A comparison of anterior adhesive areas and secretions in Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis (Monogenea : Monocotylidae) from the gills of the shovelnose ray, Rhinobatos typus (Rhinobatidae)

This study continues the collection of data on the anterior adhesive areas and secretions of monopisthocotylean monogenean (flatworm) parasites and begins an investigation of their phylogenetic usefulness. Here, two species of parasitic worms from an elasmobranch, Troglocephalus rhinobatidis (Monocotylidae: Dasybatotreminae) and Neoheterocotyle rhinobatidis (Monocotylidae: Heterocotylinae), are compared and contrasted. It has been suggested in recent literature that these two taxa are more closely related than is currently recognised. Our data support this view. Both species have multiple apertures on the ventral anterior margin through which adhesive is secreted. Two types of secretion exit from multiple adjacent duct endings terminating in each aperture: rod-shaped (S1) and spherical-shaped (S2) bodies. S1 bodies of both species show nano-banding of similar size and are membrane bound. Ultrastructure of the glands, ducts, duct endings and secreted adhesive is similar for both species, but aperture shape differs. Away from the adhesive areas, tegumental inclusions are found to differ between the two species and another, apparently non-adhesive, secretion is found in N. rhinobatidis.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Preliminary characterisation and extraction of anterior adhesive secretion in monogenean (platyhelminth) parasites

Secreted anterior adhesives, used for temporary attachment to epithelial surfaces of fishes (skin and gills) by some monogenean (platyhelminth) parasites have been partially characterised. Adhesive is composed of protein. Amino acid composition has been determined for seven monopisthocotylean monogeneans. Six of these belong to the Monocotylidae and one species, Entobdella soleae (van Beneden et Hesse, 1864) Johnston, 1929, is a member of the Capsalidae. Histochemistry shows that the adhesive does not contain polysaccharides, including acid mucins, or lipids. The adhesive before secretion and in its secreted form contains no dihydroxyphenylalanine (dopa). Secreted adhesive is highly insoluble, but has a soft consistency and is mechanically removable from glass surfaces. Generally there are high levels of glycine and alanine, low levels of tyrosine and methionine, and histidine is often absent. However, amino acid content varies between species, the biggest differences evident when the monocotylid monogeneans were compared with E. soleae. Monogenean adhesive shows similarity in amino acid profile with adhesives from starfish, limpets and barnacles. However, there are some differences in individual amino acids in the temporary adhesive secretions of, on the one hand, the monogeneans and, on the other hand, the starfish and limpets. These differences may reflect the fact that monogeneans, unlike starfish and barnacles, attach to living tissue (tissue adhesion). A method of extracting unsecreted adhesive was investigated for use in further characterisation studies on monogenean glues.

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton [1-4]. In the context of stable adhesion [1], cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis [2]. Here actin polymerization has been proposed to drive cell surfaces together [5], although F-actin reorganization also occurs as cell contacts mature [6]. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere [7]. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.

E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts

Classical cadherins mediate cell recognition and cohesion in many tissues of the body. It is increasingly apparent that dynamic cadherin contacts play key roles during morphogenesis and that a range of cell signals are activated as cells form contacts with one another. It has been difficult, however, to determine whether these signals represent direct downstream consequences of cadherin ligation or are juxtacrine signals that are activated when cadherin adhesion brings cell surfaces together but are not direct downstream targets of cadherin signaling. In this study, we used a functional cadherin ligand (hE/Fc) to directly test whether E-cadherin ligation regulates phosphatidylinositol 3-kinase (PI 3-kinase) and Rac signaling. We report that homophilic cadherin ligation recruits Rae to nascent adhesive contacts and specifically stimulates Rae signaling. Adhesion to hE/Fc also recruits PI 3-kinase to the cadherin complex, leading to the production of phosphatidylinositol 3,4,5-trisphosphate in nascent cadherin contacts. Rae activation involved an early phase, which was PI 3-kinase-independent, and a later amplification phase, which was inhibited by wortmannin. PI 3-kinase and Rae activity were necessary for productive adhesive contacts to form following initial homophilic ligation. We conclude that E-cadherin is a cellular receptor that is activated upon homophilic ligation to signal through PI 3-kinase and Rae. We propose that a key function of these cadherin-activated signals is to control adhesive contacts, probably via regulation of the actin cytoskeleton, which ultimately serves to mediate adhesive cell-cell recognition.

Anterior adhesive areas and adjacent secretions in the parasitic flatworms Decacotyle lymmae and D. tetrakordyle (Monogenea: Monocotylidae) from the gills of stingrays

The monogeneans Decacotyle lymmae and D. tetrakordyle (Monocotylidae: Decacotylinae), from gills of the dasyatid stingrays Taeniura lymma and Pastinachus sephen, respectively, have a single aperture for adhesive secretion on each side of the anterior ventrolateral region. Rod-shaped bodies (S1) and electron-dense spherical secretion (S2) exit through specialised ducts opening adjacent to one another within these apertures. The S1 bodies are 230 +/- 11 nm wide and greater than or equal to4 mum long in D. lymmae and 240 +/- 9 nm wide and greater than or equal to3.3 mum long in D. tetrakordyle. The S2 bodies have a diameter of 88 +/- 7 nm in D. lymmae and 65 +/- 6 nm in D. tetrakordyle. The apertures are unusual in being extremely small (internal diameter, 3-5 mum). Each aperture has a slit-like surface opening as small as 160 nm wide, surrounded by muscle fibres indicating that they may be opened and closed. The aperture is also surrounded and underlain by muscle fibres that may aid in secretion from, or even eversion of, the tissue within the aperture. Sensilla/cilia are also found within the apertures. Additional secretions from anteromedian and anterolateral glands (body glands), each containing granular secretions, occur in profusion and exit anteriorly and posteriorly to the position of the apertures, through duct openings in the general body tegument. These granular secretions do not appear to be associated with anterior adhesion. Both species show similarities in aperture, underlying tissue, sense organ, and secretion detail, in accordance with findings from other monogenean genera, and which supports the importance of such data for phylogenetic studies.

Mechanism of adhesion and detachment at the anterior end of Merizocotyle icopae (Monogenea : Monocotylidae) including ultrastructure of the anterior adhesive matrix

The anterior adhesive mechanism was studied for Merizocotyle icopae (Monogenea: Monocotylidae). Adult anterior apertures can open and close. In addition, duct endings terminating within the apertures are everted or retracted depending on the stage of attachment. Adhesive in adults is synthesized from all 3 secretory types (rod-shaped, small and large spheroidal bodies) found within anterior apertures. All exit together and undergo mixing to produce the adhesive matrix, a process that depletes duct contents. A greater number of ducts carrying rod-shaped bodies is depleted than ducts containing spheroidal bodies which changes the ratio of secretory types present on detachment. Detachment involves elongation of duct endings and secretion of additional matrix as the worm pulls away from the substrate. The change in secretory type ratio putatively modifies the properties of the secreted matrix enabling detachment. Only after detachment do ducts refill. During attachment, individual secretory bodies undergo morphological changes. The larval and adult adhesive matrix differs. Anterior adhesive in oncomiracidia does not show fibres with banding whereas banded fibres comprise a large part of adult adhesive. The data Suggest that this is the result of adult spheroidal secretions modifying the way in which the adult adhesive matrix forms.

A comparison of the anterior adhesive system in the oncomiracidium and adult of the monogenean parasite Merizocotyle icopae (Monocotylidae)

The anterior adhesive system of the oncomiracidium and adult of Merizocotyle icopae (Monogenea: Monocotylidae) were compared. The oncomiracidium has one ventrally placed aperture on either side of the head near the anterior extremity. In the adult, there are three ventrally placed apertures on either side of the head region. Both systems have three types of electron-dense secretory bodies opening into each aperture. A rod-shaped secretion (S1) and a small electron dense ovoid secretion (S2) are common to larvae and adults. The third secretion type differs: in adults, it is a large, spherical (S3) type but in larvae, it is an ovoid (S4) body. S4 bodies do occur in adults, but appear to be secreted as a general body secretion. An additional anteromedian secretion (S5) is also present in the oncomiracidium, but is not secreted into the anterior apertures. Homology and function of secretions are discussed.

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Perforated corneal hydrops treated with sulfur hexafluoride (SF6) gas and tissue adhesive

Purpose: To report a case of a perforated acute hydrops in a mentally retarded patient that was successfully managed with intracameral sulfur hexafluoride gas and cyanoacrylate tissue adhesive. Methods: Interventional case report. Results: A 14-year-old mentally retarded male patient with keratoconus presented with a perforated acute hydrops. A bandage contact lens was applied. However, following a large emesis 2 days later, the aqueous leak worsened with shallowing of the anterior chamber. Under general anesthesia, sulfur hexafluoride was injected to reform the anterior chamber and cyanoacrylate tissue adhesive was applied to the perforated site and covered by a bandage contact lens and temporary tarsorrhaphy. A follow-up examination at 1 month showed a formed anterior chamber with tissue adhesive in situ and no aqueous leak. Conclusions: The successful use of intracameral sulfur hexafluoride and tissue adhesive in the management of perforated acute hydrops may avoid emergency tectonic penetrating keratoplasty and reduce potential complications in the poorly cooperative patient.

Suctions, fracture energy, and plastic cracking of cement mortar and concrete

Plastic cracking of cement mortar and concrete is primarily attributable to desiccation by evaporation from unprotected surfaces. This causes high suctions (negative pressures) to develop in the pore water adjacent to these surfaces. Dissolved salts in the pore water can also contribute significantly to suctions. Quantitative expressions are available for all of the components of the total suction. The development of suctions over time is illustrated by the results of desiccation tests conducted on cement mortars, supplemented by data from the literature. It is shown that ambient conditions conducive to plastic cracking can arise almost anywhere, but that the extremely high suctions that develop in mature cement mortar and concrete do not imply that compression failures should occur A high value of fracture energy is derived from data from the desiccation tests that implies that plastic cracking is characterized by a significant zone of plastic straining or microcracking.

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts.