scholarly journals Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment.

1996 ◽  
Vol 132 (6) ◽  
pp. 1189-1198 ◽  
Author(s):  
M F Champliaud ◽  
G P Lunstrum ◽  
P Rousselle ◽  
T Nishiyama ◽  
D R Keene ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Tight Binding ◽  
Human Amnion ◽  
Laminin 5 ◽  
Integrin Alpha6beta4 ◽  
Epithelial Basement Membrane ◽  
Stable Association ◽  
Membrane Components

Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.

scholarly journals Role of the Corneal Epithelial Basement Membrane in Ocular Defense against Pseudomonas aeruginosa

2009 ◽  
Vol 77 (8) ◽  
pp. 3264-3271 ◽  
Author(s):  
Irania Alarcon ◽  
Lesley Kwan ◽  
Chong Yu ◽  
David J. Evans ◽  
Suzanne M. J. Fleiszig
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Basement Membrane ◽  
In Vitro Models ◽  
Corneal Epithelial ◽  
Barrier Effects ◽  
Epithelial Basement Membrane ◽  
Transmission Electron ◽  
Superficial Epithelium

ABSTRACT Pseudomonas aeruginosa can invade corneal epithelial cells and translocates multilayered corneal epithelia in vitro, but it does not penetrate the intact corneal epithelium in vivo. In healthy corneas, the epithelium is separated from the underlying stroma by a basement membrane containing extracellular matrix proteins and pores smaller than bacteria. Here we used in vivo and in vitro models to investigate the potential of the basement membrane to defend against P. aeruginosa. Transmission electron microscopy of infected mouse corneas in vivo showed penetration of the stroma by P. aeruginosa only where the basement membrane was visibly disrupted by scratch injury, suggesting that the intact basement membrane prevented penetration. This hypothesis was explored using an in vitro Matrigel Transwell model to mimic the corneal basement membrane. P. aeruginosa translocation of multilayered corneal epithelia grown on Matrigel was ∼100-fold lower than that of cells grown without Matrigel (P < 0.005, t test). Matrigel did not increase transepithelial resistance. Matrigel-grown cells blocked translocation by a P. aeruginosa protease mutant. Without cells, Matrigel also reduced traversal of P. aeruginosa and the protease mutant. Fluorescence microscopy revealed a relative accumulation of bacteria at the superficial epithelium of cells grown on Matrigel at 3 h compared to cells grown on uncoated filters. By 5 h, bacteria accumulated beneath the cells, suggesting direct trapping by the Matrigel. These findings suggest that the basement membrane helps defend the cornea against infection via physical barrier effects and influences on the epithelium and that these roles could be compromised by P. aeruginosa proteases.

scholarly journals A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Alina Akhbanbetova ◽  
Shinichiro Nakano ◽  
Stacy L. Littlechild ◽  
Robert D. Young ◽  
Madara Zvirgzdina ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Basement Membrane ◽  
Endothelial Damage ◽  
Epithelial Basement Membrane ◽  
Fibril Structure ◽  
Consistent Manner ◽  
Stromal Edema ◽  
Epithelial Basement

Purpose. To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. Methods. A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. Results. The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemet’s area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. Conclusions. Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction.

scholarly journals Polymorphonuclear leukocyte migration through human amnion membrane

1981 ◽  
Vol 91 (2) ◽  
pp. 459-467 ◽  
Author(s):  
RG Russo ◽  
LA Liotta ◽  
U Thorgeirsson
Keyword(s):  
Basement Membrane ◽  
Single Layer ◽  
Previous Method ◽  
Epithelial Surface ◽  
Human Amnion ◽  
Morphological Studies ◽  
Collagenous Stroma ◽  
Amnion Membrane

A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma. The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.

Kalinin: A new epithelial basement membrane adhesion molecule

1991 ◽  
Vol 49 ◽  
pp. 178-179
Author(s):  
Douglas R. Keene ◽  
Gregory P. Lunstrum ◽  
Patricia Rousselle ◽  
Robert E. Burgeson
Keyword(s):  
Basement Membrane ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Keratinocytes ◽  
Positive Staining ◽  
Human Amnion ◽  
Epithelial Basement Membrane ◽  
Type Vii Collagen ◽  
Keratinocyte Cell ◽  
Epithelial Basement

A mouse monoclonal antibody produced from collagenase digests of human amnion was used by LM and TEM to study the distribution and ultrastructural features of an antigen present in epithelial tissues and in cultured human keratinocytes, and by immunoaffinity chromatography to partially purify the antigen from keratinocyte cell culture media.By immunofluorescence microscopy, the antigen displays a tissue distribution similar to type VII collagen; positive staining of the epithelial basement membrane is seen in skin, oral mucosa, trachea, esophagus, cornea, amnion and lung. Images from rotary shadowed preparations isolated by affinity chromatography demonstrate a population of rod-like molecules 107 nm in length, having pronounced globular domains at each end. Polyacrylamide gel electrophoresis suggests that the size of this molecule is approximately 440kDa, and that it is composed of three nonidentical chains disulfide bonded together.

scholarly journals Shift in collagen type as an early response to induction of the metanephric mesenchyme.

1981 ◽  
Vol 89 (2) ◽  
pp. 276-283 ◽  
Author(s):  
P Ekblom ◽  
E Lehtonen ◽  
L Saxén ◽  
R Timpl
Keyword(s):  
Basal Lamina ◽  
Type Iv Collagen ◽  
Early Response ◽  
Type I ◽  
Metanephric Mesenchyme ◽  
Type Iv ◽  
Interstitial Collagens ◽  
Membrane Components

Conversion of the nephrogenic mesenchyme into epithelial tubules requires an inductive stimulus from the ureter bud. Here we show with immunofluorescence techniques that the undifferentiated mesenchyme before induction expresses uniformly type I and type III collagens. Induction both in vivo and in vitro leads to a loss of these proteins and to the appearance of basement membrane components including type IV collagen. This change correlates both spatially and temporally with the determination of the mesenchyme and precedes and morphological events. During morphogenesis, type IV collagen concentrates at the borders of the developing tubular structures where, by electron microscopy, a thin, often discontinuous basal lamina was seen to cover the first pretubular cell aggregates. Subsequently, the differentiating tubules were surrounded by a well-developed basal lamina. No loss of the interstitial collagens was seen in the metanephric mesenchyme when brought into contact with noninducing tissues or when cultured alone. Similar observations were made with nonnephrogenic mesenchyme (salivary, lung) when exposed to various heterotypic tissues known to induce tubules in the nephrogenic mesenchyme. The sequential shift in the composition of the extracellular matrix from an interstitial, mesenchymal type to a differentiated, epithelial type is so far the first detectable response of the nephrogenic mesenchyme to the tubule-inducing signal.

scholarly journals Reading and Function of a Histone Code Involved in Targeting Corepressor Complexes for Repression

2005 ◽  
Vol 25 (1) ◽  
pp. 324-335 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Youngsok Choi ◽  
Philip A. Cole ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hormone Receptors ◽  
Critical Role ◽  
Pericentric Heterochromatin ◽  
Histone Code ◽  
Stable Association ◽  
And Function

ABSTRACT A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

Chapter 56 Human amnion membrane as a substratum for axonal elongation in vitro and in vivo

1988 ◽  
pp. 435-438 ◽  
Author(s):  
Scott N. Blaker ◽  
George E. Davis ◽  
Marston Manthorpe ◽  
Eva Engvall ◽  
Silvio Varon ◽  
...  
Keyword(s):  
Human Amnion ◽  
Axonal Elongation ◽  
Amnion Membrane

Interactions between mesoderm cells and the extracellular matrix following gastrulation in the chick embryo

1991 ◽  
Vol 99 (2) ◽  
pp. 431-441
Author(s):  
A.J. Brown ◽  
E.J. Sanders
Keyword(s):  
Extracellular Matrix ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Primitive Streak ◽  
Cell Binding ◽  
Fab Fragment ◽  
Fibronectin Receptor ◽  
In Vitro Methods

In the gastrulating chick embryo, the mesoderm cells arise from the epiblast layer by ingression through the linear accumulation of cells called the primitive streak. The mesoderm cells emerge from the streak with a fibroblastic morphology and proceed to move away from the mid-line of the embryo using, as a substratum, the basement membrane of the overlying epiblast and the extracellular matrix. We have investigated the roles of fibronectin and laminin as putative substrata for mesoderm cells using complementary in vivo and in vitro methods. We have microinjected agents into the tissue space adjacent to the primitive streak of living embryos and, after further incubation, we have examined the embryos for perturbation of the mesoderm tissue. These agents were: cell-binding regions from fibronectin (RGDS) and laminin (YIGSR), antibodies to these glycoproteins, and a Fab' fragment of the antibody to fibronectin. We find that RGDS, antibody to fibronectin, and the Fab' fragment cause a decrease in the number of mesoderm cells spread on the basement membrane, and a perturbation of cell shape suggesting locomotory impairment. No such influence was seen with YIGSR or antibodies to laminin. These results were extended using in vitro methods in which mesoderm cells were cultured in fibronectin-free medium on fibronectin or laminin in the presence of various agents. These agents were: RGDS; YIGSR; antibodies to fibronectin, fibronectin receptor, laminin and vitronectin; and a Fab' fragment of the fibronectin antiserum. We find that cell attachment and spreading on fibronectin is impaired by RGDS, antiserum to fibronectin, the Fab' fragment of fibronectin antiserum, and antiserum to fibronectin receptor. The results suggest that although the RGDS site in fibronectin is important, it is probably not the only fibronectin cell-binding site involved in mediating the behaviour of the mesoderm cells. Cells growing on laminin were perturbed by YIGSR, RGDS and antibodies to laminin, suggesting that mesoderm cells are able to recognise at least two sites in the laminin molecule. We conclude that the in vivo dependence of mesoderm cells on fibronectin is confirmed, but that although these cells have the ability to recognise sites in laminin as mediators of attachment and spreading, the in vivo role of this molecule in mesoderm morphogenesis is not yet certain.

Analysis of elongating morphogenesis of quail anterior submaxillary gland: absence of localized cell proliferation

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 229-239
Author(s):  
Hiroyuki Nogawa
Keyword(s):  
Cell Proliferation ◽  
Basement Membrane ◽  
Regional Differences ◽  
Submaxillary Gland ◽  
Mesenchymal Cells ◽  
Submaxillary Glands ◽  
Parallel Direction ◽  
Recombination Experiments

Quail anterior submaxillary glands elongated extensively without branching (more than sevenfold) from 8 to 10 incubation days. Investigation of mitotic activity of the rudiments in vivo showed no localized cell proliferation throughout the rudiments, and recombination experiments in vitro to examine regional differences in mitogenic activity of the surrounding mesenchyme also showed that no mesenchymal region specifically stimulates the epithelial cell proliferation. Histological observation of the rudiments showed that epithelial cells did not lengthen in a parallel direction to the long axis of the rudiment, and that mesenchymal cells encircled the epithelial cord perpendicularly to its axis. The basement membrane was obscure in the distal end of the rudiments, while it was easily detected in the other part of the rudiments. These results suggest that the elongating morphogenesis of the anterior submaxillary rudiments is not achieved by localized cell proliferation but by almost uniformly distributed cell proliferation, and mesenchymal cells surrounding the rudiment or the basement membrane may be involved in the controlling mechanisms of the elongating morphogenesis.

Preparation Of Antithrombin, High Activity Heparin And A Heparin-Antithrombin Complex By A Rapid Two-Step Affinity Method

1981 ◽  
Author(s):  
R Jordan ◽  
T Zuffi ◽  
M Fournel ◽  
D Schroeder
Keyword(s):  
Ionic Strength ◽  
High Activity ◽  
Tight Binding ◽  
Therapeutic Benefit ◽  
Purification Scheme ◽  
The Matrix ◽  
Low Ionic Strength ◽  
Potential Therapeutic Benefit

The tight binding affinity of antithrombin for heparin makes possible a relatively selective purification scheme based on salt elution from heparin-Sepharose. We have found, however, that purity can often be greatly increased if the elution is carried out with soluble heparin instead. This heparin can be removed from the antithrombin, either in whole or part, by a second affinity step on Concanavalin A Sepharose. The antithrombin, which binds to the matrix through its glycosidic moieties, retains its ability to bind heparin at physiological ionic strengths. Thus, the complex of antithrombin and heparin is readily isolated free of unbound heparin species. The complex can be eluted intact with low ionic strength buffers containing sugars which compete for binding to the lectin. Alternatively, the high activity heparin (400–500 units/mg) can be obtained separately by a 1 M NaCl wash which is then followed by a carbohydrate wash to obtain the purified antithrombin.We have made certain preliminary biochemical and anticoagulant characterizations of these materials. Not unexpectedly, both the high activity heparin and its complex with antithrombin show significantly greater in vitro potency in comparison to unfractionated heparin. In vivo anticoagulant efficacy, as evaluated in a rabbit infusion model, confirmed the in vitro findings and further suggests some potential therapeutic benefit may be derived from infusion of a preformed heparin-antithrombin complex.

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