking both physical and chemical cues of the extracellular space.
PMCID: PMC2639948  PMID: 19404439
22.  The Role of the Extracellular Domain in the Biology of the Coxsackievirus and Adenovirus Receptor 
The Coxsackievirus B and Adenovirus Receptor (CAR) plays a dual role as a homotypic junctional adhesion protein and as a viral receptor. CAR is a transmembrane protein and a member of the Immunoglobulin (Ig) superfamily with two extracellular Ig-like domains. The most distal Ig-like domain (D1) mediates the homophilic interaction and is also responsible for the high-affinity binding of the adenovirus (Ad) fiber protein. Currently, no activity has been ascribed to the proximal Ig-like domain (D2). To further understand the function of the extracellular domain in the biological activities of CAR, we created extracellular deletion mutants and evaluated cellular localization, adhesion, and viral infection. Deletion of any segment of the extracellular domain results in loss of adhesion and mislocalization as explained by a model, termed “diffusion trapping,” that suggests adhesion is the driving force in junctional localization. Loss of junctional localization and adhesion was particularly apparent in polarized human airway epithelia, where mutant CAR expression was basolateral but not limited to the lateral junctions between cells. Surprisingly, the D2 domain was required for adenovirus fiber-knob binding and infection. In summary, the entire extracellular domain of CAR is of vital importance to the biology of this highly conserved and important protein.
PMCID: PMC2715320  PMID: 15778494
adenovirus; CAR; Coxsackievirus; extracellular domain; receptor
23.  Cell Adhesion to Unnatural Ligands Mediated by a Bifunctional Protein 
This paper describes a molecular strategy to restore adhesion of cells to surfaces that otherwise do not present ligands that can mediate adhesion. The approach is based on a carbonic anhydrase fusion protein that binds benzenesulfonamides and that also includes the RGD peptide motif that can bind to cell-surface integrin adhesion receptors. In this way, the fusion protein can bind to a monolayer that presents the benzenesulfonamide ligand, thereby positioning the RGD peptide at the surface, where it can mediate the adhesion and spreading of cells. This strategy may provide a general method for promoting the adhesion of cells to non-natural surfaces or to defective biological matrices.
PMCID: PMC2907716  PMID: 20583796
24.  Supported bilayers at the vanguard of immune cell activation studies 
Journal of structural biology  2009;168(1):152-160.
Biological adhesion between cells is critical for development of multicellular organisms and for the function of the adaptive immune system of vertebrates. A gap in understanding of adhesion systems arises from the difficulty of collecting quantitative data on the molecular interactions underlying adhesion, which is typically studied by population statistics such as percent adhesion in the presence of empirically defined forces to separate less adherent cells. Supported lipid bilayers formed on glass surfaces offer a useful model system in which to explore some basic features of molecular interactions in adhesive contacts. We have exploited that lateral mobility of molecules in the supported planar bilayers and fluorescence microscopy to develop a system for measurement of two-dimensional affinities and kinetic rates in contact areas. Affinity measurements are based on a modified Scatchard analysis. Measurements of kinetic rates are based on fluorescence photobleaching after recovery at the level of the entire contact area. This has been coupled to a reaction-diffusion equation that allows calculation of on-and off-rates. We have found that mixtures of ligands in supported planar bilayers can effectively activate T lymphocytes and simultaneously allow monitoring of the immunological synapse. Recent studies in planar bilayers have provided additional insights into organization principles of cell-cell interfaces. Perennial problems in understanding cell-cell communication are yielding to quantitative measurements based on planar bilayers in areas of ligand driven receptor clustering and the role of the actin cytoskeleton in immune cell activation. A major goal for the field is determining quantitative rules involved in signaling complex formation.
PMCID: PMC2762084  PMID: 19500675
adhesion; bilayer; signaling; immunology; cytoskeleton; affinity; receptors
25.  Protein-based underwater adhesives and the prospects for their biotechnological production 
Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives.
PMCID: PMC3086556  PMID: 20890598

Results 1-25 (66127)